Crystaiiographic studies of the formimino tramferase domain from the

bifunctional enzyme formimhotransferase-cyclodeaminase.

By Darcy John Reinard KohIs

Department of Bioctiemistry

McGill University

Moatréai

Short Title: Structurai studies of the formiminotransferase domain.

A thesis submitted to the Faculty of Graduate Studies and Research in partid

fdfillment of the requirements for the degree of Master of Science,

Q Darcy IR KohIs, August 1999

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Abstract

The strucnire of the formiminotramferase (FT) domain of

Formiminotransferase-cycIodeaminase (EC 2.12.5) - (EC 4.3.1.4) has been solved in

cornplex with the product analogue, lolinic acid, and in comptex with the substrate

analogue, C5,CIû-dideazatetrahydrofolate. The protein is arranged as a homodimer,

with each subunit comprishg two a /p subdomaius which adopt a novef protein fold.

Wittiin each subunit, an electrostatic tunnel which traverses the width of the molecule

is observeci and comprises the ligand binding sites. The distnïution of charged

residues in the tunnel enables us to propose the mode of binding for the natural

substrate, te~ydropteroylpolyglutamate. ModeIing studies indicate that a y-Iinked

triglutamate form of the tetrahydrofolate substrate can be accommodated thcough the

length of the tunnel. The electron density also indicates ttiat a single molecule of

glyceroI is bound to each protomer at the base of a second tunnei contacthg the main

electrostatic tunnel. This second tunneI is the expected entrance for

formiminoglutamate to the active site of the enzyme. The structure has enabled us to

propose that the residue His82 may be important in ihe cataiytic mechanism of the

transferase reaction,

Résumé

La structure du domaine formiminotrausfera~e (FT) de la

formiminotransferase-cyclodeaminase (EC 2.1.2.5) - (EC 43.1.4) a été résotue en

présence d'un analogue de produit, l'acide folinique, ainsi qu'en présence d'un

analogue de substrat, le C5,ClOdideazatetrahydrofolate. La protéine existe sous la

forme d'un homodimère dont chaque sous-unite est composée de deux sousdomaine

de type a@ qui adoptent un repliement protéique unique à ce jour. On observe, a

t'intérieur de chaque sous-unite, un tunel éléctrostatique qui traverse la molecuIe en

largeur en incluant les sites de liaison des ligands. La distriiution des résidus chargés

le long du le tunel, nous permet de proposer un mode de liaison pour le substrat

naturai: le tetrahydropteroylpolyglutamate. Des études de modélisation on indique

que la forme triglutamate (liaison gamma) du substrat, le tetrahydrofolate, peut être

accomodée dans le sens de la longueur du tunel. La densité éléctronique indique aussi

qu'une moiecule de glycerol est liée à chacun des protomères; elle se retrouve aussi i

la base d'un second tunel qui communique avec le tunel éléctrostatique principal. Ce

second tunel est une entrée vers le site actif probable pour le fomiiminog[utamate.

Cette sûucnire nous permet de propose le résidue His82 comme étant un élément

important dans le rnéchanisme catalitique de la réaction de tramferase.

Acknowiedgments

I'd like to express my heartfelt gratitude to my thesis supervisor, Dr. Mice

Vrielink, for her support, encouragement and guidance in this endeavour. She has

provided me with many opporhmities to expand my scientific abilities. 1 am also

appreciative of her prompt and careh1 reading of this thesis.

1 would also like to thank my colIaborators Dr. Robert E. MacKenzie, Dr.

Enrico Purisima and Traian S u l a for their advice and assistance in this project.

I am indebted to put and present members of tfie VrieIink laboratory for

creating an enjoyable envimament in which to work. Thanks to Jaime Cheah,

Nathdie Cmteau, Paula Lario, Kimberiey Yue, Rakesh Khanna and Enrico Schlieff

who had first wekomed me to the !ab, provided me with technical assistance and

animated scientific discussions. 1 am gratefiil to Dr. Bijan SobeI-Ahvazi for his

Enendship and advice. I woutd aiso hie to acknowledge the camaraderie of Alpesh

Patel, Rupert Abdalian and Meak Chhuom.

1 would dso Iike to thank René Coulombe for translating my abstract into

French as well as pmviding assistance in the laboratory. AIso thanks to Dr. M. Cygler

and members of his group for their assistance in structure soIving.

i am gratefd to Hervé Hogues and Stephane Raymond for providing excellent

technicd support with the cornputers. I wouid ais0 Iike to acknowledge ai i members

of the laboratory of Dr. K. Gehring, in particular, N.S- and A S . for making the

arduous trek to BR1 easier to bear.

I would Iike to thank my famiIy for their inspiration, encouragement and

support all of which is appreciated. They have provideci me with so much

understanding and patience. Thanks to al1 my &ends in Montreal and Vancouver

who have also provided support.

1 would like to express the sùicerest gratitude to my fiancée, Jaime Cheah,

whose love and passion bolsters rny spirit and who provided Iimitless encouragement

and support.

Thanks to the Medical Research Council, Canada (research grant to Dr. A.

Vrielink) for financial support.

Contributions of authors

Portions of the work presented in this thesis have been published in the

following journais:

Crystallization and preliminary X-ray andysis of the formiminotransferase

dornain h m the bifictional enzyme fomiminotransferase£yclodeaminase. Kobls,

D., Croteau, N., Mejia, N., MacKenzie, R.E. & Vnelink, A. (1999). Acta Crysr. D 55,

1206-1208.

The crystal structure of formiminotransferase domain of

fonniminotransferase-cyclodeaminase: implications for substrate channeling in a

bifhctional enzyme. Kohls, D., Sulea, T., Purisima, E.O., MacKenzie, R.E. &

Vrielink, A. (2000). Smtcture 8,3546.

All of the work presented in this thesis is my own, with the following

exceptions. N. Croteau determineci the initiai conditions for obtaining crystals of FT

domain, N. Mejia originally provided pure protein for crystaliization trials and later

provided me with a protocol for protein purification. T. Suiea and E. Purisirna

perfomed the molecuiar modeling studies on the substrate (6s)-

tetrahydropteroyltridutamate-Nme, and pmvided the respective section 2.6 in

Methods and Materiais entitled "Substrate docking" as well as the Section 3.10 in the

Results and Discussion entitled "Product analogue versus substrate bùiding" which is

part of the manuscript listed above. R.E. MacKenzie provided me with the C5,CIO-

dideazatetrahydrofolate as well as advice and reviewed manuscripts as appropnate for

coiiaborator. k Vrïelink revised the manuscripts and provided support and guidance

as her roIe as research supervisor.

Table of Contents

PAGE

ABSTRACT

RÉsUMÉ

ACKNOWLEDCEMENTS

CONTRIBUTiON OF AUTHORS

TA8LE OF CONTENTS

ABREVLATIONS

CHAPTER ONE - INTRODUCTION

1.1 Folate metaboIism

1.2 Formiminotransferasei:yclodeaminase

13 Structures of other enzymes exhibithg channehg

CHAPTER TWO - MATERIALS AND EXPERIMENTAL METIIODS

2.1 Protein purification and concentration 22

23 Crystallizahon with foihic acid 22

2.3 ûther qstakation experiments 24

2.4 Heavy atom screening and preparation 27

2.5 Data coIIection and structure determination 28

3.5.1 Crystal ni cornplex with f o l k acid 28

2.53 Crystal in compIex with C5,C IO-dideazatetmhydrofolate 33

CHAPTER TFIREE - RESULTS AND DISCUSSION

3.1 Crystallization of formiminoiransferase domain

3.3 Other crystallization experiments

3.3 Phase determination

3.4 Phase irnprovement by density modification techniques

3.5 Mode1 building and refinernent

3 -6 Overd structure

3.7 The structure of the protomer

3.8 Dimeric interface

3.9 Ligand binding sites

3.10 Product d o g u e versus subsûate binding

3.1 1 CataIytic mechanism

3.12 Daia collection and mode1 rehement of FT domain in complex

with C5,C IOaideazatetrahydrofo[ate

3-13 Sîructine of FT domain in complex with

CS, C 1 O-dideazatetrahydrofolate

CHAPTER FOUR - CONCLUSION AND FUTURE PERSPECTIVES 110

Abbreviations

A

AiCAR

BME

BSA

CD

O C

D U S

DHFR

dUMP

EDTA

FIGLU

FPGS

FT

Fr-ddf

Angstrom*

5-aminO-eimidazole-~arboxamide ninucleotide

B-mercaptoethanol

bovine senun albumin

cyclodeaminase

degree(s) Celsius

dehydrogenase / cyclohydroIase 1 synthetase

dihydrofolate reductase

dideoxyuracil-monophosphate

ethyienediaminetetraacetic acid

formirninoglutamate

folylpolyglutamate synthetase

formirninotransferase

formiminotramferase domain in complex with C5,C 10-

dideazatetrahydio folate

formirninotramferase domain in complex with folinic acid

formiminomferase-cyclodeaminase

giycinamide n'bonucleotide

tetrahydrofolate

tetrahydrofolate with n giutamates attached

Kelvin

kiloDaIton(s)

M

mg

mL

mM

rnm

P

MFTR

MIR

MOPS

NAD

NADP

NCS

Ni-NTA

PCMBS

PLP

SDS PAGE

SHMT

S R

Tris

TS

viv

m o h

miIligram(s)

rniiii tiire(s)

millirnolar

rnillimetre(s)

micrometre(s)

NSfi10-methylenetetrabydroptmylpolyglut reducrase

multiple isomorphous replacement

39-morpholino) propanesirlfonic acid

nicotinamide adenine dinucleotide

nicotinamide adenine dinucieotide phosphate

non-crystaIlographic symmetry

nickel-cheIated nitriIoacetic acid ma&

p-chlorornercurobefl~oicsulfonate

pyridoxal5'-phosphate

revolution(s) per minute

sodium dodecytsulfate poIyacryIamide sel ekctrophoresis

serine hydroxymethyltransfera~e

single isomorphous reptacement

tris@ydrovethyi)methylamine

thymidylate synthase

volume per voIume

Chapter one

Introductioa

Folate rnetabolism is of great importance to both prokaryotic and eukaryotic

cells as it provides a pool of one-carbon uni& that serve in a multitude of biochemical

pathways. One-carbon uni& are c d at different oxidation States by various

derivatives of tetrahydrofolate (El&eGlu) (Figure 1.1). These derivatives cary the

one-carbon unit at either the N5 or Nt0 position, or bndged between the NS and NI0

to give a cyclic fom of the derivative. Cells can interconvert these derivatives with

relative case. in addition, these folate derivatives are polyglutamylated within cells,

generaily with four to nine y-linked glutamate residues.

Falates serve in pathways for the synthesis of nucIeic acids, regmeration of

methionine and thymidylate synthesis. In the synthesis of purines, two one-carbon

units are used h m NlO-fonnyü&PteGlu, in two separate reactions. Glycinamide

nbonucleotide (GAN transformylase catdyzes the mausfer of the îkst one-carbon

unit, while 5-amin0-4-imidazole-chxarnide ninucleotide (AICAR)

transformylase advity catalyses the transfér of the second one-cahn unit (Mueiier

and Benkovic, t982). GAR transformylase have been found to exist as a covalent

multibctional enzyme complex with the additionai aciïvities of GAR synthetase and

arninoimidazok nbonucleotide synthecase (Daubner et uL 1985; Aimi et al. 1990) in

eukaryotic sources. Proknryotic GAR transformylases are found as monofimctionai

Figure 1.1

Chernical structure of tetrahydropteroylpolygIutamate. One carbon units are carcied at

the N5 or NI0 or bridged between the two. Polyglutamylation occurs with glutamates

added in y-luikage (modifieci h m Murley, 19%)-

enzymes. A bacterial GAR transfonnytase (purT), which, instead of using N10-

formyiH&eGlu, uses formate, has also been identified (Nygaard & Smith, 1993;

Marolewski et al. 1994). GAR transformylase h m E. coli shows a similar sequence

to the eukaryotic form. The crystaI structure of E. coli GAR transformylase has been

solved (Chen et al. 1992; Almassy et al. 1992; Klein et al. 1995) revealing two

domains which are joined by a central P-sheet. The N-terminal domain adopts a

characteristic Rossmann fold with a phosphate ion bound to the C-terminal end of the

first B-strand. The structure of GAR transformylase complexed with various folate

analogues has led to the development of various anti-cancer dmgs (Vamey et al.

1997). The E. coli crystd structure of GAR synthetase has also been solved (Wang et

al. 1998) which reveals an d B structure that can be divided into four domains.

Interestingiy, the structure of GAR synthetase shows structurai similarity to the N-

terminal region of GAR transfomylase.

