Architecture of B-Hellices Parallel)

8/8/2019 Architecture of B-Hellices Parallel)

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Progress in Biophysics & Molecular Biology 77 (2001) 111175

Review

The architecture of parallel b-helices and related folds

John Jenkinsa,*, Richard Pickersgillb

a Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UKbBiological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK

Abstract

Three-dimensional structures have been determined of a large number of proteins characterized by a

repetitive fold where each of the repeats (coils) supplies a strand to one or more parallel b-sheets. Some of

these proteins form superfamilies of proteins, which have probably arisen by divergent evolution from a

common ancestor. The classical example is the family including four families of pectinases without

obviously related primary sequences, the phage P22 tailspike endorhamnosidase, chrondroitinase B and

possibly pertactin from Bordetella pertusis. These show extensive stacking of similar residues to give

aliphatic, aromatic and polar stacks such as the asparagine ladder. This suggests that coils can be added or

removed by duplication or deletion of the DNA corresponding to one or more coils and explains how

homologous proteins can have different numbers of coils.

This process can also account for the evolution of other families of proteins such as the b-rolls, the

leucine-rich repeat proteins, the hexapeptide repeat family, two separate families of b-helical antifreeze

proteins and the spiral folds. These families need not be related to each other but will share features such as

relative untwisted b-sheets, stacking of similar residues and turns between b-strands of approximately

901often stabilized by hydrogen bonding along the direction of the parallel b-helix.

Repetitive folds present special problems in the comparison of structures but offer attractive targets for

structure prediction. The stacking of similar residues on a flat parallel b-sheet may account for the

formation of amyloid with b-strands at right-angles to the fibril axis from many unrelated peptides.r 2001

Elsevier Science Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

1.1. Scope of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

1.2. Nomenclature, definitions and general features of parallel b-helices . . . . . . . . . . . . 116

1.2.1. Parallel b-helix and its b-sheets . . . . . . . . . . . . . . . . . . . . . . . . . . 116

*Corresponding author.

E-mail address: [email protected] (J. Jenkins).

0079-6107/01/$ - see front matterr 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 7 9 - 6 1 0 7 ( 0 1 ) 0 0 0 1 3 - X


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1. Introduction

1.1. Scope of the review

In 1993 Yoder et al. (1993a) reported the structures of the first parallel b-helix and Baumann

et al. (1993) that of the first b-roll, which were at first regarded as revolutionary in using parallel

1.2.2. Coils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

1.2.3. Stacks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

1.2.4. Turns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

1.2.5. Packing ofb-sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

2. Description of known structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

2.1. Pectinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

2.1.1. The extra-cellular pectate lyase family . . . . . . . . . . . . . . . . . . . . . . . 126

2.1.2. Polygalacturonases and rhamnogalacturonase A . . . . . . . . . . . . . . . . . . 130

2.1.3. Pectin methylesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

2.1.4. PelL from Erwinia chrysanthemi . . . . . . . . . . . . . . . . . . . . . . . . . . 134

2.1.5. Pectate lyase Pel-15 from Bacillus sp. strain KSM-P15 . . . . . . . . . . . . . . 134

2.2. The P22 phage tailspike endorhamnosidase . . . . . . . . . . . . . . . . . . . . . . . . . 135

2.3. Chrondroltinase B from flavobacterium hepinarum . . . . . . . . . . . . . . . . . . . . . 138

2.4. P69 pertactin from Bordetella pertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

2.5. Glutamate synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

2.6. The antifreeze protein from Tenebrio molitor . . . . . . . . . . . . . . . . . . . . . . . 1412.7. The leucine-rich repeat family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

2.7.1. Ribonuclease inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

2.7.2. The GTPase-activating protein Ma1P from Schizosaccharomyces pombe . . . . . 144

2.7.3. Human insulin-like growth factor receptor. . . . . . . . . . . . . . . . . . . . . 144

2.7.4. Human spliceosomal protein U2A0, Rab geranylgeranyltransferase and the

mRNA export factor TAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

2.7.5. Internalin B from Listeria monocytogenes . . . . . . . . . . . . . . . . . . . . . 146

2.8. Left-handed parallel b-helix structures . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

2.8.1. Left-handed parallel b-helix structures containing hexapeptide repeats . . . . . . 147

2.8.2. The left-handed parallel b-helix antifreeze protein from spruce budworm . . . . . 150

2.9. Parallel b-rolls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

2.10. Spiral folds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

3. The prediction and design of parallel b-helix structures . . . . . . . . . . . . . . . . . . . . . 152

4. Are amyloid fibrils related to parallel b-helices? . . . . . . . . . . . . . . . . . . . . . . . . . 156

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

5.1. Evolutionary relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

5.2. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

J. Jenkins, R. Pickersgill / Progress in Biophysics & Molecular Biology 77 (2001) 111175112


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b-sheet alone to form complex structures by repeating individual coils. Subsequently the group of

proteins built using similar principles, for which Jurnak et al. (1994) used the term coiled folds and

Kobe and Deisenhofer (1995a, b) used solenoid proteins, has greatly extended. However, new

discoveries do not remain shocking for long and structural biology has rapidly absorbed thelessons of these structures. Purely parallel b-structures are no longer described as unstable and the

parallel b-helix has simply taken its place amongst the globular proteins with all b-folds. This is

justified because the parallel b-helix architecture is simply one of many ways to form a globular

folded protein, which are stabilised by the same types of interactions. Thus we can easily imagine

a parallel b-helical enzyme and one with a different architecture converging to use the same

catalytic mechanism.

However, structures with the parallel b-helix architecture do possess some unusual common

features and the ambiguity of finding a unique solution when aligning these structures does

pose special problems when comparing them. The unusual simplicity of the architecture also

suggests that understanding their evolution or predicting their occurrence may be unusuallyeasy. This is not unique and may also apply to folds such as the b-propeller fold (F .ull.op and

Jones, 1999).

Parallel b-helix domains appear to fold as a single co-operative unit as do the domains of other

globular proteins. However, there is a sense in which parallel b-helices and other coiled folds are

intermediate between most globular enzymes and structures made up of domains arranged as

beads on a string, where the folding unit is the individual repeating unit. In the parallel b-helices

the unit of folding is the domain while the unit of evolution may have been the individual coil

(see Section 1.2.2 below).

Parallel b-helices also form a bridge between globular and fibrous proteins. The rather flat

(untwisted) parallel b-sheets and the stacks of similar or alternating residues are special and

probably related features of these folds and we may expect to find similarities between the parallelb-helix architecture and other systems where untwisted b-sheets pack against each other. This has

led to analogies being made between parallel b-helices and some models of amyloid.

Kobe and Kajava (2000) have recently briefly reviewed the whole family of coiled folds or

solenoid proteins and identified 18 solenoid folds (see http://cmm.info.nih.gov/kajeva/solenoidta-

ble.html). The structures containing only a-helices have been recently reviewed by Kobe et al.

(1999) and we will focus on the structures with parallel b-sheets, which are listed in Table 1, and

especially the parallel b-helical proteins. Even this is a long list and is growing rapidly (for

example two different parallel b-helical antifreeze protein families have been published in 2000,

glutamate synthase shows a parallel b-helical domain and the abstract by Rozwarski et al. (1999)

appears to promise a new family). The main focus will be the architecture of these proteins ratherthan the details of their function. All these parallel b-sheet containing proteins have some

common architectural features. These may also share a common mechanism of evolution (as

distinct from a common ancestor). They show the more general properties of solenoid proteins

identified by Kobe and Kajava (2000). However, the focus on the parallel b-sheet containing

proteins makes it hard to discuss Kobe and Kajavas suggestion that evolution may relate

the leucine-rich repeat (LRR) proteins discussed below with the LRR variant family with a

3/10-helix and an a-helix per coil (Peters et al., 1996). We will also include a brief description of

the evidence for proposed amyloid structures, especially on the suggestions of similarity to parallel

b-helices.