In the synthesis of thymidylate, NSJ10-methyleneH4PteGlu, donates a one-

carbon unit to dideoxyuracil-monophosphate (dUMP) in a reaction cataIyzed by

thymidylate synthase (TS). The pteridine ring becomes oxidized to dihydrofolate in

this transfer which is then reduced by dihydrofolate reductase (DHFR). in

bacteriophages, fiin@, prokaryotes, mammalian v-es and vertebrates TS and DHFR

are separate monofiinctional enzymes, However, in protozoa (Ferone & Roland,

1980; Ivanetic & Santi, 1990) and some plants (Ceiia et al. 1991 ; Lazar et al- 1993)

both TS and DHFR activities are found encoded on a single polypeptide chah

Interestingly, TS-DHFR shows the ability to chamiel and crystal structure anaiysis has

provided insight to the mechanisrn of channeling (Knigfiton et al. 1994) (Further

discussed in section 1.3).

u1 methionine synthesis, N5,NlO-methyleneCtpteGlu, reductase (MFTR)

irreversiily catalyses the reduction of N5,NIO-methyleneHJ'teGIu, to N5-

rnethyl&.PteGlu,. Methionine is then formed by the transfer of the methyl group

h m 5-rnethylH&eGlu, to homocysteine by methionine synthase, which uses

vitamin Biz as a cofactor. Vitamin B12 has been impiicated in post-transcriptional

regdation of methionine synthase (Gulati et al. 1999). This also effectively removes

homocysteine, which, at high levels, is indicated to be a significant nsk for

cardiovascular diseases (Refsum et al. 1998).

in order to maintain an adequate supply of folate derivatives to the various

pathways, the ceIl must be able to interconvert folate derivatives into a variety of

usab le forms. N5,N 1 O-methyleneH&eGlun, NS,N 1 O-methenylH&eGlu, and N 1 O-

f o m y ~ e G l u , can be interconverted by the trifunctional ~ ~ ~ ( ~ ) + - d q e n d e n t

enzyme, N5,N 1 O-methyleneH&eGlu, dehydrogenase (D), NS ,N IO-

methenyWeGlu, cyciohydrolase (C) and N10-formyllt9teGlu, synthetase (S).

D U S is thought to be regulated as a housekeeping enzyme @xi and MacKemie,

1991) and is expressed at low levels in aii tissues (Thigpen et al. 1990; Peri and

MacKenzie, 199 1). Interconversion of N5,Nl O-rnethyIeneHgteGlu, and NI 0-

forrnyH&eGlu,, are thought to be kept near equiiibrium (Pelletier and MacKenzie,

1995). Recently, the crystal structure of a dimer of the D/C domain h m the cytosolic

irifunctiond enzyme has been solved (Maire et al. 1998). The structure shows that

the NADP' cofactor binds to one waii of a wide cleft fonned by two a / p domains.

This NADP' binding site is shown to have a characteristic Rossrnann fold which was

not predicted by amino acid sequence analysis. The opposite waü of this cleft is the

expected binding site for the ligand, N5,NIO-metheny~eGlu,. These structural

studies support previous implications that the dehydrogenase-cyclohydrolase substrate

binding sites overlap (Tan & MacKenzie, 1977; Schirch, 1978; Dnunmond et al.

1983; Appling & Rabinowitz, 1985).

Serine is the major contributor of one-carbon units in the ce11 (reviewed by

MacKenzie, 1984). Carbon-3 of serine is transférred to H4PteGlu, by serine

hydroxyrnethyltransferase (SHMT), producing NS,N10-methenyi.H.,PteGlu, and

glycine. SHMT and DIUS are capable of converting NSJlO-meîheny~eGlu, to

NS-formyEWteGlu, (Stover and Schirch, 1990) with low specific activity. N5-

formyiH&eGlu, has an inhibitory tünction to several folatedependent enzymes

(reviewed in Stover et al. 1990) and couId be a storage form of folate ( h c h w i t z et

al. 1994). NS-formylH&eGlu, can be converted back to NS,NI O-methenyH@teGlu,

through an ATP-dependent reaction catalyseci by N5J1O-methenyH@teGlu,

synthetase. Recently, it has been shown that mitochondrial and cytoplasmic SHMT

isozymes kom Saccharomyces cmaisoe Cunction in different directions dependhg on

the nutritional environment of the ceH (Kastanos et al. 1997). It was found that the

cytosolic SHMT is the primary provider of folates for purine synthesis when the cells

are grown in a medium with serine as the primary one-carbon source. Mitochondrial

SHMT was the dominant isozyrne in cataiyzing the synthesis of serine h m one-

carbon units when ceus were grom on giycîne. The crystd structures of both human

and rabbit cytosotic SHMT have been solved (Renwick et ai. 1998; Scarsdale, et al.

1999) in complex with pyridoxal S'-phosphate (Pm) bound in the active site. Both

structures show an overail fold typicd for the a class of PLP dependent enzymes.

These structures provide insight into the mechanism of the transfer reaction. Since

serine is the major conûibutor of one-carbon units in the ce11 (reviewed in

MacKenzie, 1984), human SHMT provides a target for the design of

chemotherapeutic agents to inhibit the pathways of purine synthesis.

Tetrahydrofolate can be reconstituted h m two activities of 10-

fonnylH.@GIu, dehydrogenase-hydro lase. With the fmt activity, tetrahydro folate

and COz are produced from an NADP'-dependent dehydrogenase reaction, while the

second hydrolase activity, releases the o n e a b o n unit as formate. This enzyme was

shown to bind a polyglutarnylaied form of tesahydrofolate quite strongly (Cook &

Wagner, 1982; Min et al. 1988), with the strongest binding occuning with the

pentaglutamylated form. The enzyme is composed of four identicai subunits (Schirch

et al. 1994) and binds one molecule of the.HSteGIu~ per subunit (Kim et al. 1996).

Wagner and colleagues (Cook es al. 1991) have shown through arnino acid sequence

analysis that the N-terminal domain possesses the hydrolase activity while the C-

terminal domain has a dehydrogenase activity. Recently, crosslinking experiments

have reveded that the tight binding folate site is on the N-terminal domain and is

separate h m the cataiytic site (Fu, et al. 1999).

AIthough folates are transportecl uito the ceil in a monoglutarnylated form

(Nahas et al. 1972; Hoffbrand et al. 1973), poIygiutamylation of folates play a crucial

part in metaboikm (reviewed in Schirch & Smng, 1989; Shane, 1989; Lin et al.

1996). Polygiutarnylation of folates is necessary for the retention and efficient use of

this coenzyme within the cell- Elongation of the poIyglutarnate tail is perforrned by

the ATPdependent enzyme, folylpolyglutamate syathetase (FPGS), in mammalian

tissues, both cytosol and rnitochondna isozymes exist. The crystal structure of FPGS

h m Lactobacilhs casei has been solved (Sun et al. 1998) showing ihat the protein

consists of two domains, one domain has a mononuclmtide-phosphate binding fold

while the second domain is similar to that of dihydrofolate reductase.

PoIygIutamylation of h1ates has been shown IO improve the catdytic efficiency of

most enzymes involved in iolate metabolism, as these enzymes show preferential

binding for certain polygIutmylated substrates or inhibitors (Matthews & Baugh,

1980) and in some cases act to improve the binding of other non-folate substrates

(Matthews, 1984; Findlay et al. 1989). The length of the poiygiutamate tail has b e n

implicated in controlling the flux of folates through the various metabolic pathways

skce different enzymes have different preferences for folates of specific

polyglutamate tait length (Baggott & Knundieck. 1979). FinaIly, polyglutamate chain

length is important in substrate channeling as in the case of the bihinctionai enzyme,

focmiminotransferaçe~yclodeaminase (FTCD), where optimal channeling occurs with

the pentagiutamylated fom of its substrate (MaciCemie & Baugh, 1980; Paquin et al.

I985) (Further discussed in section 1.3).

Fonnate and histidine serve as a minor source of one-carbon units in the folate

pool (BIakely, 1969; reviewed in MacKenzie, 1984). During the degradation of

histidine a one-carbon unit is rescued (reviewed in Shane & Stokstad, 1984). The

k t step in histidine degradation is cataIyzed by histidine arnmonia-lyse where the

a-amino group is e b a t e d , d t i n g in both an a-$ unsaturateci tmns-urocanate

and amrnonia. Recently, the crystal structure of a histidine arnrnonia lyase fiom

Pseudomonas putida has been solved to 2.1A resolution (Schwede et al, 1999)

reveding that an autocataiytic cyclization and dehydration of residues 142-144 (Ala-

Ser-Gly) produces the electrophile 4-methylidene-imidazole-5-one which is necessary

for cataiyzing this first step. The next step is catalysed by the enzyme urocanase

which produces in imidazolone proprionate. Formiminoglutamate (FIGLU), which is

the initial substrate of the bifunctionai enzyme FTCD, is then produced by the

hydroLysis of imadozolone. The degradation of FIGLU is carried out through a

fotatedependent reaction (Tabor & Rabinowitz, 1956; Tabor & Wyngarden, 1959).

The bifiinctionai enzyme, fomiminotransferasecyclodeaminase (FTCD),

catalyses two independent but sequential reactions in the histidine degradation

pathway in mammalian liver. The transferase activity of FïCD transfers the

formimino group of formiminoglutamate to the N5 position of tetrahydrofolate

producing 5-forrniminotetrahydrofolate and glutamate. The cyclodeaminase activity

catdyses the cyclization of the formimino goup yieIding NS, NIO-methenyl-

tetrahydrofolate and releases arnrnonia (see Figure 1.2). The enzyme displays the

ability to channel poIyglutamyiated substrates (discussed below).

The formiminotransferase (FT) and cyclodeaminase (CD) activities were first

obtained simultaneously h m hog Iiver acetone powder (Tabor & Wyngarden, 1959;

Slavik et al. 1974). This study first showed that while the activities are pirrified

Figure 1.2

The two sequentiai reactions catalyzed by bifwictional formimino-transferase

cyclodeaminase. The first reaction is the transfer of the formimino group fiom

formiminoglutamate to the NS position of tetrahydropteroylpolyglutamate. The

second reaction is the cyclodeamination of N5-foxmimh~otetrahydropteroyl-

polyglutarnate yielding NSJIO-methenyltetrahydropteroylpolyglutamate Atorn

nurnbering used in text is shown.

together, treatment with ammonium hydroxide at pH 10.5 inactivates the transferase

activity and leaves the cyclodeaminase activity intact, while treatment with

chyrnotrypsin inactivates the cyclodeaminase activity leaving the most of the

tramferase activity intact.

It was k t postulated that F K D is an ohgomer of 7 to 9 subunits, as the

monomer has an approximate molecuIar mass of 62 kDa as demonstrateci by SDS-

PAGE (Dmy et al. 1975) while equilibrium sedimentatioa determined that the native

rnolecular weight of FTCD is approximately 540 kDa. These studies indicated that

FTCD could be either a multifiuictional enzyme or a multienzyrne complex. The

existence of FTCD as a multifiinctional enzyme was confirmeci by isoelectric focusing

and cyanogen bromide cleavage (Beaudet & MaciCemie, 1976). ïhese studies

demonstrated that FTCD is cornposed of identical subunits. ï h e number and

arrangement of the subunits dat ive to each other was indicated through electron

microscopy with rotational reinforcernent of negatively stained molecules. These

electron microscopy studies demonstrated that the mcilecuIe consists of eight subunits

arranged as a planar ring.

Proteolysis with chymotrypsin in the presence of the inhibitor, folic acid,

produces an 80 kDa hgment with the transfefase activity, which shows the presence

of a single species when subjected to SDS-PAGE (MacKenzie et al. 1980). This

transfemse active hgment loses specificity for poIyglutamylated substrates.

Experiments with the crossLinking ragent dithiobis(succinEnidy~ proprionate) support

that native FTCD is an octamer, whiie experiments with the short bifunctional

ceagent. difluorodinitrobenzene, produces predominantly even-numbered oiigimeric

states. These results indicate the presence of two types of subunit interactions,

suggesting that the native stnicture would be more aptiy descnïed as a tetramer of

dimers. These results, in conjunctioo with the chemicai modification to selectively

inactivate the transferase or deaminase activity (Tabor & Wyngarden, 1959; Dniry &

MacKenzie, 1977; MacKenzie et ai. 1980), suggest that the transferase activity and

cyclodeaminase activity are located at distinct sites on the polypeptide chah.

Chernical modification studies have also shown that the two activities c m be

inactivated separately. Use of 5,s'-dithiobis(2-nitrobemic acid) can selectively

inactivate the cyclodeaminase activity (Dnrry & MacKenzie, 1977). This inactivition

conrelates with the modification of two sulniydryl groups per subunit. The inhibitor,

folk acid, provides protection against inactivation, indicating that a cysteine residue

rnay be important in the cyclodeaminase reaction. The transferase activity is

selectively inactivated when ETCD is subjected to treatment with

diethylpyrocarbonate (MacKenzie & Baugh, 1980), which Ied to the postulation that a

histidine residue is important in the d i e reactioa

Denaturation and renaturation studies using urea as a denaturant indicated that

upon increasing the concentration of denaturant, FTCD denatures in a rnuIti-step

sequentiai fashion, as monitored by enzymatic activity, fluorescence spectroscopy and

subunit association through cross-linking expaiments (FindIay & MacKenzie, 1987).