J. Jenkins, R. Pickersgill / Progress in Biophysics & Molecular Biology 77 (2001) 111175 113


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Table 1

Structures with repetitive folds containing b-sheetsa

Name and origin of the protein Short name PDB code Resolution in (A,

reference

Comments

Right-handed parallelb-helix proteins

Pectate lyase PelC from Erwinia PelC 1AIR 2.20 [1]

chrysanthemi 1PLU 2.20 [2] Lu3+ complex

Pectate lyase from Bacillus subtilis Bspel 1BN8 1.80 [3]

2BSP 1.80 R279K

Pectate lyase PelE from Erwinia

chrysanthemi

PelE 2.20 [2,4]

Pectin lyase PnlA from Aspergillus niger PnlA 1IDJ 2.40 [5] pH 6.5

1IDK 1.93 [5] pH 8.5

Pectin lyase PnlB from Aspergillus niger PnlB 1QCX 1.70 [6]

Rhamnogalacturone A from Aspergillus RGase A 1RMG 2.00 [7]

aculeatus

Polygalacturonase PehA from Erwinia

carotivora

PehA 1BHE 1.90 [8]

Polygalacturonase II from Aspergillus

niger

PG II 1CZF 1.68 [9]

Pectin methylesterase from Erwinia

chrysanthemi

PemA 1QJV 2.40 [10]

Pectate lyase Pel-15 from Bacillus

sp. strain KSM-P15

Pel-15 1EE6 2.30

Salmonella P22 phage tailspike TSP 1TSP 2.00 [11,12]

endorhamnosidase 1TYU 1.80 [13] Complex

1TYV 1.80 [13] Complex

1TYW 1.80 [13] Complex

1CLW 2.0 [14] V331A

1QA1 2.0 [14] V331G1QA2 2.0 [14] A334V

1QQ1 1.8 [15] E359G

1QRB 2.0 [15] T326F

1QRC 2.5 [15] W391A

Chrondroitinase B from Flavobacterium 1DBG 1.70 [16]

hepinarum 1DBO 1.70 [16] Complex

P69 pertactin from Bordetella pertussis Pertactin 1DAB 2.60 [17]

Glutamate synthase from Azospirillum

brasilense

1EAO 3.0 [18]

Antifreeze protein from Tenebrio molitor TmAFP 1EZG 1.40 [19]

Leucine-rich repeat proteins

Porcine ribonuclease inhibitor RI 2BNH 2.30 [20]

1DFJ 2.50 [21] RibonucleaseComplex

Human ribonuclease inhibitor HRI 1A4Y 2.00 [22] Angiogenin

complex

Human insulin-like growth factor receptor IGF 1IGR 2.60 [23]

Human spliceosomal protein U2A0 U2A0 1A9N 2.4 [24] U2b00-U2A0U2

RNA complex

Rab geranylgeranyltransferase RabGGT 1DCE 2.0 [25]

Human mRNA export factor TAP TAP 1FO1 2.9 [26]

GTPase-activating protein Ma1P from

Schizosaccharomyces pombe

Ma1P or

RNA1P

1YRG 2.66 [27]

Internalin B from Listeria monocytogenes InlB 1DOB 1.86 [28]



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Table 1 (continued)

Name and origin of the protein Short name PDB code Resolution in (A,

reference

Comments

Left-handed parallelb-helix hexapeptide

repeat proteins

UDP-N-acetylglucosamine

acyltransferase from Escherichia coli

LpxA 1LXA 2.60 [29]

Carbonic anhydrase from

Methanosarcina thermophila

Cam 1THJ 2.80 [30]

Tetrahydrodipicolinate

N-succinyltransf erase

DapD 1TDT 2.20 [31]

2TDT 2.00 [32] Complex

3TDT 2.00 [32] Complex

Xenobiotic acetyltransferase from

Pseudomonas aeruginosa

PaXAT 1XAT 3.20 [33]

2.25 [34]

N-acetylglucosamine 1-phosphate uridyltransferase

from E. coli (truncated)

2.30 [34] Complex

N-acetylglucosamine 1-phosphate

uridyltransferase from E. coli

1HV9 2.1 [35] Complex

N-acetylglucosamine 1-phosphate 1G95 2.33 [36]

uridyltransferase from 1G97 1.96 [36] Complex

Streptococcus pneumoniae 1HMO 2.3 [37]

1HM8 2.5 [37] Complex

1HM9 1.75 [37] Complex

Left-handed parallelb-helix pentapeptide repeat proteins

Antifreeze protein from spruce budworm sbwAFP 1EWW NMR [38]

b-roll proteins

Alkaline protease from Pseudomonas

aeroginosa

1KAP 1.64 [39]

50 kDa metalloprotease from Serratia

marcescens

1SAT 1.75 [40]

Spiral folds

4-Chlorobenzoyl coenzyme A dehalogenase 1NZY 1.80 [41]

Enoyl-coenzyme A hydratase 1DUB 2.50 [42]

2DUB 2.40 [43] Complex

ATP-dependent Clp protease from

Escherichia coli

ClpP 1TYF 2.20 [44]

aPublications describing new or refined structures are given as [1] above. These are 1. Yoder et al. (1993a); 2. Lietzke

et al. (1994); 3. Pickersgill et al. (1994); 4. Lietzke et al. (1996); 5. Mayans et al. (1997); 6. Vitali et al. (1998); 7. Petersenet al. (1997); 8. Pickersgill et al. (1998); 9. Van Santen et al. (1999); 10. Jenkins et al. (2001); 11. Steinbacher et al. (1994);

12. Steinbacher et al. (1997); 13. Steinbacher et al. (1996); 14. Baxa et al. (1999); 15. Schuler et al. (2000); 16. Huang et al.

(1999); 17. Emsley et al. (1996); 18. Binda et al. (2000); 19. Liou et al. (2000); 20. Kobe and Deisenhofer (1993); 21.

Kobe and Deisenhofer (1995b); 22. Papageorgiou et al. (1997); 23. Garrett et al. (1998); 24. Price et al. (1998); 25. Zhang

et al., 2000; 26. Liker et al. 2000; 27. Hillig et al. (1999); 28. Marino et al. (1999); 29. Raetz and Roderick (1995); 30.

Kisker et al. (1996); 31. Beaman et al. (1997); 32. Beaman et al. (1998a); 33. Beaman et al. (1998b); 34. Brown et al.

(1999); 35. Olsen and Roderick (2001); 36. Kostrewa et al. (2001); 37. Sulzenbacher et al. (2001); 38. Graether et al.

(2000); 39. Baumann et al. (1993); 40. Baumann (1994); 41. Benning et al. (1996); 42. Engel et al. (1996); 43. Engel et al.

(1998); 44. Wang et al. (1997).



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1.2. Nomenclature, definitions andgeneral features of parallelb-helices

1.2.1. Parallelb-helix and its b-sheets

The term parallel b-helix was introduced by Yoder et al. (1993a) when reporting the structureof the pectate Lyase PelC, the first protein structure displaying this fold. It is important to note

that the parallel b-helix is not related to the term b-helix, used earlier to describe the

structure of gramicidin (Wallace and Ravikumar, 1988; Langs, 1988). The publication of the first

left-handed structure of UDP-N-acetylglucosamine acyltransferase by Raetz and Roderick (1995)

required that parallel b-helix be further extended to either right-handed parallel b-helix or

left-handed parallel b-helix. The nomenclature generally used to describe the basic architecture

of the parallel b-helical proteins was essentially fixed by Yoder et al. (1993b), who introduced the

nomenclature PB1, PB2 and PB3 for the three parallel b-sheets of pectate lyase and T1, T2 and T3

(turn) for the regions following the b-sheets. This was revised to include PB1a and implicitly T1a

by Petersen et al. (1997) when the structure of rhamnogalacturonase A from Aspergillus aculeatusclearly showed a fourth b-sheet. Figs. 1 and 2 illustrate this convention. An alternative

nomenclature was proposed for a fragment of the P22 phage tailspike protein by Steinbacher et al.