When the enzyme is exposed to conditions in a potassium phosphate buffer with

between 2 and 3 M urea, it fVst dissociates mto dimers as indicated by the 105s of both

activities and a decrease in the intensity of the ûyptophan fluorescence spectm. A

red shifl in the wavelength of maximum fluorescence emission marks the second

transition that occurs when the concentration of urea is between 3 and 4 M. This red

shift was interpreted as a physical change in the dimers. The dimers then dissociate to

monomers when the concentration of urea is increased to p a t e r than 4 M. The

presence of different subsmte analogues protected the two activities and, by varyuig

the conditions, two different types of dimers could be isolated hrther confirming that

the tramferase and deaminase active sites are separate. Very different hgmentation

patterns are observed upon proteolysis of the transferase and deaminase active dimers,

suggesting that dimers with sûucturally distinct subunit interfaces can be isolated.

Denaturation and senaturation studies with guanidine hydrochlonde showed that

FTCD recovers greater than 90% of its activity including the ability to channel within

48 hours of dilution (FindIay & MacKenzie, 1988). The proteolytically denved

transferase-active Fragment renatures under the sarne conditions as the fiill-length

native enzyme. This suggests that the transferase fhgment could fiinction as an

independent foIding unit.

The deduction of the nucleotide and amino acid sequence and cloning of

porcine FTCD (MurIey et al. 1993) confirmed the size and ailowed for fiirther

experiments to probe the structure of the two activities. Deletion mutagenesis has

shown that each subunit consists of an N-terminai transferase active domain and a C-

terminal deaminase active domain which are separateci by a short linker sequence

(Murley & MacKenzie, 1995). The linker sequence is ail that keeps the domains in

close proximity to each other as attempts, such as crosslinking eXpertments, to

determine if there were any interactions between the domains Iacking the linker

segion, were unsuccessfiil. Loading one of the domains on a Ni-NTA column with the

0 t h domain previously bound does not slow the elution of that domain. Also,

channehg does not occur between domains when both domains are combined at hi&

concentration. i appm that the linker region is important in maintaining domain-

domain interactions. Through the separation of the domains Mudey and MacKenzie

(1995; 1997) were able to confirm that the CD domain retains specificity for

poIygIutamylated substrates. Further denaturation and maturation studies were

pursued (Murley & MacKenzie, 1997) which supports the previous work of

MacKenzie and coworkers (1 988).

Recentiy, FTCD has been implicated in having a second bct ion in liver celIs.

initiaIIy, an unknown 58 kDa protein (58K) was impIicated in binding to the Golgi

apparatus and providing a subsequent anchorage site for ihe microtubule binding in

rat liver (Bloom & Brashear, 1989). 58K was found to be capable of stimdating

polymerization of tubulin and was later identifiai as the rat homologue of FTCD

(Bashour & Bloom, I998; Gao et al. 1998). The chicken homologue of 58WFTCD

has also been shown to bind to the Golgi apparatus and micronibules (Hennig et al.

1998). These authors have speculated that FKD binds to microtubdes that have

been post-translationdly modifieci by the addition of polyglutamylated tails. Figure

t -3 shows a sequence alignrnent of porcine FTCD with chicken FTCD and the known

sequence of rat FTCD. Sztul and coworkers (Gao et al. 1998) have postuIated that a

possible reason the metaboh enzyme, FTCD, associates with the Golgi membranes is

tbat other enzymes in the histidine degradation pathway are also associated with the

Golgi apparatus. This would

Figure 1.3

Alignment of amino acid sequence of porcine FTCD with chicken and rat protein

homologues. Regions that are identical have ken shaded. The amino acid sequence

For rat FTCD has not been M y detennined.

produce an assembly Iine able to perforrn sequential reactions. Currently, there are no

data to support this postulation. Anothw possible reason, as speculated by Sztd and

coworkers (Gao et al. 1998), include specialized regdation of FTCD acûvities.

Recently, FTCD has been implicated as possibly being usefiil in eariy detection of

autoimmune hepatitis (LaPiene et al. 1999) as it was found to be a liver specific

autoantigen.

It has been proposed that FTCD uses a rapid equilibrium random kinetic

rnechanism (Beaudet & MacKenzie, 1975) in that either FIGLU or

tetrahydropteroyipolyglutamate may bind to the enzyme k t . Both the

formiminotransferase and cyclodeaminase bind polygiutamylated substrates better

than monoglutarnylated substrates, thereby in&ng the catalytic efficiency of the

enzyme (MacKemie & Baugh, 1980; Paquin et al. 1985). Findlay et al. (1 989) have

dso found that polyeJutarnylated substrates improve the bhding of the non-folate

substrate, FELU, as marked by an approximate ten-fold decrease in the K, value.

FTCD can channel y-linked polyglutamylated NS-fomiminotetrahydrofoIate

beween the transferase and deaminase active sites (MacKenzie & Baugh, 1980;

MacKenzie, 1979). The efficiency of this channeling is dependent on the length of

the polyglutamate tail, with optimal channeling observai for the pentagiutamate tom

of tetrahydrofolate (MacKenzie, 1979). This observation led to the postulate hat the

polyglutamate chain acts to anchor the substrate to the octamer thus aIIowing the

substrate to move between active sites (MacKemïe et al. 1980) in a 'swinging ami'

mechanism. Binding studies have shown that there are four polyglutamate bhding

sites per octamer, lending firrther support for this mode1 (Paquin et al. 1985). The

crystal structure of the monofiuictional FT domain with the bound ligand, folinic acid,

provides an initial view of the channeting mechanian of this enzyme.

1.3. Structures of other entvmes ex hi bit in^ channeiing

Substrate channeling is an important phenomenon that enabIes enzymes to

directly transfer metabolic intermediates between distant catalytic active sites rather

than by diffusion through the solution (teviewed in Srere & Ovidi, 1990; Ovadi,

1991; Ovadi & Srere, 1992). The channeling of intemiediates has a nurnber of

advantages. It prevents the Ioss of intennediates by diffision to the aqueous

environment, protects cheaicalIy unstable intennediates h m degradation during the

transfer between distant active sites, and decreases the tirne needed to transfer the

intermediate between active sites hence increasing the catalytic efflciency of an

enzymatic pathway. Multiîünctional enzymes involved in substrate channeling

between distinct active sites have been studied both biochemicdly and stnicnirally for

a number of years. The focus of many of these studies has been on addressing the

molecular rnechanisms that mediate the channehg activity (reviewed in Miles et al.

1999). Examples of enzymes involved in channeling activity include tryptophan

synthase, thyrnidylate synthasedihydrofolate reductase, carbamoyl phosphate

synthetase and more recently, E i ,2-oxoisovaIerate dehydrogenase. With tryptophan

synthase the intermediate, indole, is transferred h m the a-site to the P-site through a

25A long tunnel (Hyde et al. 1988). This m effect sequesters the non-polar

intermediate h m the aqueuus environment and increases the efficiency of overall

catalysis. Further cqstallographic studies have revealed confornational changes to

the structure as a result of monovalent cation binding, which affects the interactions

between the a- and P-subunits (Rhee et al. 1996). in addition, studies have s h o w

that the channeling and the coupling of'activities of the two active sites are contmlled

by allosteric signals that cause the two catalytic cycles to occur in phase (Pan et al.

1997).

The structure of carbarnoyl phosphate synthetase h m E. coli reveaIs a tunnel

of 96A through which the enzymatic intemediates are passed between three active

sites (Thoden er al. 1997). This effectively resuhs in intermediate channeling with

100% efficiency as well as protection of the IabiIe intermediates, carboxytphosphate

and carbarnate, from decomposition. Recent structwal studies have shown that the

active sites communkate with each other via domain movements as a result of the

binding of a nucleotide triphosphate (Thoden et al, 1999). EL.2-oxoisovaferate

dehydrogenase aiso shows the presence of a long hydrophobie tunnel where the E2

IipoyI-lysine arm teads to the active site and ailows channeling of the enamîne

intermediate (Ævarsson et al. 1999).

In contrast to the tunnels observeci in the structures of tryptophan synthase,

carbamoyl phosphate synthetase and E l,2-oxoisovaierate dehydrogenase, the structure

of the bihctional enzyme thymidylate synthase-dihydrofolate reductase reveaIs that

the transfer of dihydrofoIate between the active sites occurs by movement of the

Ligand across the surface of the protein (Knighton et al. 1994). The u n d surface

charge distniution is believed to account for the channeiing of the intermediate

beîween active sites. This charged surface Iinking the thymidylate synthase active site

and the dihydrofolate reductase site, 400A away, has been temed the electrostactic

highway (Stroud, 1994).

The crystal structure of FTCD will provide insight into the mechanism by

which this enzyme channels and will provide evidence for the location and number of

Iigand binding sites. information for the cataiytic mechanism of both the transferase

and deaminase reaction cm also be gleaned fiom an analysis of the three dimensiond

structure of FTCD. The anaiysis of the three dimensionaI structure of FT domain

provides an initiai view of the cataiytic mechanism for the tramferase reaction and

provides a basis for enhancing our understanding of the channeling mechanism.

Cbapter two

Materials and Experimentai Methods

2.1. Protein m d h t h o n and concentration

Hexahistidine-tagged formiminotransferase domain was overexpressed using

the pBKe-Cm1 expression vector in Escherichia coli strain BL21tDE3. Purification

was performed as describeci previously (Murley & MacKenzie, 1995) omitting the last

DEAE Sepharose column. The pooled fractions containhg activity for the Fï domain

were then dialyzed into 25mM MOPS pH 8.2, lûrnM K2S04 pH 7.3,35rnM BME and

10% (vlv) glycerol with the addition of ZûmM EDTA to remove any N?+ which may

have leached h m the colurnn. The protein was dialyzed again in the same buffer

excluding EDTA to remove any chelated Pli'' and EDTA. The protein solution was

then concentrated to 8mg m~-' using a Centriprep 10 and Centricon 3 (Amicon, Inc.).

Protein concentrations were detemiied using a Bradford assay with BSA as the

standard.

2.2. Cwstallization with folinic ocid

CrystaIIization conditions were screened by the hanghg-&op vapour diffusion

technique (McPherson, 1990). Protein for these trials was pudied as above. Prior to

these trials, the protein soIution was transferred to a 0.22 pm Eppendorf mter

e (Mïilipore Corporation, Bedford, MA) and filtered by spinning the sampie at hi@

speed (14,000 rpm) in an Eppendorf centrifuge 54132 for 2 minutes at 4OC to remove

any particulate matter which may interfere with crystallization. initial trials using a

sparse-matrix screen, descnbed by Jancarik & Kim (1991), showed a promising

p u l a r precipitate with some precipitants (ammonium suifate, sodium formate,

sodium citrate, polyethylene glycol 2000 and 8000). Further crystallization

experiments around these conditions did not resuit in any significant improvement. A

source of structural heterogeneity is that many proteins have considerable

conformation flexibilty. Such flexibility may act to inhibit crystallbtion. This

flexibility is especially prevalent in muitidomain proteins where the interdomain

contacts may be flexible (Sousa, 1997). It was thought that the introduction of a

ligand to the crystallization trials of FT domain may act to enhance crystailization,

particularly if such conformational flexibility exists. The ligand may 'lock' the

protein in a single conformational state, thus rendering the protein more amenable to

crystalIization. For this reason, it was decided to screen crystailization conditions of

Fï domain in the presence of various substrate and product analogues. in particdar,

the product analogue analogues 2mM folinic acid and 2mM glutamate were added,

either together or independently, to the conditions h m the randorn screen which gave

the most granular precipitates. X-ray diffraction quaIity crystals were obtaïned using

1M Na3citrate, IOOmM Tris pH 8.0 and 15% giycerol as mother Iiquor with the

addition of 2mM foluiic acid to the protein buffer describeci above. Tt should be noted

that some variation in the conditions would still yield crystais. Specifically, the

concentration of Na3citrate couid Vary between 0.95M to 1. tM. The glycerol

concentration could Vary h m 10% to 20%. It was Iater noted that the pH of the

@ mother liquor was approximately 9, despite the pH of the Tris buffer added. It is

thought that the relatively hi& concentration of Na3citrate would saturate any effect

that the Tris buffer would have. Equal volumes of mother liquor and protein were

mixed in a cirop and the ûays were incubated at 290K for two weeks, after which

crystals with a typical size of 0.7 x 0.25 x 0.25 mm and in a fin-like morphology

appeared.

Crystallization conditions of FT domain with substrate andogues were

pursueci in order to obtain a data set that would yield a structure that would give more

information on the binding of a substrate. Crystailization experiments were attempted

with folic acid and C5,ClO-dideazatetrahydrofolate as well as CO-crystdlization

experiments with folinic acid and glutamate together and C5,Clû-

dideazatetraùydrofolate together with formirninogiutamate. Figure 2.1 shows the

chemicaI structures of the compounds used in this study. Since the stnrcture h m the

crystals with folinic acid revealed the binding of glycerol (discussed below),

crystallization experiments were also attempted in the presence and absence of

glycerol. Al1 crystallization experiments were perfomed as describeci in Sections 2.1

and 2.2 with regards to pmtein solution, incubation temperature and the ratio of

mother liquor to protein solution used. Crystais coufd be grown in the absence of

gIycemI and either in the presence of 3mM folinic acid or in the presence of 2mM

C5,C 1 O-dideazatetrahydrofolate. These crystais appeared within two weeks and were

both of smaii size and poor quaiity.

Figure 2.1

Chemicai structures of a) tetrahydrobIate, b) folinic acid and c) C5,CIO-

dideazatetrahydrofolate.

Crystals were also produced in the presence of glyceroi with the addition of

2mM folinic acid and IOOmM giutamate, or, with the addition of 2mM C5,CIO-

dideazatetrahydrofolate. These crystaIs were of a bipyramidal morphology with

largest dimensions obsewed being 0.25 x 0.20 x O.OSmm and generally appear in four

to eight weeks. Crystals with a morphoIogy identicai to crystals obtained in Section

2-2 were observed to grow in the same drops as the bipyramidai crystals. These

crystals have approxirnate dimensions of 020 x 0.15 x 0.15rnrn and appeared in four

to eight weeks.

2.4. Heavv atom derivative screenin~ and prewratikn

Heavy atom derivatives were screened by soaking crystals in a solution of

mother liquor and the heavy atom to be screened. The concentration of the heavy

atom and the duration of the soak were var id in an attempt to optimize conditions to

produce a successfu1 derivative. To test whether a crystai soaked in a particular heavy

atom solution was a successful derivative five to ten X-ray diffraction images were

collected h m the soaked crystai. These images were then processed and scaied to a

native data set using the program SCALEIT h m the CCP4 suite of programs

software (Collaborative Compuwtional Pmject, 1994). Possiile derivatives would be

indicated by having an above 12%. Data collection was aiiowed to continue on

derivatives that showed promisîng RdarvS.

Isomorphous difference Patterson maps and, once an initiai set of phases had

been determineci, difference Fourier maps, were caicuiated h m these heavy atom

derivative data sets using programs h m the CCP4 software (Collaborative

Computational Project, 1994). The coordinates for the position of these heavy atoms

were detemineci h m these maps and relined with MLPHARE, h m the CCP4 suite

of programs. A total of three heavy atom derivatives were found. By çoaking crystaIs

in pchlorornercuriobenzoate suIfonic acid (PCMBS) (FI& Chemie, AG CH-9470

Buchs) at a concentration of ImM for approximately 4 hours, a mercurial derivative

was successhlly obtained. A platinum derivative was obtained by soaking crystais in

mother Iiquor containing LmM K2m(CN)4] (Fluka Chemie, AG CH-9470 Buchs) for

17 to 24 hours. Finally, a goid derivative was obtained by soaking crystals in mother

liquor containing 6mM K[Au(CN)2] (Aldrich Chem Co.) for 40 to 44 hours.

2.5. Duta collection and m a r e determinmion

2.5.1. Crysral in complerr wath/olinic acid

Data were collected at 83K on a MAR image plate detector mounted on a

Rigala RU-200 rotating anode X-ray generator (CuKa radiation). Synchrotron data

were collected at 1.04A for the goid derivative and 1.07A for the high resolution

native data on bearnline X8-C (NSLS, Brookhaven NationaI Laboratory, New York).

The X-ray images were processeci using DEEiZO m the HKL suite of software

(ûtwinowski, 1993; Minor, 1993). ScaIing and merging of the data was continued

with SCALEPACK h m the EML suite of software (Ohrinowski, 1993; Minor,

1993). The intensity data were sorted and structure bcîor amplitudes caldated

using the progams SORTMTZ and TRUNCATE h m the CCP4 suite of software

(ColIaborative Computationd Projeci, 1994). The hi@ resolution native data set,

collected at the synchrotron radiation faciiity was ody 73% comp1ete in the lowest

resolution bin (50.0 - 3.66A) due to spot intensity ovdow. In ordw to complete the

data, the high tesoiution (1 .74 and low resolution (2.8A) data sets were merged

using the program SCALEPACK h m the HKL suite olsoftware.

The structure was soivcd by the rnethod of multiple isomorphous replacement

using 3 heavy atom derivatives. Difference Patterson syntheses were usai to identiîy

the heavy atom positions for the mercuriai derivative and an initid set of phases was

calculated using the program MLPHARE (Collaborative Computationd Project,

1994). Positions of the other heavy atom derivatives (gold and platinun) were

determined h m difference Fourier maps ushg the initiai set of phases h m the

mercuriai derivative. The anornaiou signal h m the gold derivative was obtained

h m data colkcted at the synchrotron facility and used together with the isomorphous

signa1 h m the three derivatives in order to obtain the best set of MIR phases. The

MIR phases were fwther optimized by sdvent flattening and histogram matcbg

using the program DM (Collaborative Computationd Ehject, 1994), with a solvent

content of 50% (assuming two mofecuies per asymmetcic)- The electron density map

calculated h m the improved phases clearly deiineated ttie two protomers in the

asymmetric unit and showed elements of secondary structnre which were related by

ttonnystallographic symmetry (NCS). A prelnnniary mode1 was constructed for a B-

strand and an a-helix in both protomers and the atoms in the mode1 as welI as the

heavy atom positions used to obtain the non-crystallographic symmetry matrices. A

mask was built around one of the protomers and, using the non-crystallographic

symmetry matrices, two-fold averaging was perfomed using the RAVE software

(Jones, 1992; Kleywegt & Read, 1997). The initial mode1 for a single protomer was

built h m the resulting electron density map ushg the program O (Jones et al. 1991).

This Lirst model consisted of 286 of the 328 residues with 77% of the full amino acid

sequence. However, only an alanine side chah was included in the model when the

electron density was unclear.

Crystallographic refinement was initially perfomed with the program XPLOR

(Brünger et al. 1987) and, in later stages, the program CNS was used (Briinger et ai.

1998)- Initially constrained refinernent was carried out to 2.8A resolution. Once the

resolution was extended to 2.2A the constraints were removed and the protomers were

refined as separate molecules with no non-crystallographic symmetry imposed. Each

cycle of refinement was followed by a manual rebuild using the program O (Jones et

ot 1991). SIGMAA weighted maps calculated with coefficients 3Fo-2Fc and Fo-Fc

were used for the model rebuilds. In the h a 1 stages of refinement 2 b F c maps were

used. The difference electron density for the f o h c acid ligand appeared clearer for

one of the two pmtomew however both were included in the model. Figure 2.2

displays the electron density map for the two separate ligands. Water moIecuies were

built where difference electron density above 3a was observed and where hydrogen

bond contacts were made to other polar atoms. In the final stages of refinement,

mulriple conformations for the side chains of 24 residues were modeleci and refined

with CNS. The h a 1 mode1 consists of residues 2 - 326 for one protomer and 2 - 207

0 and 214 - 326 for the second protomer, two molecules of folinic acid and 771 water

Figure 2.2

The difference in the quality of the 2Fo-Fc electron deasity map at 1.7A resolution,

contoured at I.3a, for the folinic acid ligand and giycerol molecule modeled in a)

protomer "A" and b) protomer "B" (produced with the program, SETOR, Evans,

1993).

molecules. Mer the final round of refinement, the program PROCHECK (Laskowski

et al. 1993) was used to calculate a Ramanchadran plot which indicated that al1 of the

residues are in favorable regions of cp/W space (Figure 2.3). Coordinates have been

deposited in the Bmkhaven Protein Data Bank (Bernstein et al. 1977) (accession

number I QD 1).

2.5.2. Crystal in cornplex with CS, CI O-dideazatetrahydrofolate

Data were also collected at 83K on a MAR image plate detector mounted on a

Rigaku RU-200 rotating anode X-ray generator (CuKa radiation) with double

focussing mirrors (Supper Ltd.). The X-ray images were also processeci, scaled and

merged using DENZO and SCALEPACK h m the HKL suite of software

(Otwinowski, 1993; Minor, 1993). The data were sorted and structure factor

amplitudes calculated using the pmgrams SORTMTZ and TRUNCATE h m the

CCP4 suite of software (CoIlaborative Computational Project, 1994). Data were then

convmed to a format usable by CNS (Briinger, 1998). Since the space group is the

same and the ce11 dimensions of this crystal are identical to the crystai complexed

with folinic acid, difference Fourrier techniques was used to solve the structure of the

C5,ClMdeazatetrahydrofolate complexed crystal. Prior to rehement with this

model, the folinic acid, the glyceroi and the water molecules were removed h m both

protomers. in order to prevent model bi s , Ioop regions that either made contact with

the folinic acid, or were near the binding site of folinic acid, were aIso removed h m

Figure 2 3

The Ramanchandran plot (Ramakrishman & Ramanchandran, 1965) of the main-

chain dihedral angles for 558 non-glycine and non-proiine residues modeleci in the

structure of FT domain in cornplex with folinic acid, as calculated by PROCHECK

(Laskowski et al. 1993). GIycine residues are represented as triangles. A total of 5 13

residues (91.9%) are in the most energeacally favoumble regions (A,B,L). A total of

45 residues (8.1%) are in the energeticaity les favourable regions (a,b,l). No residues

fa11 in either of the generously albwed regions or disaIlowd regions.

both protomers. Specificaily, the loop regions removed consisted of residues 37-

48,78-85,137-142, l75-183,224-23 1 and 267-272. Also, the loop region consisting

of residues 208-213 was also removed due to the fact that density could ody be seen

in one of the protomers h m the original model. Refinement was carried out using

the data collected h m the C5,ClO-dideazatetrahydrofolate complexed crystal against

the modified model. Restrained rigid-body refinernent, sirnulateci annealing, least-

squares minimization and group temperature factor refinement were initidly

performed with this new data. SIGMAA weighted electron density maps, calculated

with coefficients 3Fo-2Fc and Fo-Fc, were used for rnodel rebuilding. The loop

regions that were previously removed were rebuilt Uito the new electron density. The

density conesponding to both the C5,ClMideazatetrahydmfoIate molecuIe and

glycerol molecule was present but of fairly poor quaiity, therefore another round of

restrained refinement was performed as descri'bed above. The electron density maps

caIculated h m this refinement resulted in significant hprovements for the ligand

regions of the sûuchue. ïhe mode1 was manually rebuilt again and both the gIyceroI

molecule and C5,ClO-dideazatetrahydroflate molecule were included and a final

mund of refinement was performed. Figure 2.4 shows a Ramachmdran plot which

indicates that most residues lie in favourable regions of q/y space.

2.6 Substrate docking

The (6S)-tetrahydropteroyI-trigiutamte-Nme andogue of the naturai substrate was

@' docked into the binding site of the FT-domain using Sybyl6.5 molecular modeiing

Figure 2.4

The Ramanchandran plot (Rarnakrishman & Ramanchandran, 1965) of the main-

chah dihedral angles for 562 non-glycine and non-proIine residues modeled in the

structxe of FT domain in cornplex with C5,CIO-dideazatehahydrofolate, as

calculated by PROCHECK (Laskowski et al. 1993). Glycine residues are represented

as triangles. A total of 496 residues (88.3%) are in the most energetically favourable

regions (A,B,L). A total of 64 residues (11.4%) are in the energeticaily l e s

favourable regions (a,b,l) and 2 residues (0.4%) fdl are in generoudy ailowed regions.

Glul45, which is one of the residues in the genemusly ailowed region, is slightIy

strained as it is part of a loop structure bridging two short a-helices. The electron

density allows for unambiguous modeling of tbis strained conformation. No residues

are in disalIowed regions.

-1b - 3 5 -40 4s 4'5 Phi (degrees)

software (Tripos, Inc. St, Louis, MO). Structural refinement was performed in Sybyl

6.5 by energy minimization using AMBER 4.1 all-atom force-field with a Powel

minimizer, distance dependent (4r) dielectric constant and an 8A non-bonded cutoff.

The energy tninimization was carried out until the mot-rnean-square of the gradient

was smaller than 0.05 kcal/molk

The coordinates of the protomw with better defined electron density were used as a

starting point for the molecular docking. The folinic acid, glycerol and al1 water

molecules were removed. The hydrogen atoms ad AMBER 4.1 point charges for the

protein part were added with the Biopolymer module in Sybyl 6.5. For consistency

with the force-fieId employed in energy minimization the atomic partial charges of the

substrate molecule were determined on the "fragment-additivity" bais using (6s)-

teîrahydropteroyl and y-linkable glutamate as hgments. Charge calculations were

performed on the neutrai (6s)-tetrahydropteroy1-Nme and negatively charged Ace-

yGlu-Nme rnolecuIes in an extended conformation at the 6-3 IG* ab initio level using

Gaussian 94 (Gaussian, inc., Pittsburgh, PA) without geometry optirnization and with

subsequent fitting to the electrostatic potentiai. Missing atom types as welI as

m d e h e d equilibrium vaIues and force constants for the ligand moiecule were

assigneri by analogy with those parametenzed in the AMBER 4.1 force-field.