(1994) in which the PB1 is equivalent to b-sheet C, PB2 equivalent to A and PB3 equivalent to B.

Van Santen et al. (1999) in describing polygalacturonase II from Aspergillus niger use PB2a and

PB2b as an alternative nomenclature corresponding to PB1a and PB2 used for RGase A and

PehA.

1.2.2. Coils

As turn was used by Yoder et al. (1993b) to describe the regions between the b-stands by

analogy with b-turns in hairpin loops, there is a need for an unambiguous term for a complete

turn of the parallel b-helix and the term coil is used in this review. However, there remains the

difficult problem of whether to count the coils using topological arguments or to accept only coils

with the regular three-stranded parallel b-helical structure. This ambiguity results in great

confusion on how many coils occur in known structures and in how to number them. A good

example is provided by the family of hydrolases containing RGase A, PehA and PG II because the

reasoning for the different choices between 10 and 13 coils can be explicitly described. RGase A

might be said to have 12 coils starting with the first PB2 (residues 2022) and finishing with the

c

Fig. 1. Ribbon diagrams and views of the cross-section of single coils running clockwise for various types of repetitive

fold. The atoms of the cross-section are coloured by type: black for carbon, blue for nitrogen, red for oxygen and yellow

for sulphur. (a) Part of the b-roll of the alkaline protease from Pseudomonas aeroginosa (1 kap) with the tandem

repeated sequence GGXGXDXLX giving rise to the b-roll architecture. The two b-sheets are coloured red and green.

Calcium ions are shown as yellow balls. The single coil runs clockwise starting with the sequence IleLeuTyr. (b)

Bacillus subtilis pectate lyase (1bn8) with the N-terminal end of the parallel b-helix to the left and the T3 region above.

The three b-sheets PB1 (ThrAspAla), PB2 (IleThrMet) and PB3 (TyrTyrHis) are coloured yellow, green and red.

An asparagine of the asparagine ladder can be seen at T2 (just before PB3) and a residue of the aromatic stack can be

seen on PB3. (c) Porcine ribonuclease inhibitor with the b-sheets in red and the helices in green. In the ribbon diagram

the chain direction or direction of the superhelix is anti-clockwise. However, the single coil runs clockwise and shows

the leucines from the a-helix and b-sheet packing in the hydrophobic core. (d) The left-handed UDP-N-

acetylglucosamine acyltransferase from Escherichia coli with the N-terminal end of the parallel b-helix to the right.

The coil shows the isoleucines that form its hydrophobic core.



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last PB1a (residues 353356) as shown in Table 1 of Petersen et al. (1997), which lists 12 PB2

stands. Petersen et al. suggest that there are 13 coils because the main chain forms a very tight coil

with residues 354 and 356 forming hydrogen bonds to the previous PB2 at residues 331333 (i.e.

the last PB1a acts as both a PB1a and a PB2), forms a disulphide to Cys 350 in the previous PB1

Fig. 2. Ribbon diagrams of some of the longer right-handed parallel b-helices. The three common b-sheets PB1, PB2and PB3 are coloured yellow, green and red, while the extra sheet PB1a found in the polygalacturonase family is

coloured blue. The N-terminal end of the right-handed parallel b-helices are to the right. (a) The Salmonella P22 phage

tailspike endorhamnosidase. (b) P69 pertactin from Bordetella pertussis showing the longest known parallel b-helix. (c)

The polygalacturonase PehA from Erwinia carotavora showing the PB1a sheet within the T1 region.



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from Cys 359 and then reverses direction to give a last PB1 strand (residues 369375), so that the

topological winding number is 13. However, the strand of residues 369375 is anti-parallel to the

previous PB1 making the nomenclature PB1 rather confusing, so that we might reject the residues

after 356 as not being part of the parallel b-helix. We might also reject the first coil because itcontains a single b-strand (PB2) and an a-helix by arguing that this is not a parallel b-helix,

leaving us with 11 coils. Finally, using the original definition of Yoder et al. (1993b) and requiring

that each coil starts with PB1 and ends with PB3 would leave 10 coils, which is how the very

similar structure of PehA is described by Pickersgill et al. (1998). PehA forms the same final PB1a

to PB2 hydrogen bonds but does not form the disulphide or the anti-parallel structure. PGII does

not form the PB1a to PB2 hydrogen bonds as it forms a rather different structure with proline 357

in approximately aR conformation at the end of PB1a (or PB2a), terminates by forming the

disulphide without any anti-parallel extension and is also described as having 10 complete turns

(van Santen et al., 1999). What is more significant than the number of coils used to describe the

structure is that these hydolases have two complete coils and a PB1 and PB1a more than thepectate lyases or the pectin methylesterases and three fewer coils than the P22 phage tailspike

endorhamnosidase. It is important to check the number of coils visually rather than to accept

statements in the literature since an unexpected definition may have been used.

1.2.3. Stacks

Yoder et al. (1993b) first discussed the stacking of similar residues at the equivalent positions

in neighbouring coils and this was extended by Raetz and Roderick (1995), who revived the term

cupped stacks for the most common stacking of valines and isoleucines. Petersen et al. (1997)

introduced the useful distinction between stacked and aligned residues. Stacked residues

had to be similar residues with similar w angles while aligned described the same or chemically

similar residues packing with different w angles (especially w1). The distinction is between regularand irregular structures but stacks that mix valines and isoleucines with leucines or methionines

with similar w angles do occur. The stacks of valines and isoleucines packing on each other tend to

be more regular. There are at least three different types of stacks found in the parallel b-helix

proteins as illustrated in Figs. 3 and 4. Aliphatic stacks such as the cupped stacks are the most

common and are found in every protein, aromatic stacks are formed by the offset face to face

packing of phenylalanine and tyrosine side chains (Hunter et al., 1991) and polar stacks include

the well-known asparagine ladder (Yoder et al., 1993b), several variations of which are shown in

Figs. 4 and 5.

Aligned residues are ubiquitous in b-sheet containing proteins and occur in both parallel and

anti-parallel sheets. Cupped stacks of valines and isoleucines were discussed by Richardson(1981) long before the parallel b-helix was observed, who noted that the type of packing seen in

the stacks will not be stable in anti-parallel b-sheet because it would demand that the w1 angles

differ by 1801. However, such stacking is rather rare except in the repetitive folds. In a highly

twisted parallel b-sheet, stacking will be restricted to a single ridge if it occurs, which may explain

its rarity in TIM barrels. An example of a single ridge is seen in glyceraldehyde-3-phosphate

dehydrogenase from Sulfolobus solfataricus, PDB code 1B7G (Isupov et al., 1999) where residues

Ile 50, Val 31, Val 6, Val 82, Ile 105, Ile 134 and Ile 117 form a ridge and Val 6 to Ile 134 clearly

form a cupped stack. By contrast, the relationship between stacking and the repetitive folds is one

of the most basic features of their architecture so that no parallel b-helix has been observed



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without stacking. However, the source of the relationship is less obvious. The average spacing

between coils is about 4.8 (A which means that the distances between the equivalent atoms are

slightly longer than would give ideal packing of valines or isoleucines. However, in general each

Fig. 3. Stacking of non-polar residues in parallel b-helices. Carbons are drawn in light grey with oxygens and nitrogens

in dark grey. (a) Aliphatic cupped stacks of isoleucines 212, 214, 240 and 242 and valines 265 and 267 on PB2 of the

polygalacturonase PehA. (b) The internal aromatic stack of residues phenylalanine 159 and 201 and the tyrosines 242,

273 and 295 on PB3 of Bacillus subtilis pectate lyase. The tyrosine oxygens form hydrogen bonds with threonine 226,

tryptophan 310 and two buried waters inside the parallel b-helix.