Dockhg of the substrate rnolecuie was carried within a "ligand-C~s"' stepwise

protocol. The (6s)-tetrahydroptmyl-Nme rnolecuie was positioned in the binding

site in the sirnilar fashion with corresponding fiagrnent of the crystailized (6R)-folinic

acid and relaxeci in the h e d protein environment. Each of the foliowing y-linkabIe

giutamate units was then joined-up in two steps, 6rst as an aminobutyrate and

subsequently as a complete y-Glu-Nme. The conformation of the added fragment was

selected manuaIIy by considering several stmctural features of the enzyme binding

site such as steric allowance, polarity, H-bonding capabiIities and position of water

moïecules in the original crystal structure as well as conformation strain in the ligand

molecule. Following energy minimization with the protein atoms constrained to their

crystallographic positions the next hgment was added to this docked partid substrate

molecule. M e r accommodation of the complete substrate analogue, four

crystallographic water molecules that allow H-bonding with the ligand molecule were

added and relaxed in the fixed complex environment. Finally, the ligand and water

molecule dong with the protein residues 8A fiom the ligand were dlowed to move

during energy minimization.

Chapter three

Results and Discussion

The formiminotransferase domain with the product analogue, folhic acid,

bound was crystallized in the orthorhombic space gruup P2i212r, with unit ce11

dimensions a+.4A b=103.7A c=1223A (Figure 3.1). Using a molecular mass of

74kDa (for the homodimer) and assuming the presence of one dimer per asymmetric

unit, a Vm value of 2.95A3~a-' was obtained. m-s vaiue is witùin the range observed

by Matthews (1968) and corresponds to a solvent content of 50%.

Crystals with foünic acid w m aiso obtained with identical conditions reported

above with the omission of glycerol. These crystais grew in tight sphencal clusters

and had jagged edges, characteristic of crystaIs growing too fast, where individuai

motecules are unable to orient ttiemselves quickly enough to form an ordered crystaI

Iattice. These crystals were not of X-ray diffraction quaiïty. No fitrttier improvernent

of these crystals was attempted. Perhaps the addition of additives 0th than giycerol

such as plutamate or FIGLU andlot decreasing the CryStaIIization temperature may act

to sIow the qsîahation process, thus improving the quality of crysîais obtained,

S m d bipyramidal crystais were obtained m Simrlar experimentd conditions

as above, except the absence of giyceml and in the presence of 2mM CS,C10-

Figure 3.1

A crystal of FT domain grown in the presence of 2mM (6RIS)-folinic acid and 10%

(vlv) glycerol by vapour d i m o n using the hanging drop method. The crystal size is

approximately 0.7 x 0.25 x 0.25mm.

dideazatetrahydrOpten,yhono~utamate. These crystals appeared within two weeks.

Attempts to mamseed these crystais in order to obtain larger crystals were not

successfiii and streak seeding crystais onIy yieIded more crystals of a similar size.

Two crystal morphologies were obtained using conditions similar to those

with folinic acid and giycerol. In the presence of either 2mM C5,ClO-

dideazatetrahydrofolate or 2mm folinic acid and 80mM glutamate, bipyrarnidal

crystals were grown. A satisfactory data set has yet to be obtained h m crystals with

this morphology. The highest resolution data collected to date for this crystai form is

approximately 3.1& aithough crystals have shown potentiai to diffiact X-rays near

Z ~ L resolution using a synchrotron Iight source.

A complete data set has been collected h m a crystal grown in the presence of

2mM folinic acid and IOOmM glutamate. This data set yielded an ambiguity in the

space group as the data could be successfuIIy processecl in either the orthorhombic

crystal system, with ceIl dimensions of a=10 l.4A b=101 .SA c=l36.1& or the

tetragonal crystal systern, with ceIl dimensions of a=b=10 1 .SA c=136.l A, using the

HKL software (Otwinowski, 1993; Minor, 1993). Upon scaiing the data, the

orthorhombic space group seems more Iikely to be correct as the R-mage obtained is

17.2% as opposed to 26.9% for the tetragond space group. WhiIe these R-merge

values are very large indicating that these data sets will not be usehl for caicuiating

an electron density map, the difference between the two values is significant and may

be used as an indication for the correct space group. Further attempts to work on the

structure will solve this ambiguity as weU as provide more information on the binding

of the substrate, FTGLU. Further work may benefit fiorn screening a different

cryoprotectant as the data collected suffered h m excessive mosaicity (>1.5),

Rod-like crystals were also obtained with FT domain in complex with 2mM

C5,Cl O-dideazatetrahydrofolate. These crystals are of the same space group and ce11

dimensions as those grown with foiinic acid. The resolution bits to which it

d i f i c t s are also similar to that of the crystais grown with f o l k acid. A complete

data set was coIIected to 2.8A resolution. (Further discussed in Section 3.1 1)

3.3. Phase deîermination

ï h e î h t heavy atom to be identified was a mercuriai derivative using the

heavy atom reagent pchloromerc~benzenesulfonic acid (PCMBS). Two Hg-atom

positions were identified h m the Patterson vector superposition method in SHELXS

(Sheldnck et al. 1993). The heavy atorn parameters (coordinates, occupancy and

isotropic- temperature factor) for these two sites were refmed using the program

WHARE h m the CCP4 suite of software. These initiai phase estimates revealed

that the rnercuriai derivative is weakly occupied, as seen by the phasing power 0.91

and the Rd& of 0.79 (see Table 1 and 2 for a summary of statistics). This initiai set

of phases allowed for the determination of the other heavy atom derivative sites usiug

the difference Fourier rnethod.

A second derivative was identifiai utiliang ImM &(Pt(CN)4] soaked for 17

hours (See Table 1). Using the singie isomorphous repIacement (SIR) set of phases

fiom the mercurial derivative, two platinyl sites w m determined h m difference

Table 1

Data Collection Slatistics for Fonniminotransferase Domain complexed witli foliiiic acid.

7 MAR, MAR Rcsearch X-ray plato detector with a double mirror focussing system, mounted on a Rigaku RU200 rotaiing anode gencrator using CuKa radiation. $ SRS, Synchrotron radiation light source al wavelength 1.0397A, beamline X8C Brookhaven National Light Source, Upton, New York. 1 R-nierge = C C - 11 1 C C Ih,, (summed over al1 intensities) 8 R-deriv - C lFderivh - Fnathl/C F nath (resolution range 40A Io 2.8 A) Y In the lowest resolution bin (50.0 A Io 3.66A) data was only 73.1% complete thus the data was scaled and merged witli a low resolution native daln set. *In the lowest resoluiion bin (50.0 A to 3.66A) data is now 94.8% complete.

Table 2

Heavy Alom Refinement Statistics for Formiminoiransferase Domaiii complexed with folinic acid.

1 Resolution Range (A) ( R-cullist 1 Phasing Powerf 1 No. of sites 1 Occupancy

PCMBS 1 1 0.79 0.9 1 2 0.407

1 1 1 1 1 0.230 t R-cullis = Et, (IFPI, f Fpl - FII(CIIIC)I 1 Zt, lFp,l *Fp( $ Phasing power = r.m.s, heavy-atam structure factor / r.m.s. lack oîclosure. Overall Figure of Mcrit is 0.57 using al1 reflections frorn 15.0A lo 2,8A

Fourier qntheses. With the hown positions of the platinum atorns a set of phases

could be calculated with MLPHARE (CCP4 suite, 1994). Two platinyl derivative

data sets were collected with different soak times to obtain differently occupied heavy

atom sites, and slightly different phase information (See Tables 1 and 2).

A third derivative was identified by soaking native crystals in 6mM

K[Au(CN)2] for 40-45 hours. Two gold sites were Iocated by difference Fourier

techniques with phases obtained h m the mercurial derivative. ùiterestingiy, the

coordinates for the gold sites were equivaient to those of the platinum derivative.

These new gold sites were aiso refined as above using MLPHARE (CCP4 suite, 1994)

to give an initial estimate of the phases. The occupancies and B-factors of the gotd

and pIatinum derivatives were aiso reflned sirnultaneously. Possi'bly due to the

difference in occupancies between the gold and platinum derivative, the concurrent

refmernent of the two derivatives yielded satisfactory statistics.

Finally, the heavy atom parameters (coordinates, occupancy and isotropie

temperature factors) for each of the fou. derivatives, were rehed using MLPEMRE

(CCP4 suite, 1994) and an electron density map was calculated. The finai Figure of

Ment (FOM) after heavy atom refinement was 0.50. Upon visualization of this rnap

in O (Jones et al. 1991), as well as the bones atorns comûucted fivm this map, some

areas of secondary structure were visible.

A second gold denvative data set was coiiected, mcluding the anomaious

signai, using a synchrotron light source using a wavelength corresponding to the

absorption edge of gold (1.04A)- This data set was included in the heavy atom

cefiement to give a FOM of 0.57. map was slightiy improved when compareâ

with the electron density rnap caiculated without the anornaIous signal and reveals

larger regions of interpretable stmcture. Further improvement of this map is

discussed in Section 3.4. Figure 3 2 displays the impmvemwts observed in the

etectron densiîy map.

3.4. Phase improvement bv densitv mdification teciiniuues

Although the electron density map had regions that were clearly interpretable,

density modification methods was undertaken in order to furthe; improve the phases.

To this end, solvent Battening and histogram matching (using a solvent content of

SPA) were perfonned As seen in Figure 3.2 this had significantiy improved the

electron density map and allowed some regions of the mode1 to be built. Since the

asymmetric unit contains two molecules, it was thougbt that two-fold averaging

would dso improve the electron density map. Figure 3.3 shows the mask that was

built around one of the protomers for two-fold averaging- Upon two-fold averaging,

using the RAVE software (Jones, 1992; KIeywegt & Read, I997), a M e r improved

etectron density map was observed, with regions of we11-dehed etectron density for

both the polypeptide main chah and side challis (See Figure 32).

3.5. Model Buildin~ and Retlnement

M e r soIvent flattering, histogram-matcbg and two-fold averaging, the

eIectron density map was of sufncient quality to allow the initial mode1 to be bu&

Figure 3.2

A view at 2.8A resolution of the 3Fo-2Fc electron density map, contoured at 1.3 cr, in

a P-sheet region of the FT domain in complex with folinic acid. The electron denisty

map shows improvernent as denisity modification and averaging techniques are

appIied. The electron density map was calcuiated sequentiaIly h m a) the phases

determined h m the heavy atom refiement with out contribution h m the anmaIous

signai, b) with contriiution of the anomaious signai, c) after solvent flattening and

histograrn matching and d) f ier two-fold averaging.

Figure 3 3

A mask built around se!ected bones atoms for two-fold averaging The mask was

calculated and improved using the program MAMA (Kleywegt & Jones, 1994). The

matrices used in two-fold averaging were improved with the program iMP (Kleywegt

& Jones, 1994)

One protomer was built by fitting the amino acid sequence, as detemined by Murley

& MacKenzie (I995), into the electron density rnap. The other protomer was then

generated by using the NCS matrices. This initial model was subjected to a strict

NCS rehement using XPLOR (Briinger, 1987). SimuIated aanealing, least-squares

minimization and group temperature factor refinement were included. The first

rounds of refinement were canied out using data to 2.8A resolution. SIGMAA

weighted 3Fo-2Fc and Fo-Fc electron density maps were calcutated with the phases

h m the refined model and used for the manual rebuilds.

Once the high resolution (1.74 native data set was coIlected, the refinement

was extended to 2.2A resolution. During these stages, the NCS constraints were

loosened such that the two protomers wouId be refined independently of the other and

the ligands, folinic acid and glycerol, were included in the model. in the Iater stages

of refinement to 1.7A resolution, the NCS restraints were removed, and 2Fo-Fc

electron density maps were used for manual rebuilds. 771 water molecules were

included into the mode1 using differwce Fourier eIectron density maps. In addition,

alternate conformations for 24 side chains were modeled with quai occupamies

assigned. The folinic acid Iigand was assigned hl1 occupancy in both protomem. The

h a 1 refinernent statistics are shown in Table 3. Figures 3.4 and 3.5 give examples of

the electron density d e r the Iast round of refinement.

Table 3

Modei refinement statistics for mode1 with folinic acid.

Resolution Range (A) 50.0 - 1.7 R-factor 19.1 R-free * 213 R.m.s.d. bond Ieagtbs (A) 0.005 R.m.s.d. bond angles (3 1.25 Number of non-hydrogen atoms 5035 Number of water moIecules 77 1 Average B-factors (2) - Overall 2221

- Protein atoms 20.19 - Water rnolecuies 34.69

* 10% of the reflections was used to calculate R-fiee.

Figure 3.4

A s t e m view of the 2Fo-Fc electron density rnap depicting the folinic acid ligand and

glycerol molecule for a single protomer, Some side c h a h and water molecules are

also displayed. The rnap is contoured at 1 . 3 ~ (produced with the program, SETOR

Evans, 1993).

Figure 3.5

A stereo view at 1.7A resolution of the SFo-Fc electron density map depicting the two

conformations of Val303. The map is c o n t o d at 1 . 3 ~ @roduced with the program,

SETOR, Evans, 1993).