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1.2.4. Turns

The b-sheets of the right-handed parallel b-helical proteins often change direction sharply with

a single residue in aL-conformation interrupting the residues in b-conformation. To avoid

confusion it is important to distinguish between the angle through which the sheet is turned from

its extended direction and the interior angle (taking a single coil as a polygon). The chain turns 801

at the T2 turn between PB2 and PB3 of the pectate lyases, giving an interior angle of 1001. By

contrast, in a typical b-hairpin turn the chain changes direction by 1801. Many aL turns occur in

the right-handed parallel b-helix proteins but can be subdivided into at least two forms. The most

common form is shown in Fig. 4a. This was the form initially identified in the structure of PelC by

Yoder et al. (1993b). This turn involves two residues not forming all the possible main chain

hydrogen bonds to the neighbouring coils. However, the simplest arrangement is that illustrated

in Fig. 4b which occurs at the start of PB2 in BsPel and extensively in RGase A and the

polygalacturonases. The T2 turn of TSP also shows this regular hydrogen bonding towards the

carboxy-terminus of the parallel b-helix. This type of hydrogen bonding was identified as possible

Fig. 5. Asparagine ladders and T2 turns in leucine-rich repeat proteins. Carbons are drawn in light grey with oxygensand nitrogens in dark grey with some residue numbers indicted to the right of the a-carbons or carbonyl carbons.

Hydrogen bonds from the main chain of the central coil and the central asparagine are indicated by thin lines. The

hydrogen bonding of the main sheet (PB2) above is regular but the residues below form less regular hydrogen bonds. (a)

Stereo view of the asparagine ladder of insulin-like growth factor receptor domain 3. (b) Stereo view of the asparagine

ladder of U2A0. Only three of the four asparagines are shown.



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by Chothia and Murzin (1993). The turn shown in Fig. 4a can be derived from the simpler

Chothia and Murzin turn by rotating the amides of the residue after the aL residue (denoted

therefore as residue i 1) inwards. At T2 in TSP at the amino terminal end of the parallel b-helix

these amides form hydrogen bonds with buried water molecules although the NH of 352 canhydrogen bond to Og of Ser 384. Similar structures also occur at T1 in TSP. In pertactin Ser 121

and Ser 142, and in pectin methylesterase Ser 249 and Thr 294, can hydrogen bond to the inward

pointing NH from the previous coil. In the lyases a much more elaborate structure has evolved,

the asparagine ladder (Fig. 4a) in which the main chain NH of residue i 1 hydrogen bonds to the

carbonyl of the asparagine side chain and the NH2 of the asparagine side chain hydrogen bonds to

the main chain carbonyl of the residue i 2: Ribonuclease inhibitor shows a CysAsn ladder

related to the asparagine ladder (Kobe and Deisenhofer, 1995b) but with the difference that the

asparagines (and thus also the cysteines) only hydrogen bond to the main chain atoms. These have

a peptide rotated away from b-conformation so that the i 2 carbonyls can bond to the

asparagine NH2 group rather than with the main chain from neighbouring coils. The humanspliceosomal protein U2A has ladders with only asparagines but follows ribonuclease inhibitor in

that the asparagine NH2 groups now bridge between each pair of i 2 carbonyls, which are again

directed inwards (Fig. 5b). However, the insulin-like growth factor receptor (Garrett et al., 1998)

shows asparagine ladders resembling those of the pectate lyases with main chain hydrogen bonds

along the helix axis.

The left-handed parallel b-helices also have aL-containing turns but their coils are much closer

in shape to an equilateral triangle than are those of the righthanded parallel b-helices. Thus they

have turns that give approximately 1201 changes of direction and 601 interior angles. These turns

often form the 14 hydrogen bond of the classical b-turn but are clearly different from the latter as

the chains subsequently diverge. Interestingly, a similar turn also occurs in pertactin and Figs. 6a

and b show that these turns are locally similar in parallel b-helices with the opposite hand.The turns seen in pectate lyase were termed a distorted gbE turn by Yoder et al. (1993b) after the

classification of Wilmot and Thornton (1990). Chothia and Murzin (1993) viewed the turn as a

kink in a single continuous b-sheet, similar to the b-bulge (Richardson et al., 1978; Chan et al.,

1993), whilst Pickersgill et al. (1994) identify the occurrence of aL-bounded b-strands as a new

motif. The turns in the left-handed parallel b-helices have generated less controversy, possibly

because they were assumed to resemble those of the right-handed family. A Wilmot and

Thornton definition for them might be bPgL; which is the classical type II turn of Richardson

(1981) rather than bEgL: However, this turn also involves four residues rather than two outside the

normal b-sheet conformation and only one of the three peptides between them can form hydrogen

bonds along the axis of the parallel b-helix.So far turns involving an external aR residue have only been found in pertactin (Fig. 7a) but

turns in which the aR residue is inside the parallel b-helix (and the surface is therefore concave)

occur in all the right-handed parallel b-helix enzymes at the start of PB1 (Fig. 7b). These can also

form hydrogen bonds along the axis of the parallel b-helix. The groove formed by this turn is

normally part of the active site of these enzymes.

1.2.5. Packing ofb-sheets

The packing of two b-sheets in a sandwich is a common feature of many protein folds and has

been extensively studied Chothia et al. (1997). There is a correlation between the optimal relative



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orientation of the b-sheets and their twist, with low twist allowing the sheets to pack with their

strands (anti)parallel. Most b-sandwich proteins have more twisted sheets and the strands are

rotated by an angle of about 301, or pack approximately orthogonally. In the right-handed

parallel b-helices, PB1 and PB2 form such a sandwich in RGase A and the polygalacturonases.

Fig. 6. Stereo views of LpxA (UDP-N-acetylglucosamine acyltransferase from E. coli) together with P69 pertactin after

superposing all the main chain atoms of residues 116123 of LpxA on residues 352359 of pertactin with an RMSD of

0.78 (A. The side chains pointing out of the parallel b-helix towards the viewer are reduced to alanines for clarity. (a)

Stereo view of the bonds of LpxA drawn as cylinders. Carbons are drawn in light grey with oxygens and nitrogens in

darker grey. Some LpxA residues are numbered. (b) Stereo view of the bonds of LpxA around residues 121 drawn as

thick grey lines together with the bonds of pertactin drawn as thinner black lines. Some pertactin residues are

numbered. (c) Stereo view of the bonds of pertactin drawn as cylinders. Carbons are drawn in light grey with oxygens

and nitrogens in darker grey. Some pertactin residues are numbered.



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The helix becomes more triangular in shape in the other proteins and especially in pertactin and

pectin methylesterase, while the left-handed parallel b-helices are close to an equilateral triangle in

cross-section. In RGase A and the polygalacturonases, PB1 and PB2 are rather flat and pack

almost anti-parallel. When viewed down the helix axis, the g-carbons from PB1 and PB2 arenearly aligned so that they approach each other rather than packing as knobs into holes.

However, this sandwich is unusual in being formed from a purely parallel sheet. The g-carbons are

slightly off the perpendicular to the helix axis with those of PB1 directed slightly towards the

C-terminus while those of PB2 are directed towards the N-terminus. This allows opposing

residues to avoid steric clashes between the sheets by simply having w1 near 1801 which also avoids

clashes within the sheet. The g-carbons from both sheets are generally directed towards the N-

terminus. However, there is no steric clash because the b-carbons from PB1 of one coil slot

between the g-carbons from PB2 of the same coil and the equivalent residue in the next coil. Thus

in this case the local preference for w1; cupped stacking, the rather flat b-sheets, and the optimal

Fig. 7. Stereo views of right-handed a-helical residues forming turns in the right-handed parallel b-helix family.

Carbons are drawn in light grey with oxygens, nitrogens and a sulphur in darker grey. (a) The turn from T3 into PB1 atthe active site of the polygalacturonase PehA viewed from along PB1. The active site residues Asp 202 and Asp 224 are

shown but Asp 205 is omitted. (b) Part of the stack of a-helical residues at T3 near the C-terminal end of the pertactin

parallel b-helix. Hydrogen bonds of the main chain are indicated by thin lines. The hydrogen bonding is not completely

regular with two protons being donated to the carbonyl of Asn 442 (note that the residue numbers are to the left of the

residues in this image only).



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anti-parallel packing combine to stabilise the right-handed parallel b-helix architecture. However,

the other, more triangular structures are also stabilised by the packing of stacked residues from

two or three b-sheets. The b-roll protease structures (Section 2.9) all show almost exact anti-

parallel packing but the sheets pack unusually close together so that the residues mustinterdigitate unless one is a glycine. Thus the pleating in these b-sheets is anti-correlated between

the sheets. In the thicker b-roll like section of pertactin (Section 2.4) the packing involves a

tryptophan from each coil packing against two leucines from the other sheet. The right-handed

antifreeze protein from Tenebrid molitor has similar spacing to the b-roll structures with cysteines

on opposite sides forming disulphide bridges but only two adjacent sides of the helix are b-sheet

and thus only one of the opposed cysteines comes from a b-sheet.

2. Description of known structures

2.1. Pectinases

2.1.1. The extra-cellular pectate lyase family

The family contains most known pectate lyases, including all those with published three-

dimensional structures starting with the archetype PelC (Yoder et al., 1993a), and all known

pectin lyases (Henrissat et al., 1995). Henrissat and Coutinho have developed a web site, http://

afmb.cnrsmrs.fr/Bpedro/CAZY/lya.html, displaying lists of polysaccharide lyases classified into

homologous families where this family is listed as family 1.

The X-ray structures of five members of this family have been published (see Table 1) and

crystals of one more have been reported (Doan et al., 2000). The known structures comprise three

rather distantly related pectate lyases, PelC and PelE from Erwinia chrysanthemi and the BacillusSubtilis pectate lyase, and the two pectin lyases A and B from Aspergillus niger, PnlA and PnlB,

which are more closely related with more than 60% sequence identity. The overall fold of these

structures is shown in Fig. 8. There is a structurally conserved core comprising the parallel b-helix,

the N-terminal helix and the N- and C-terminal extensions. The shape of the parallel b-helix is

formed by three b-sheets and is very similar in all five enzymes. It is often described as L shaped,

which arises because strands PB2 and PB3 are relatively long and make a slightly obtuse interior

angle at T2 (about 1001) and PB1 is often preceded by a residue in (aR conformation and then

runs roughly antiparallel to PB2 (Figs. 1 and 7b).

All five structures are similar in the general pattern of long and short T1 and T3 loops

especially in having long T3 loops in the coils to the N-terminal end of the parallel b-helix.These T3 loops are sometimes long enough to bury some hydrophobic residues as well as forming

hydrogen bonds. Thus this region sometimes appears to form a non-contiguous domain.

However, there is no information to suggest that any of these structures can fold independently

of the parallel b-helix. It is also worth noting that there is little similarity in the detailed structures

of the T3 and T1 loops between these enzymes except between the two pectin lyases. For example,

although each of the pectate lyases has a helix in its first T3 loop, this coil in PelC does not

have a PB3 but after a few residues in irregular conformation starts an a-helix which packs

against PB3 of the next two coils, while PelE and BsPel have a PB3 and thus leave the coil

at a different angle. The T3 loop is much shorter in PelE (residues 5874) than in BsPel



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(residues 64121) where the loop continues out through irregular structure and two turns of a-

helix (residues 8591) before returning as a long helix (residues 104121) which overlaps with a

shorter helix in PelE for the last 3 turns (residues 6774). The pectin lyases have long loops which

roughly but not exactly overlap the loop of BsPel but consist of irregular structure and a long b-

hairpin.

The T1 regions include both extended irregular structure and shorter chains in mostly extended

conformation resembling short b-strands as part of the parallel b-helix, which, however, do not

form all the hydrogen bonds needed for the region to be classified as a b-sheet. In fact the lyases

Fig. 8. Comparison of the pectinase folds, showing that even the obviously homologous lyases have very different loop

regions. Bacillus subtilis pectate lyases with a single bound calcium at (b), Aspergillus niger pectin lyase A at (d), Erwinia

chrysanthemi pectin methylesterase, PemA, at (a) and the Erwinia carotivora polygalacturonase PehA at (c) are shown.

The three common b-sheets PB1, PB2 and PB3 are coloured yellow, green and red, while the extra sheet PB1a of the

polygalacturonase family is dark blue. The b-hairpins in the T1 region of pectin methylesterase are cyan. All the active

sites are towards the viewer and the N-terminal ends of the parallel b-helices are at the top.



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are intermediate between the esterases where this region is definitely not a b-sheet and the

hydrolyases where it forms PB1a as shown in Fig. 2.

Jenkins et al. (1998) compared the lyases except for PnlB and determined that PelE and BsPel

have 3134% identical residues after structural alignment (depending on whether alignment wasautomatic or assisted by human intervention), while the other pairs had between 13.6% and 17%

identical residues after structural alignment (PnlA was most different from the pectate lyases).

This low level of sequence identity allows us to have complete confidence that these are

homologues but makes alignment difficult without knowledge of the structure. For example,

Henrissat et al. (1995) used the three pectate lyase structures to align all the then known sequences

but chose not to align several regions of the fungal pectin lyase sequences, including one active site

T3 region, because there was insufficient similarity. Examination of the aligned sequences suggests

that there may also be a bacterial pectin lyase family, which has diverged as far from the pectate

lyase and the fungal pectin lyases as these have from each other. Pissavin et al. (1998) recently

convincingly suggested that PelZ from Erwinia chrysanthemi is another even more remotehomologue.

The structurally aligned primary structures show rather few identical residues overall but there

are two clusters of conserved residues. One represents the active site as expected but the second,

including the sequence W(I/V)DH, is on the opposite side of the parallel b-helix from the active

site near T2 of the coils bearing the active site residues. This second cluster does not seem to be

associated with enzymic activity but may be critical for folding or stability. This region is at least

partially buried by the N-terminal extension and the C-terminus is also nearby. The X-ray

structures tend to show low temperature factors for that region and Jurnak et al. (1996) found

that mutation in that region impaired folding. The conservation of some other residues, such as

the asparagine ladder, shown in Fig. 4, is more simply explained. Some other conserved residues

are involved in the folding of the N-terminal extension, while one asparagine forms a hydrogenbond to the main chain of the C-terminal region. Kamen et al. (2000) have studied the

denaturation and renaturation of PelC and find that the protein may unfold in two structural

blocks, but these could not be clearly identified in the structure. The unfolding transition is highly

co-operative and the protein was described as unusually stable.