The structure of the FT domain forms a homodimer; the two protomers are arranged

such that the dimeric unit adopts a 'W7-shaped morphology (Figure 3.6). The two

protomers withh the dimer are related to each other by a non-crystalIographic two

fold rotation axis. The overall dimensions of each protomer are 50A x 43A x 35A.

The N- and C-termini of each protomer are Iocated in close proximity to each other

but due to the non-crystailographic two fold rotation axis, the termini on one protomer

are located on the opposite face of the molecule than the termini of the second

protomer wiihin the dimeric unit. The coordinates for a singIe protomer were

submitted to the Daii server (Hoim & Sander, 1993) in order to identiQ any

topological similarities with previously identified protein motifs. No significant

stnicturaI sirnilarity was observeci indicating that the FT domain adopts a novel

protein hld.

3. Z The structure of the protomer

The protomer is made up of two a@-units comprishg an N-terminal and a C-

tenninal domain. A topology diagram showing the secondary structure elements in

each domain is shown in Figure 3.7. Figure 3.8 shows an Ca trace of the pmtomer

with numberhg for every 25 residues- Each domain consists of a P-sheet with a-

helices Iocated on the extemal sinface (Figure 3.9). The $-sheet of the N-terminal

domain fxes that of the C-terminai domain to form a double P-sheet layer between

Figure 3.6

Ribbon diagram of the dimer of formirninotransferase domain. The different

protomers are a light and dark grey. The product analogue, folinic acid, is depicted in

a bail-and-stick representation. The dashed lines correspond to residues 208-214

which were not rnodeled due to the poor quality of eIectron density @roduced with

the program MOLSCRIPT, KrauIis, 199 1).

Figure 3.7

Topology diagram for FT domain with p-strands and a-helices numbered in the order

they appear in the primary sequence. The N-terminal domain is depicted in light grey

and the C-terminal is depicted in dark grey. The arrows represent the ~-strands with

their directionality, whiIe the a-heIices are represented as cylinders.

Figure 3.8

A stereo view of the Ca trace for a single protomer of FT domain with every 2 5 ~

residue labelleci. The dark grey trace repments the N-terminal domain and the C-

terminal domain is in light grey. Folinic acid and giycerol are show in a black stick

representation (produced using the program MOLSCRIPT, Krauiis, 1991).

Figure 3.9

Riibon diagram of single protomer of FT domain displayed in stem representation.

The domains are shaded such that light grey represents the N-terminal domain and

dark grey depicts the C-texminal domain (produceci with the program MOLSCRIPT,

KrauIis, 199 1).

the a-helices. The a-helices in the C-terminal domain forrn the bottorn d a c e of the

"V-shaped dimer while those in the N-terminal domain make up the top sides of the

dimer (Figure 3.6). A cIeft making up the binding site for the ligand, folinic acid, is

Iocated between the P-sheets of each domain.

Due to the high remlution of the native data (1.7A) used for crystai1ographic

rehemew the two protomers were refined without irnposing any non-

crystaliographic symmetry (NCS) restraints. Superposition of the alpha carbon trace

for the two protomers, yields a mot-mean-square (r.m.s.) difference of 0.45A between

the 3 18 structuraily homologous Ca atoms indicating no signifiant difference in the

overall fold of the two protomers. Upon superposition of the two protomers, it was

noted chat the best fit was observed in the B-sheet regions of the structure. The

largest differences were found in a number of loop regions of the structure (residues

223 - 232,310 - 314, 318 - 326 and 204 - 214) and at the a4 helix (tesidues 131 -

146). The Ioop region between residues 310 and 314 is involved in dimer contacts.

Movements in this region, away h m exact two fold symmetry, may act to optimize

the interactions between the two protomers. Further differences in the twp region

between residues 223 and 232 may be correiated with the ptedicted

fomiminogIutamate bmdmg site (to be firrther discussed beIow).

The N-terminal domain of the protomer consists of resihes 1-178. It is made

up of a six stranded mixed fi-pleated sheet (pl - 86) and five a-heIices (a l - a5)

(Figure 3.7). Strands BI - B3 are arranged in an anti-pdleI fashion whereas 84 - 86

are parailel. The five a-helices are arranged on the extema1 surface of the p-sheet.

An extended hop between helix a2 and strmd a4 is observe& This loop folk back

over the structure, from the extemal surface of the dimer, m s s the f!-sheet in the N

terminal domain and lies near the giutamate portion of folinic acid. A second

extended loop is seen between residues 128 and 138, on the surface of the molecule

where the cyclodeaminase domain is expected to lie. A glycine cesidue at position

127 and a proline at position 139 enabte the loop to fold back h m the glutamate

portion of the folinic acid ligand, This region of the structure may interact with the

cycIodeaminase domain.

The C-terminal domain consists of residues 182 - 326 and also folds into a

mïxed a@ structure similar to the N-temiinal domain but with a four stranded anti-

parailel P-sheet (B7 - p 10) ( s e Figure 3.7). The topology of this four stranded B-

sheet and two a-helices (a6 and a7) is similar to strands PI - P4 and helices ai and

a2 of the N-terminal domain. A superposition of 67 structurally homologous alpha

carbon atoms comprising the secondary structure elements of this region of the C-

terminal domain with the equivalent region in the N-terminal domain resulted in a

rms. difference of 2.9A. A major difference is seen m the orientations of the h t

heIix in each domain (al and a6) dative to the position of the P-sheet and the

second a-helix. Also, the relative orientations of the Ioop regions between strands B2

and B3 and strands $8 and P9 is significantIy different in the superposition of the two

domains. Finally, the loop region h m residues 260 - 266 is much shorter than the

equivalent bop (residues 73 - 89) in the N-terminal domain; the latter loop extends

across the p-sheet to lie over the foIinic acid ligand Sequence c o ~ s o n s between

residues 2 - 95 of the N-temiinal domaiu and residues 182 - 270 of the C terminal

domain did not show any significaat sequence homology. The h a i two heIices in the

C-terminal domain, a8 and a9, are located near the dimer interface and have residues

involved in intersubunit interaction.

Residues 208 - 214, which are in a Ioop region between a6 and a8, are poorly

defined in the electron density map and codd ody be modeled for one of the two

protomers. The temperature factors in this region of the structure are significantly

higher than observed in the rest of the structure suggesting some conformational

ffexibility in this region of the structure. The C-terminus of the molecule adopts a

short 310-heIix. This region of the structure is the expected entry-point into the

cyclodeaminase domain.

The two domains are separated by a short linker region (residues 179 - 18 1).

The side chain of Ars1 79 makes hydrogen bonding contact to the y-carboxylate group

of folinic acid. The temperature factors in this Iinker region are not significantly

higher than in other regions of the protein chah indicating that the linker is not more

flexible than the remainder of the molecule.

The dirner interface has been implicated as important for the hmction of the

FI' domain, since dissociation of the domain into protomers results in a Ioss of

catdytic activity (Murley & MacKenzie, 1997). Using the program GR4SP

(Nicholls et al. 1991), the buried surface area between the two protomers was

calcuiated to be approximately 1901 A'. The mterface is made up solely of residues

in the C- terminai domain. Three loop regions, between 07 and P6 (residues 189 -

192), P8 and P9 (residues 229 - 230) and a 7 and $10 (residues 260 - 266) and

residues 288 - 316 in the C-terminal a-helix make hydrogen bond contacts as welI as

hydrophobie interactions across the dirner interface. This C-terminal helix is

comprised of a poIar face made up of residues Gln295, G1397, His298, Arg301,

As11305 and Arg306. The side chains of al1 of these residues with the exception of

Arg301 make hydrogen bonding contacts with residues in the two loop regions

between p8 and P9 and behveen a7 and Plo. At the central region of the dimer

where the NCS two-fold symmetry axis is located a pocket of water molecules

rnaking contact to both protomers. Interestingly, a water molecule is present exactly

where the NCS two-fold symmeay axis lies. This waler molecule makes hyd&en

bonding contact with the main chah oxygen atom of Asn3O5 of each protomer as well

as well as two other NCS related water molecules. (Figure 3-10)

3.9. Ligand bindinp sites

The folinic acid binding site lies between the two domains of a protomer and

makes extensive contacts with residues in both domains. Significant diffmces were

observed in the positions of the foiinic acid ligand in the two protomers, particuiarly

the p-aminobenzoyl portion of the iigand. The electron density for the ligand in one

protomer was considerably weaker than obsened in the second protomer. Figure 3.4

shows the electron density for foIinic acid as well as select residues and water

molecules in the vicinity of the ligand in one monomer. IntereStingiy, the co-

q crystallization was carrieci out using a racemic murture of foiinic acid- Since the

Figure 3.10

A stereo view of the dimeric interface with the water moIecule (1abeId wat) at the

NCS two-fold symmetry axis. Some of the residues present at the dimeric interface

are also displayed. (producd using the program MOLSCRIPT, Krauf s, 1991).

physiological substrate for the enzyme is (65')-teûahydrofolate, it was expected that

the 6S enantiomer of folinic acid would bind preferentially. However, to our surprise,

it is clear fiom the electron density maps that the 6R enantiomer of folinic acid

preferentially binds to the enzyme. Attempts to mode1 and refine 6S enantiomer

clearly revealed difference electron density that confinned the presence of (6R)-folinic

acid, Co-crystdlizations were carried out with enantiomencdly pure (6R)-folinic acid

and (6s)-folinic acid (Schircks Laboratorks) using the same conditions as for the

racernic mixture. Ctystals ody appeared with (6R)-folinic acid confirming that the

enzyme that was crystallized has preferentiaI1y selected the 6R isomer of the ligand.

When the protomers are superimposed on each other it is observed that most

of the side chains which interact with the ligand adopt similar conformation in the two

protomers. The contacts between the ligand and the protein side chains differ in a

number cases (see Table 4) due largely to the differences in the position of the ligand

in the active sites of the two protomers.

in the protomer where the electron density for folinic acid is better defined, the

ligand makes 25 hydrogen bonding contacts with the protein and a M e r 7 hydrogen

bonding contacts with water motecdes. In the second protomer, the ligand makes

hydrogen bonding contacts with 24 pmtein atoms and a f i e r 5 hydrogen bonds with

water molecules. The hydrogen bond contacts made between f o h c acid and the

protein are given in Table 4. The teuahydropteridin ring system for (6R)-folinic acid

makes hydrogen bond contacts with the side ch in of Asp39, Ser40, Thr44 and

Glu228 (see Table 4). The carbanyi oxygen of the paminobemoyl moiety makes

e more extensive hydrogen bonds with the protein in the protorner (B) exhibiting the

Table 4 Hydrogen bonding contacts between folinic acid and FT domain.

*Col - glycerol Y - see Fiam 1.3 for atom name reference

Pmtomer "A" Ser4O-O

Gé1i'8-OEZ Wat588-0

Gln268-NE2 -39-ODI

Ser4O-O Thr44-W 1

Wad88-0

Asn 1 CLOD 1

hsnU7-ODI

GoI780*92 Wai829-0

HisBî-NE2 kg46-NH2

W 7 9 D I Go1780-03

AsnlO-ODI His8Z-NE-

Argl42-NH I

Asn 186-ND2 Gln233QEl Gln2684E1 l'y1 26-OH Arg t 79-NE Wat667-0

Tyr I269H Atg l %NE Arg 179-NH2

wat807-O

Distance (A) 3.04

3.20 3.50

353 2.48 3.32 3.41

2.4

3.23

2.80

3.30 3.02

3.61 3.07

3.17 3.29

352 3.45 3.26

3.24 2.86 278 2-64 3 -04 2.75 3.64 3 .14 186 2-76

folinic acid atorn nam& N 1

N3

N5 N8

NI0

OH4

N

O

O 1

0 2

0 3

OEl

OE2

Distance (A) 3.01 3.77

3.00 2.74 3.45 3.53 3.54 2.82 3.53 3.50 3.3 1 2.94 2.61 3.38 2.86 3.52 3.50 3.52 3.52 3.01

2.27

2.95 3.05 3.04 3.15 3.57

2.87 3.53 L90

266 3.06 2.69 3.55 3.21 7.97 in

Pmtomcr "Bn ser40-0

Glr268-N€2

Watl237-O Gld.28-OEI WatS66-O wat12060 (3111268-NE! @39-OD 1

Ser40-0 Asp39-43 Ars460

Thr44-OG1 Witt5660

Wat 1 290-0 Am IO-ODI Wat5 18-0

M 7 - 4 3 D I Asn237-ND2 His82-NE2 Go1780-02 - Wat909-O -

- Ars 142-NH2 Am237-OD I Go178 092 Go178 0.03 AsniO-ODI

*142-NH I Arg l 42-NH2 Asn 1 86-OD I

Tyr 1 Z-ûH Arg 179-NE W&28-û

Tyr 12WH Arg 179-NE Ar1 79-NH2 ws 1 I Z ~

better electron density for the ligand (Table 4). Furthemore, the side chahs of Val48,

His82 and Vd270, and the aliphatic portion of kg46 make favorable van der WaaIs

contacts with the ring of the p-amuiobenzoy1 moiety. Figure 3.11 shows the

interactions made by the folinic acid ligand and the protein molecule for a single

protomer.