The active sites of the pectate and pectin lyases are very different with mostly small hydrophilic

side chains forming the pectate lyase active site and many large aromatic side chains dominating

that of pectin lyase. As expected pectate lyase has a positive potential while pectin lyase is

negatively charged. Calcium is essential for pectate lyase activity but its precise role has been

poorly understood until very recently. The structure of the calcium complex of Bspel (Pickersgill

et al., 1994) showed that there was a single high affinity site and its affinity could also be measuredcalorimetrically as 0.2 mM. However, the kinetic constant Km for calcium was much higher (near

1 mM) suggesting that calcium had a more complex role (Smith, D., unpublished). Scavetta et al.

(1999) overcame many difficulties to determine the structure of a complex of the R218 K mutant

of PelC with calcium and the substrate pentagalacturonic acid. The surprising result was that four

calcium sites were found including one equivalent to that seen in BsPel. All the calcium atoms had

at least one atom from both the protein and from the substrate as ligands and thus formed

bridges. Only four galacturonates were observed implying either that the substrate had been

partially degraded or the extra site was either blocked or energetically unfavourable for binding.

The penta-(or tetra-)galacturonate bound almost parallel to the parallel b-helix along the grove in



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PB1 interacting with ligands from T3, PB1 and T1. Not all the calcium ligands were conserved

even between the three known structures of the pectate lyases but it is possible, for example, that

PelCs Asp 160 and Asp 162 might be replaced by PelEs Glu 124 or BsPels Asp 170 which come

from different T3 loops to a similar position. Thus, it is likely that Scavetta et al. have solved thebasic problem of how the extra-cellular pectate lyases bind their substrates although it is probable

that a longer substrate would bind at extra sites and possibly use extra bridging calciums. Some

differences in the substrate binding between the different pectate lyases are also suggested by the

alterations in the side-chains binding the calciums. The structure of the complex immediately

accounted for most of the available data accumulated by site directed mutagenesis. Kita et al.

(1996) had shown that mutation of Asp 131, Glu 166, Asp 170, Lys 190, Arg 218 and Arg 223 led

to reduced activity. Except for the already mutated Arg 218, all of these are ligands of either

calcium or the substrate and are generally conserved except that Glu 166 is an aspartate in most

pectate lyases.

As there was no potential base near any of the C5 atoms of the substrate, Scavetta et al.proposed a mechanism assuming that the sites occupied in the complex were 3 to +1 and that

Arg 218 would have the same position in the productive complex as it had in the free enzyme. This

would place the arginines NH2 2.6 (A from the C5 in an ideal position to act as the base removing

a proton from C5 with the b-elimination at O4 occurring in either a stepwise or a concerted

reaction. This explains the complete conservation of the arginine in the family and the dramatic

loss of activity on its mutation but has not won universal acceptance (Huang et al., 1999). The use

of an uncharged arginine as a base might be plausible if the pH optimum for activity were high

and the arginine was close to one or more calciums. However, only a very small fraction of Arg

236 of pectin lyase A would be uncharged at its pH optimum near 5.5 and no calcium ions are

required for pectin lyase activity. The review by Herron et al. (2000) gives a detailed view of the

mechanism and its relation to pathogenesis by Erwinia.The pectin lyases have a simpler active site than the pectate lyases, consistent with having a

simpler task, as pectin will easily undergo non-enzymatic b-elimination. They have no known

calcium binding, no lysine homologous with Lys 190 of PelC and only one of the three

carboxylates of the high-affinity calcium site of pectate lyase site is conserved. This carboxylate is

the one furthest from the substrate and appears to function to orient the arginine that has

replaced Glu 166 of PelC. The pectin lyases A and B of Aspergillus do present a new problem in

that activity is lost above pH 7. This may be associated with the two different structures

reported by Mayans et al. (1997) at pH 6.5 and 8.5 of PnlA from two different strains of

Aspergillus niger. At pH 6.5, Asp 186 and Asp 221 are buried within the parallel b-helix and

form a hydrogen bond while at pH 8.5, Asp 186 has turned outwards and been replacedby Thr 183 with a significant rearrangement of the T1 region. This conformational

change is probably associated with pH rather than with the small number of amino acid

substitutions between the strains, differences in glycosylation or altered crystal packing.

PnlB at pH 5.5 and high salt concentration (Vitali et al., 1998) has a very similar conforma-

tion to the pH 6.5 structure of PnlA, crystallised from polyethylene glycol. However, it is

not clear how the conformational change is related to the loss of activity or its biological

function. Jaap Visser (pers. comm.) has suggested that a pectin lyase is unnecessary above pH 7

when pectin is labile and that PnlA may change conformation to accelerate its recycling by

proteases.



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2.1.2. Polygalacturonases and rhamnogalacturonase A

The polygalacturonases and two rhamnogalacturonases were identified as family 28 of the

glycosyl hydrolyases (Henrissat and Bairoch, 1996) and have been shown to act with inversion of

anomeric configuration (Biely et al., 1996; Pitson et al., 1998). Three crystal structures ofrhamnogalacturonase A from Aspergillus aculeatus (Petersen et al., 1997), the endo-polygalactur-

onase PehA from Erwinia carotivora (Pickersgill et al., 1998) and endopolygalacturonase II or PG

II from Aspergillus niger (van Santen et al., 1999) have been determined and the crystallisation of

several further enzymes has been reported (Yoder and Schell, 1995; Federici et al., 1999; Lu et al.,

2000). Although these three enzymes are clearly homologous, they resemble the lyases in that they

have diverged beyond the level of sequence identity where accurate models can be constructed. All

were solved by using heavy atom derivatives rather than molecular replacement (for example, van

Santen et al. (1999) report that superposition of PG II and PehA gives an rms deviation (RMSD)

of 1.8 (A for 265 Ca atoms and 19% sequence identity). Despite the change of function, RGase A

is slightly more closely related to PG II than the two polygalacturonases are to each other and itmay thus have diverged from PG II after the fungi diverged from bacteria. In fact PehA is also

very slightly closer to RGase A than to PG II and superposition by O (Jones et al., 1991) gives

1.6 (A for 280 Ca atoms between RGase A and PG II, 1.67 (A for 253 Ca atoms between PehA and

PG II, and 1.67 (A for 280 Ca atoms between PehA and RGase A (using chain B for PG II).

RGase A and PehA have N-terminal extensions which both start with the amino terminus

forming hydrogen bonds to a T1a turn residue in aL conformation. In PehA this residue is Asn

264 and the OD1 also bonds to the N-terminus while in RGase A the N-terminal residue is an

asparagine and forms an internal hydrogen bond so that only the carbonyl of Asp 235 bonds to

the amino terminus. Ser 3 also makes a conserved hydrogen bond to an aL residues at T1a (Asn

185 of RGase A or Asn 211 of PehA). The nearby residues lie in the same region with Leu 2 and

Val 6 of RGase A packing in approximately the same position as Asp 2 and Arg 4 of PehA butforming very different interactions. The chains then diverge with Glu 8 of PehA forming a salt

bridge to another aL residue, Lys 189, which is at T2. At the equivalent position at T2 RGase A

has Asn 165 which forms a hydrogen bond to the C-terminal extension of RGase A. RGase A

forms a short a-helix and Cys 15 of PehA and Thr 20 of RGase A come together again near the

second residue of PG II, Ser 29 (in fact Asp 28 of PG II is close to Lys 19 of RGase A) and enter

the parallel b-helix.