During the course of the crystallographic rehement some density of unknown

origin was observed near the p-aminobenzoyl portion of folinic acid. inspection of

both the density and the crystallization conditions suggested that a single glycerol

molecule (10% in the crystallization mixture) was bound to each protomer (see Figure

3.8). As is the case with the folinic acid ligand, the quality of the electron density for

the giycerol molecules differ in the two protomers. Glycerol makes a total of four

hydrogen bond contacts with protein cesidues around the folinic acid binding pocket

(NE2-His82, NH2-Arg142, N-iie222, GSer235). In addition, the glycerol molecuIe

contacts the glutamate carboxyfate group of folinic acid (Figure 3.8).

An inspection of the molecular surface was carried out using the program

GRASP (Nicholls et al. 1 991) (see Figure 3.12 and 3.1 3). From this anaiysis we are

able to visuaiize the folinic acid Ligand buried between the two domains of the

monomer, in a tunnel which spans the width of the protein (see Figure 3.10). The

tunnel is approximately 38A long and 8A wide. The electrostatic surface of the

tunue1 reveals a concentration of negatively charged residues (e-g. Asp39. Glu228) at

the tetrahydropteroyl binding region of the protein and a trail of positively charged

residues (Arg142, Argl72, Lys180 and Lys218) where the y-Linked polyglutamate

moiety of the natural substrate is expected to bind. The folinic acid Iigand contains

Figure 3.1 1

Stereo view of the folinic acid binding site of FT domain. The main chah of the

protein is depicted in a nibon representation. The folinic acid and gLycerol moIecuIes

are disptayed in a dark baI1-and-stick styIe whiIe amino acid residues that make

hydrogen bonding contacts are displayed in Iight ball-and-stick bonds. Water

molecules are dispiayed as spheres, while the hydrogen bond contacts are displayed as

dashed bIack lines (produced using the program MOLSCRIPT, ffidis, 1991).

Figure 3.12

Mdecular surface representatian of the FT domain dimw. The electrostatic potential

of the protomer moIecules are mapped ont0 the surface between -1SkT ( r d ) and

+15kT (blue). Surface accesibk atorns of folinic acid are depicted in yelIow as a

space-fiIling mode1 (produced with the program GRASP, Nicholls, 199 1).

Figure 3.13

The cross-section through the surface representation of one the protomers tevealing

the electrostatic charges (-15 kT is represented in red and +15 kT is represented in

blue) king the tunnel. A backbone trace of the pmtein is represented as tubes, while

the ligands. (6R)-CoIinic acid and giyceroI are sbown in grey stick representation

(produced wi th the program GRASP, Nicholls, 199 1 ).

only a single glutamate group thus the remaining part of the tunnel which would

constitute the expected polyglutamate binding regions is occupied by water

molecules. Inspection of the two sequences of FTCD show that most of the residues

that make up the d a c e of the electrostatic tunnel, ùz particultir, the residues that are

thought to be important in bùiding the polyglutamate tail (specifically AsnlO, Glu128,

Arg179, Glu220, Asn237) are conserveci- This demonstrates the importance of these

residues. Studies by MurIey and MacKenzie (Murley & MacKenzie, 1995) have

s h o w that the predorninant glutamate binding site resides in the CD domain. The

base end of the tunnel, containhg the polyglutamate binding sites, lies near the same

surface as the C-terminus of the FT domain. Entry into the CD domain commences at

residue 334, with a Iinker regions of 8 residues behveen the two domains. Thus, the

location of the polyglutamate binding region should lie near the approximate position

of the CD domain. By docking a substrate andogue we were able to position a total

of three glutamate binding sites in the Rdomain.

Further inspection of the molecular surface revealed the presence of a second

tunnel approximately 9A long that intersects with major tunnel near the p-

aminobenzoyl portion of fohic acid A glycerol molede is located at the base of

tbis shorter second tunnel, where it intersects with the folinic acid tunnel. The

electrostatic surface of the tunnel is only slightIy positiveIy charged in contrast with

the surface of the main tunnel (see Figure 3.13).

3.10. Producc analome versus snbsfrate binding

Using the tetrahydrupteridin ring in the crystal structure as an anchor, we

modeled the substrate analogue, (6S)-tetrahydroptemyItriglutamate-Nme in the

binding site of FT-domain. This moIecuIe has the same chirality at the C6 position as

the natural substrate. As descnied below, this modeled complex appears to have

binding interactions with the protein that are equalIy favourable as those of (6R)-

folinic acid. Why then is the (6R) isorner of folinic acid preferred by the protein?

The answer seems to be due to the fact that folinic acts more like a product analogue

as a result of the presence of the formyl group at NS of the tetrahydropteridin ring.

This presence of the formyl group results in a steric repulsion between C9 and the

formyl oxygen thus destabtizing the bund conformation of the (65') isomer and

decreasing its binding affinity relative to the (6R) isomer which, as the c y t a i

structure shows, does not exhibit any steric repulsion. The substrate analogue is

unsubstituted at NS and is thus more readily accommodateci in the active site without

unfavorable steric interactions. One might raise the objection that the naturai

biosynthetic product of the substrate is in fact the N5-formimino derivative which is

isosteric with the fomyl group in folinic acid. We argue however, that this is not

inconsistent with the nature of the enzyme. ï h e formimino p u p is only present in

the product of the formiminomsferase reactian, Thus, upon product formation, the

unfavorable steric repulsion exhibiteci in the proteidproduct complex acts as a dnWig

force to release the ligand h m the FT binding site. Furthermore, these sterk clashes

wodd prevent the product h m rebinding to the transferase active site and would

drive the channehg of the product to the deaminase active site.

We now describe the main features of the substrate analogue mode1 (69-

tetrahydropteroyl-triglutamate-Nme. The optimum number of glutamates for

channehg of the product to the CDdomain is five, however, ody three can be

accomodated in the main fl tunnel. This suggests that the remaining two

glutamate binding sites residue in the CD-domain.

The overall charge distriiution of the docked substrate is compIemenmy to

that at the surface of the main tunnel of the domain. Positively charged pockets in the

tunnel accommodate negatively charged a-carboxylate groups of the three y-Iinked

glutamyl residues. in contrast, the region of the tetrahycîropteroyl rnoiety which

carries partid positive charges is sequestered within the opposite end of the tunneI

which is negatively charged (Figure 3.13). Molecula. eIectrostatic dipole

calculations, performed with the program GRASP (Nicholls, 1991) on the

uncomplexeri single protomer molecule are striking in that it shows a signifiant

dipole moment (237 Debye) positioned in the tunnel and directed towards the

negatively charged surface near the binding site of the tetrahydropteroyl rnoiety- This

dipole moment is anti-paralle1 to that of the isolateci substrate andogue molecule

calculated for its bound conformation, ïhus, the electrostatics in the tunnel is

expected to aid in the channehg mechanism by guiding the highly charged and

poIarïzed subsûate h m t3e primary glutamate binding site in the CD domain to the

FTdomain where the reaction is to occur.

The docked substrate analogue is predicted to establish a number of favorable

interaction contacts within the tunnel of the FTdomain (Figure 3.14). The

amphiphilic substrate molecule packs in a complernentary fasbion against the

intercalating polar and hydrophobie patches at the tunnel surface. in the mode1 of the

6S substrate analogue the methylene substituent at C6 lies in the equatorial rather than

the axial position as observed in the (6R)-foiinic acid complex. Nevertheles, the

(69-tetrahydropteroyl rnoiety of the docked ligand interacts with the protein residues

in a fashion which is sirnilar to that of the (6R)-tetrahydropteroyl part of the

crystailized folinic acid. The hydrogen bond interactions of the tetrahydroptendin

rnoiety with Asp39 and Glu228 as well as with a burieci moIecule are preserved (see

Figures 3.8 and 3.1 1). Energy minimization ailowed formation of a novel hydrogen-

bond between NI of the tetrahydropteridin ring and Gin268 residue.

The p-aminobenzoy1 fragment of the substrate anaiogue undergoes a

translation of approximateiy 1.8A W e r into the tunnel relative to its position as

seen in the compIexed folinic acid. This translation is a direct result of the more

extended structure of the equatorial versus axial conformation at the tetrahydropteroyl

C6 atom. in fact, we observed some W o m in the accommodation of the p-

aminobenzoyl p u p of the folinic acid in the two protomers.

The y-linked triglutamate part of the substrate is predicted to bind in an

extended conformation which follows the nanow channel of the enzyme domain

Translation of the paminobenzoyI fiagrnent alters the binding mode of the first

giutamate in the substrate relative to that in the folinic acid. in the substrate analogue,

the a-carboxylate of the first glutamate occupies the mean position of the y-

Figure 3.14

Stereo view of the enzyme in the region of the docked substrate, (6s)-

tetrahydropteroyltrigiutamate-Nme, ï he main chain of the protein is depicted in a

nibon representation. The substrate analogue is displayed in a dark ball-and-stick

style while arnino acid residues that make hydrogen bonding contacts are displayed in

tight ball-and-stick bonds. Water molecules are displayed as sphetes, while the

hydrogen bond contacts are displayed as dashed black lines (produced using the

program MOLSCRIPT, Ktaulis, 199 1 ).

carboxylate and a buried water molecule in the folinic a c i m domain complex. This

change in binding mode is quite reasonable, given that the y-carboxylate in folinic

acid becomes a y-linked amide in the substrate analogue. The a-carboxylate p u p

rnakes hydrogen-bond interactions with the side chains of AsnlO and kg179 as well

as with a buried water molecule. This water molecule is also hydrogen-bonded ta the

arnide NH p u p of the b t glutamate. The y-amide carbonyl interacts with the side

c h a h of Tyr126 and Arg179. in addition to the polar interactions, there is also a very

good hydrophobic packing between the aliphatic portion of the fïrst glutamate and the

side chains of Arg142 and k g 4 6 are no longer involved in salt-bridge interactions

with a-carboxylate group of the hrçt glutamate of the substrate. These positively

charged residues are now free to interact with the second substrate,

forniminoglutamate, and to stabilize a tetrahedral intermediate that woufd be fonned

during the transier of the formirnino group.

The second glutamate residue of the docked substrate analogue is also mostly

bwied in the putative binding tunnel. Its a-carboxylate makes hydrogen-bond

contacts with the side chab of Gln220 and the main chah MI group of Leu238. The

arnide NH groq interacts with a burieci water molecule. The y-amide carbnyl is

partially exposed to the solvent. The aliphatic portion of the second glutamate makes

hydrophobic interactions with the side chahs of Leu239, Leu182 and the aliphatic

portion of the k g 1 79 side chain.

The third giutamate residue is the most solvent exposed part of tûe modeIed

substrate complex. Its a-carboxylate group is hydrogen-bonded to the backbone

amides of Lys180 and Glu128, and is positioned in close proximity to the ammonium

group of Lysl80. The amide NH group interacts with a buried water molecule

whereas the y-amide carbonyf makes hydrogen-bond interaction with the side chah of

Lys 180.

3.1 1. Catafvtic mechanism

The reaction catalyzed by FTCD (Figure 1.2) transfers the formimino group

from formiminoglutamate to tetrahydrofalate and subsequently carries out a

cyclodeamination to give NS,N10-methenyl-THF- The precise mechanism for the

reactions and the residues important for catalysis and substrate binding are not yet

known. The structure of FTdomain provides us with a first view of the enzyme

active site and enable us to identify potential residues which may be implicated in the

catalytic mechanism. The N5 atom of the substrate, the nucleophile expected to

attack the irnino carbon of formiminoglutamate, is completely buied h m the

external surface of the protein. Such a buried environment will protect the labile NS-

formimino-tetrahydrofolate product of the FT reaction h m hydrolpis. The presence

of the bound glycerol molecule (a close mimetic of the product glutamate) at the base

of the second tunnel suggests that this short tunnel may be a route through which the

formiminoglutamate substrate enters and the glutamate product Ieaves. However,

examination of this route shows that access to N5 of the tetrahydropteridin ring is

bIocked by the side chain of His82- The presence of a histidine (His82) near the

submte is tantalizing, since previous studies have indicated that a histidine residue

may be important in the formimùiotransferase reaction. A posaile role for His82 is

that of a base abstracting the proton fiom N5 of tetrahydrofolate, thus increasing iis

nucleophilicity for attack at the imino carbon atom of formiminoglutarnate (see Figure

3-15), The protonated fiis82 could subsequently facilitate the breakdowu of the

intermediate by protonating the amino group of the giutamate yielding the products.

in the cunent crystal structure, His82 is positioned approximately 7 A h m NS of the

substrate which is too far to fulfill its proposed role as a base. In order for both the

formiminoglutamate and the side chain of His82 to lie in close proximity to the N5 of

tetrahydrofolate, the protein must undergo a change in conformation, including a

change in the histidine side ch in position in the substrate bound structure in order to

provide access to N5 of tetrahydrofolate. His82 c m be brought within 3.5A of N5

when Chi 1 is rotated. Other movernents in the structure would need to be made in

order to accommodate formiminoglutamate near the nucleophilic center of

tetrahydrofolate. ïhese changes in the protein may occur not just as srnail torsional

changes of side chahs but may involve large loop movements. in order to address

these questions it will be necessary to determine the structure of a subsmte analogue

complex of the Mdomain.