The parallel b-helix of PehA ends abruptly with the final strand of PB1a, while that of PG II

loops back to form a C-terminal disulphide bridge which may cap the parallel b-helix. RGase A

has a long C-terminal extension which forms a strand of anti-parallel b-sheet with the last strand

of PB1 (residues 369375) and then packs against PB3 burying several aromatic residues such asthe externally stacked pair of Tyr 215 and 242. It also partially buries the longer stack of Phe 104,

His 141, Tyr 164 and His 189 from the end of PB2 by forming a S bend (residues 386398412

421) which lies against the PB3-T3 region. Thus the C-terminal extension like the N-terminal

extension interacts with a stack ofaL turns (at T2 in this case). The main chain interactions with

the Asp 142, Asn 165 and Asp 190 may be especially significant. A final factor that may stabilise

RGase A is its glycosylation which is unusually well ordered in the crystal structure.

The first obvious distinguishing feature of the central parallel b-helix of the family 28 enzymes is

the occurrence of the fourth PB1a b-stand (PB2a) as shown in Fig. 2. In fact the conformational

differences in this region between a pectate lyase such as BsPel and the hydrolases are not as



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striking as might be imagined. RGase typically has two residues in b-conformation flanked by two

residues in aL conformation forming hydrogen bonds along the helix axis. The b-arch of BsPel at

residues 148151, 190193 and 231234 has glycines at 190, 191 and 231, which together with the

small Ser 149 and Ala 232 allows these residues to adopt an irregular conformation while the Capositions are close to the equivalent residues of RGase A. However, residues 150151, 192193

and 233234 of BsPel have essentially the same conformations and hydrogen bonds as the

equivalent residues of RGase A.

The second feature is that the structure of RGase A is generally much more regular than that of

the lyases and forms many of the hydrogen bonds needed to generate the perfect parallel b-helix

imagined by Chothia and Murzin (1993) but not actually seen in the pectate lyases. For example

at T1 Gly 70, Asp 97, Asp 134 and Thr 157 are all in the aL conformation in successive coils

forming hydrogen bonds along the parallel b-helix. This is followed by a coil with a continuous

PB1-PB1a b-sheet, and finally by three more distorted turns with one residue in aL conformation

but without regular hydrogen bonding. At T1a these coils have Ala 73, Asp 100, His 137, Asp 160,Asn 185, Asn 208, Asp 235, Asn 263, Asn 299, and Asp 330 in aL conformation forming regular

hydrogen bonds along the parallel b-helix before ending with the more continuous sheet as

described in the introduction. Finally at T2 after two irregular turns Asp 142, Asn 165, Asp 190,

Ser 213, Asn 240, Asn 268, Asn 304 and Asp 335 are in a L conformation with the regular

hydrogen bonding pattern. The differences between the type of aL turn generally seen in the

lyases and the hydrolases are illustrated in Figs. 4a and b. Some, but not all, of the asparagine

residues in these turns form regular external stacks. There is also often a residue in aR

conformation at the start of PB1 and again RGase A shows regularity with Met 92, lle 129, His

150, Gly 178 and Cys 199 in aR conformation, followed by an irregular coil, then Met 249, Ser

277 and Pro 317 in aR conformation without making all the possible hydrogen bonds and finally

a longer PB1 making anti-parallel sheet with the C-terminal extension.PehA and PG II have very similar parallel b-helices and interior residues to RGase A. As well as

slowing the evolution of the interior, the conservation of the overall shape of the coils may make

compensating mutations rather more probable than usual. For example, Met 249 in RGase

becomes Gly in PehA and Ala in PG II but Gly 271 of RGase A is replaced by Met both PehA

and PG II to conserve the volume. The four disulphide bridges between Cys 21 and Cys 47, Cys

199 and Cys 216, Cys 322 and 328, and Cys 350 and Cys 359 are conserved between RGase A and

PG II (although the last does not have the same structure) but are not found in PehA, which

however has a single disulphide between Cys 89 and Cys 99 in a T3 loop. An unusual feature of

RGase A is the cluster of three cysteines 199, 216 and 222 where Cys 222 is in the position

equivalent to Cys 199 in the next coil but does not have the same aR conformation. It is not clearif Cys 222 assists in the folding. The large cavity reported in RGase A seems to be mostly occupied

by Phe 74 in PG II and at least partially by Leu 65 of PehA.

The family 28 enzymes have very many aliphatic residues both aligned and forming stacks

and these dominate the interior of the parallel b-helices. Although there are several aromatic

residues, there are very few internal aromatic stacks: one of Phe 129, Phe 152 and Phe 182 in PG II

at the start of PB1, Phe 263 and Trp 305 at the asparagine ladder position of RGase A at the start

of PB3 as shown in Fig. 4b (Phe 101 and Phe 138 are aligned) and Tyr 296 and Phe 330 on PB2 in

PehA (Phe 162 and Phe 185 have similar conformations but do not interact strongly). There are

no internal polar stacks in RGase A or PG II but there is a single Asn 245 at the asparagine ladder



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position of PehA. This hydrogen bonds to the main chain NH of Ser 217 in the previous coil. OG

of Ser 217 in turn hydrogen bonds to the main chain NH of Thr 190 but OG1 of Thr 190 only

hydrogen bonds weakly to the carbonyl oxygen of Leu 164. To the C-terminal side, ND2 of Asn

245 hydrogen bonds to a buried water and to the carbonyl oxygen of Val 214. The buried water iscapable of bonding to three carbonyl oxygens of residues 242, 268 and 269 (as Asp 269 forms an

a L turn). The existence of a stable asparagine step suggests that the evolution of new

asparagine ladders is possible.

As discussed in the introduction, the parallel b-helices of RGase A and PG II finish with a

disulphide and may have a topological winding number of 13, while PehA finishes with the final

PB1 a and has a winding number of 12. All of the family 28 hydrolases have 10 coils as defined by

Yoder et al. (1993b). The pattern of long T3 loops especially at the N-terminal end of the parallel

b-helix and long T1 loops towards the C-terminal end is also seen in these hydrolases. Both PehA

and PG II have longer T3 and T1 loops than RGase A, so that the active sites become deeper and

narrower clefts. However, there is no detailed similarity in the loop conformations suggesting thatthis similarity reflects the requirements of polygalacturonase as opposed to rhamnogalacturonase

activity. RGase A has several unusually well-ordered N-inked sugar residues at the C-terminal

end of the parallel b-helix.

The active sites of the family 28 hydrolyases have not yet been fully identified by determining

the structure of a complex with a substrate, product or inhibitor. However, sequence conservation

and analogy with other enzymes suggests that substrates bind to the cleft formed by the T3PB1

T1 region and that catalysis involves three carboxyl residues: Asp 177 (Asp 202, Asp 180), Asp

197 (Asp 223, Asp 201) and Glu 198 (Asp 224, Asp 202), where the RGase A residue is given

followed by the PehA and PG II equivalent residue in brackets. Van Santen et al. (1999) reported

that site directed mutation of PG II gave activities of 0.01% and 0.08% for D180E and D180N,

0.01% and 0.01% for D201E and D201N, and 0.6% and 0.01% for D202E and D202Nwhile only slightly changing the Km values. The H223A mutant also had only 0.5% of the WT

specific activity and no change in Km but R256N and K258N showed 14% and 0.8% of the

WT specific activity with an order of magnitude increase in Km. Histidine 223 of PG II

(His 251 of PehA) is not conserved in RGase A but may thus have a critical role in binding the

substrate in polygalacturonases in an altered conformation to assist the catalysis. Armand et al.

(2000) report the properties of these mutants in more detail as well as mutations at Arg 256 and

Lys 258.