3.12. Data colIection and mode1 mfinement of FT domain comdaed with C5,CIû- dideazatetrah ydrofolate

Data were collected to 2.8A resolution using CuKa radiation and a MAR

image plate detector. Statistics h m the data collection are shown in Table 5. Since

the space group and the unit ce11 dimensions are identicai to those of the folinic acid

complex model (FT-fol), this model was used to soive the C5,ClO-

dideazatetrahydrofoIate complex structure (FTddf), The water molecules, Ligands

Figure 3.15

Schernatic of a possible catalytic mechanism far the fomimino-tramferase

reaction using His82 as a base catalyst.

Table 5

Data collection statistics for FT domain in complex with C5,ClO- dideazatetrahydrofolate.

Resolution (A) 50.0 - 2.8 Number of unique reflections 207 15 Total number of reflections 82 106 % complete 98.9 l/c 11.0 R-merge (%) 6.1

and severai loop regions were rernoved (as discussed in Section 2.5.2) and this edited

model was used to calculate the initial phases for the C5,CIO-dideazateûahydrofolate

complex structure. This was accomplished by refining the data obtained against the

modified folinic acid model. SIGh4AA weighted 3Fo-2Fc and Fo-Fc electron density

maps were calculated fiom the refined modified folinic acid model and used to

rebuild the rnissing loop regions as weU as the mode1 for the C5,ClO-

dideazatetmhyrofolate substrate analogue. The refinernent statistics for the FT-ddf

model are shown in Table 6.

3.13. Structure of FT domoin in com~fex with C5.C1Oilideazatetrahvdrofolate

The overall structure and fold the FTddf model is very similar to that of the

FT-fol structure. The major differences that occur between the two structures are in

and around the ligand binding site and in the actual conformation of the ligand itself.

The model consists of two protomers, each a single polypeptide chah

encompassing residues 2-327. While, in contrast to the originai model where

protomer "A" consisted of two polypeptide chahs (residues 2-207 and 214-326) and

protomer 'B" consisted of a single polypeptide chab (residues 2-326), the two

protomers in the FTddf mode1 consists of a single polypeptide chah each (nisidues

2-327). ïhis is likely due to the fact that NCS-restrahts were applied to the

refînement of the latter model, to 2.8A cesolution, while the former, to 1.7A

reoslution, was refined without imposing any NCS symmetry. The new model aiso

contains a moIecule of giycerol and a molecuie of (6R)CS,C10-

Table 6

Model refinement statistics for mode1 C5,ClO-dideazatetrahydrofolate.

Resolution range (A) 6.0-2.8 R-factor 22.7 R-fiee * 28.1 R.m.s.d. bond lengths 0.008 Rm.s.d. bond angles 1.30 Number o f non-hydrogen atorns 5057 Average B-factors (A2) - Overail 24.34 - . - -- --

* 10% of data was used to cdcuiate R-ke.

dideazatetrahydrofoIate per protomer. The eIectron density for the glycerol is not very

well defineci, due to the reiatively low resolution, making the exact torsional

conformations difficult to determine. No water molecdes were built into the model,

alsa due to the resolution of the electron density map. Figure 3.16 shows a regioa of

the elecmn density map.

Superposition of the two protomers h m FTddf mode1 resuIted in an r.ms.

difference of 0.3 1A between 326 structurally homolugous Ca-atoms- Superposition

of the first and second protomer of ihe FT-fol model ont0 the respective protomer of

the FTddf mode1 yields a r.m.s. difference of 0.31A and 030A between 318

snucturaIly homologous Ca-atorns. Thus the structures of the two models are vwy

similac Rowever, local differences are observed, for example, in protomer "B" of the

FTddf model, the loop region between residues 224-231 shi% as much as 2.5A

(Figure 3.17). The ligand, C5,CIO-dideazatetrahydrofolate, appears to be in a more

extended confoxmation that places the pterh ring closer to the extemal surface of the

protein when compareci to the bindhg of the folinic acid ligand. Such a shifl in the

Ioop region avoids unfavourable contacts that wodd occur between the pterin ring of

the ligand with the side chahs of Leu226 and Lys 229 h m this loop region (Further

discussed beIow).

InterestingIy, upoo initial inspection and cornparison of the FTddf model with

the FT-fol mode1 it would appear &at the better dehed densrensrty for the ligand resides

in the protomer "A" as opposed to protomer "B" as it did in the FT-fol model.

Attempts to model (6R)CS,Clûdideazatetrahydrofolate m the electron deLlSity of both * protomers was s u c c d - The (6R)CS,C 1 Mdeazatetrahydrofolate is analogous to

Figure 3.16

A stem view of the 3Fo-2Fc electron density map depicting the substrate analogue,

C5,ClO-dideazatetrahydrofolate, and giycerol molecule for a singie protomer. Some

side chains and water moiecules are also displayed. The map is contoured at l a

(produced with the program, SETOR, Evans, 1993).

Figure 3.1 7

Stereo view of the C5,ClMideazatetrahydrofoIate binding site of FT domain. The

main chah of the protein is depicted in a nibon representation. The substrate

analogue, 3,1 O-dideazatetrahydrofolate, and glycerol molecules are displayed in dark

grey bonds. Amino acid residues that make hydrogen bonding contacts are dispIayed

in light grey bonds, the hydrogen bond contacts are displayed as dashed black lines

(produced using the program MOLSCRIPT, Kraulis, 1991).

toi

(69 tetdydrofolate in t a s of stereochemistry (Figure 3.18). While the electron

density is not very well defined due to the relatively poor resolution ( U A resolution

for the F ï d d f modei as opposed to 1.7A for the FT-fol model), the physiologically

relevant stereoisomer can be satisfactorily modeled into the density. However, due to

the poor electron density around the C6 atom of C5,C1O-dideazatetrahydrofolate,

higher resolution data will be needed to unambiguously confirm whether the correct

stereoisorner has been modeled (Figure 3.16).

in comparing the conformation of the ligands between the two structures,

changes in the relative position of the pterin ring system are observed. There is an

inversion in the stereochernistry at Cd and a change in the pucker of the pterin ring

systern (Figures 3.17 and 3.19). This difference in ring pucker results in an equatorial

substituent at C6 rather than the axial as was observed in the FT-fol model and results

in an extended conformation that pushes the pterin ring towards the extemal M a c e

of the protein. With out a shifi in the loop region consisting of residues 224-229, this

conformation of the ligand would resuit in unfavourable contacts between the

substrate analogue and some of the side chains of residues in this Ioop region. This is

in agreement with what was predicted in the substrate docking study for pterin ring of

(65')-tetrahydrofolate ( s e section 3.10).

Similar contacts are made to the C5,ClO-dideazatetrahydrofolate ligand as to

the fotinic acid Ligand in their respective models. Interestingly, the contacts made in

protomer "B" of the FT-fol model a~ more similar to those observed in protomer "'An

of the FTddf model. For example, identical hydrogen bond contacts between OEl

and OE2 of G l m 8 and NA2 and N3 of the pterin ring are observeci for protomer "B"

Figure 3.18

Chemical structures of the naturai substrate (65')-tetrahydro fo Iate and the substrate

analogue, (6R)-5, IO-dideazatetrahydrofolate depicting the chirality at C6.

Figure 3.19

Stem view of the superimposition of FT-fol and the FTddf ligand binding sites. The

main-chah of the protein is depicted in a ribbon representation and the side chahs,

foliuic a d , C5,Cl Oaideazatetcahydro folate and glyceroI are shown in a ball-and-

stick representation. Dark grey represents the FT-fol mode1 and light grey corresponds

to the FTddf mode1 (produced using the program MOLSCRIPT, Kraulis, 199 1).

of the ET-fol model and protomer ""A" of the FT-ddf model. This suggest that

dlosteric interactions exist as the binding of a ligand to one protorner of Ff domain

appears to interfere with ligand binding in the other protomer. Further structural and

kinetic analysis is necessary to test this postulation. Table 7 shows a list of hydrogen

bond contacts made between the C5,ClOdideazatetrahydrofolate ligand and the

protein, for each protomer.

Table 7

Hydrogen bonding contacts between C5,ClO-dideazatetrahydrofolate and FT domain.

Protomer "A"

- Glü228-OE 1 Glu228-0E2

Thr44-N

Asnl O-OD 1 A~nî37-OD 1 Arg 142-NH1 Arg46-NH 1

, Arg142-NH2 Arg 1 79-NH 1 Argl 79-MI1 Tvrl26-OH

Distance (A)

- 2.77 3.35 2.95

C5,C 1 O- dideazatetrahydrofolate atom

name N1

NA2 N3

OH4

3.1 1 2.57 2.64 2.8 1 2.78 3.56 2.55 3.04

3.12 2.73 2.79 2.82 3.05 2.64 2.92

N O O1

0 2 OEl 0E2

Distance (A)

3.08 -

2.93 3.40

AsnI O-OD 1 -7-ND2 Arg 142-NH 1 Arg46-NH 1 Arg 142-NH2 Arg 1 79-NH 1 Tyr1 26-OH

Protomer "B"

AJn268-NE2 -

M 6 8 - O E 2 Asn 186-ND2

Chapter four

Conclusions and future perspectives

Substrate channeling has been described as a kinetic process where the

product of one reaction is directly transferred from one enzyme active site to the

another active site, without allowing the product of &he k t reaction to accumulate.

Channeling is considered to be a beneficid process for a number of reasons (reviewed

by Ovadi and Srere, 19%; Clvadi, 1991; Srere and Ovidi,1990) . Channehg

decreases the time it takes for the intermediate to be ûansferred between active sites,

thereby increasing the overd1 efficiency of the metabolic pathway. Aiso, if the

intemediates are labile, channehg has the advantage of protecting these

intermediates h m degradation, by either sequestering or converting the intermediate

to a more stable product. Sequestering an intemediate also prevents the loss of the

intermediate by difiion. Structurai studies intended to examine and cornprehend the

mechanisms of channeling have shown that charge distrhtion via d a c e

etectmstatics or sequatering via an intramolecular tunnel are two mechanisrns by

which channeling occurs. It has been shown that FïCD has tbe ability to cbannel

intermediates (MacKenzi and Baugh, 1980) and it tias been postulated that the

mechanism is a swinging ami mechanism. Crystaiiographic siudies of FT domain

have provideci an initial of view of the mechanism of channeling for FTCD, and wiII

enable compatison with the rnolecular mechanisms of other b h c t i o n d enzymes.

The two structures of FT domain with different ligands has shown that it is

composed of two sub-domains both adopting a novel a /p foId One structure binds a

moleeule of (6R)-folinic acid and a glycerol molecuie per monomer, while in the

other structure a molecule of (6R)-C5,ClO-dideazatetrahydrofolate and a molecule of

glycerol are bound to each protomer, The FT-fol model provided a high resolution

view of the structure and a starting point for addressing the molecuIar mechanisrn of

channeling as well as the mechanism of the actual transferase reaction. The structure

of the FT-ddf model provides confirmation to the substrate docking and molecular

modeling study. The structures provide an initial foundation for testing the

mechanisms by site-directeci mutagenesis. Mutagenesis of the His82 residue in FT

domain, with kinetic analysis of the resulting mutant enzyme, will test the hypothesis

of the role of this residue. Mutagenesis of residues implicated in the binding of

tetrahydrofolate, such as G1338, Arg46, Arg142, Arg179 and other arginine and

lysine residues implicated in the binding of the polyglutamate tail, in conjunction with

kinetic and stnictural analysis, will test the role of these residues in the binding and

channehg of the polyglutamylated folates.

Further crystailographic studies using other substrate analogues, such as

FIGLU and glutamate, will provide information on the residues important in their

respective binding. Crystallization of the CD dom* which is undenvay in the

laboratory, and its subsequent stnictural analysis wiIl aiso funush fiirther

understanding in the channeling rnechanism of FïCD.

The crystal structure presented in this thesis has provided detaiIed information

on one di-c interface in FTCD. ïhere has been some speculation that the active

site for the FTCD is located at the duneric interface, since its integrity is necessary for

acbvity. The crystal structure of FT domain has shown that this is not the case and

that the active site lies between hvo subdomains in the protomer. Thus we suggest

that the d i m e c interface maybe necessary to stabilize the active site. The crystal

structure has also provideci an initial view of the mechanism of the

fomiminotransferase reaction, alIowing the molecuIar mechanisms of FTCD to be

probed by site-directeci mutagenesis.

Other crystallization attempts with substrate analogues for both

tetmhydrofolate and formiminoglutamate may yield crystals which wiil in turn Iead to

new models which will fiirther the understanding of the binding mode of both

substrates. Also crystallization trials of CD domain are underway. The structure of

CD domain will give information on the binding specificity of the polygiutamate tail,

a s weI1 as provide the other haif of the picture for the channeling mechanism.

ültimatefy, crystals of the native FïCD will provide a view of how both domains

interact and provide a complete picture of the channekg mechamanism.

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