As this family of enzymes was known to catalyse hydrolysis with inversion of anomeric

configuration, both Pickersgill et al. (1998) and van Santen et al. (1999) noted conserved water

sites in contact with two of the conserved carboxylates. Similarities to the active site of the P22phage tailspike endorhamnosidase were noted as well as the fact that the distances between the

carboxylates did not fit the rules used previously for identifying inverting and retaining

glycosidases. However, these rules were developed for b-linked sugars such as the substrates of

xylanases and cellulases where the lone pair on the glycosidic oxygen which is protonated by the

acid catalyst is on the opposite side of the sugar to the attacking nucleophile. The 78 (A distance

for these inverting family 28 hydrolases suggests that the lone pair is not pointed directly away

from the nucleophilic water in these enzyme substrate complexes. Van Santen et al. are more

explicit in proposing the water in contact with and activated by Asp 180 and Asp 202 (water 2 of

PehA and water 37 of RGase A) as the nucleophile attaching at C1. Asp 201 of PG II is assumed



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to act as an acid to protonate O1 (i.e. the leaving group). This is plausible as Asp 202 is in contact

with the positively charged Arg 256 and is likely to be charged.

Pag"es et al. (2000) reported the properties of the mutants N186E and D282 K, which implied

that the substrate binds with its reducing end towards the C-terminus of the parallel b-helix as isseen in PelC by Scavetta et al. (1999) and that N186 was part of subsite 4 while D282 was part of

subsite +2. Mutations were also reported of residues forming the more central subsites and these

often showed greater effects on the overall activity. Examples include D183N at subsite 2, and

Y291L or Y291F at subsite +1. Finally the mutation E252A allowed PG II to accept a

methylated substrate more easily.

As might be expected given the same substrate, the general nature of the residues at the active

sites of these enzymes resembles those at the active sites of the pectate lyases. However, calcium is

not required for substrate binding or activity. Some aromatic residues are found such as Phe 151,

Trp 182, Tyr 276 and Trp 284 of RGase A, Phe 175 and Tyr 231 of PehA and Tyr 130, Tyr 254,

Tyr 283 and Tyr 326 of PG II (as the exact binding site is unknown we cannot assert that all theseresidues are in contact with the substrate in a productive complex). None of these aromatic side

chains are conserved in all three structures.

2.1.3. Pectin methylesterase

The structure of the pectin methylesterase PemA from Erwinia chrysanthemi has been reported

by Jenkins et al. (2001) at 2.4 (A resolution. It has the same number of coils as the pectate and

pectin lyases and the coils of this right-handed parallel b-helix have the same general shape. PemA

has an a-helix at the N-terminal end of the parallel b-helix, long T3 loops and long T1 loops

towards the C-terminal end of the parallel b-helix, and a C-terminal extension. The C-terminal

extension interacts with PB2 rather than PB3 as seen in the lyases and is longer than that of the

lyases. It also makes interactions with the parallel b-helix in the region which interacts with the N-terminal extensions of the lyases and the hydrolyases.

In terms of the shape of the coils, the structure represents a divergence from the lyases in the

opposite direction from the hydrolyases with little trace of the PB1a sheet and a short T1 arch

region. Stacked aR residues are again found at the start of PB1 but the angle between PB2 and

PB3 is less obtuse than in the other pectinases so that the distance between PB3 and the start of

PB1 is shorter. This arises because there is no internal stack of aromatic residues on PB3 as seen in

the lyases. The overall shape of the coil is similar to that at the N-terminal end of the parallel b-

helix of pertactin (see Section 2.4).

PemA shows many internal aliphatic stacks and an internal aromatic stack, which occur on

PB2. An external asparagine stack is found but the asparagine ladder position of the lyases isfrequently a cysteine in the pectin methylesterases. In PemA the disulphide between Cys 192 and

Cys 212 appears to be partially formed but the crystals had been treated with the reducing agent

DTT as 100 mM DTT did not inhibit the activity (K. Worboys, unpublished observations). It is

not clear if the cysteines in PemA and in its homologues generally form disulphides or stacks of

cysteines although some sequences contain cysteines which cannot form disulphides.

There is a deep cleft along the parallel b-helix formed the T3PB1T1 region which contains the

most conserved sequences and corresponds to the substrate binding site of the lyases. The T3 and

T1 loops of PemA have no detailed similarity to those of the lyases and the cleft is rather deeper

(as is the active site cleft of the polygalacturonases). Two of the T1 loops form b-hairpins and with



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one hydrogen bond between the hairpins almost form a four stranded anti-parallel b-sheet. The

cleft resembles the pectin lyases in having many aromatic residues including an external aromatic

stack of Tyr 158, Tyr 181 and Phe 202 on PB1. The similarity between pectin methylesterases and

pectin lyases results from convergent evolution to bind the same substrate because only onearomatic residue, Phe 202 of PemA and Tyr 215 of PnlA, occurs at an equivalent position.

Only the sequence conservation (Markovic and Jornvall, 1992; Laurent et al., 1993) and the

observed pH activity profile gives us any information on the catalytic mechanism of PemA. The

enzyme has a broad roughly bell-shaped pH optimum from about pH 59 (Pitk .anen et al., 1992).

However, if the active site has been correctly identified, the only conserved potentially catalytic

residues are Asp 178, Asp 199 and Arg 267, and the last two form an ion pair. As Asp 199 is likely

to be deprotonated, only Asp 178 is likely to act as an acid and either Asp 178 after it has donated

a proton or Asp 199 or both may activate a water molecule to act as a nucleophile. PemA is thus

the first member of a new class of aspartyl esterases.

2.1.4. PelL from Erwinia chrysanthemi

The structure of PelL from Erwinia chrysanthemi is currently being determined in our

laboratories. This is a member of family 9 in Henrissat and Coutinhos classification of the

polysaccharide lyases. Although the sequences are unrelated (Lojkowska et al., 1995), PelL

resembles the extra-cellular pectate lyases in requiring calcium for activity. PelL is an endo-

pectate lyase and makes a significant contribution to tissue maceration in vivo. However, its

expression in Erwinia is not regulated by the same mechanism that controls expression of the

extra-cellular family enzymes. Crystallographic refinement has not yet been completed but the

current model with an overall R factor below 17% and a free R factor below 19% to 1.6 (A

resolution allows almost all the residues to be clearly defined.

The architecture of PelL is a right-handed parallel b-helix and the overall shape of the coils issimilar to that of the extra-cellular lyases with three b-sheets, PB1, PB2 and PB3. The T1 region

resembles BsPel, generally with an aL turn at the start of PB2 but without a regular PB1 a sheet

as seen in the polygalacturonases and rhamnogalacturonase A. However, there are 10 coils as in

polygalacturonase rather than eight, as in the other lyases. There is an N-terminal a-helix as in all

the right-handed parallel b-helix proteins except pertactin (Emsley et al., 1996). There is a short

N-terminal extension of residues 2639, numbering residues from the gene so that Ala 26 is the

first residue of the mature enzyme, and a long C-terminal extension of residues 357425 which

packs against PB3. PelL has 12% of asparagine in its sequence and as initially suggested by

Lojkowska et al. (1995), these form both internal and external stacks. However, the internal

asparagine ladders occur at the start of PB2 (T1a) and at T3 rather than at T2. There is a clusterof conserved residues including Asp 209, Asp 233, Asp 234, Asp 237 and Lys 273 in the T3PB1

T3 region where all the known pectinase active sites are located and this region can be tentatively

identified as the active site of PelL.

2.1.5. Pectate lyase Pel-15 from Bacillus sp. strain KSM-P15

Akita et al. (2000) have reported the crystallization of a pectate lyase Pel-15 from Bacillus sp.

strain KSM-P15 with a very alkaline pH optimum of 10.5 and although no article has yet been

published describing the structure, the coordinates have been deposited to the Protein Data Bank

as accession 1EE6. This structure is the first for a member of the family 3 lyases according to



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Henrissat and Coutinhos classification. The structure is a right-handed parallel b-helix with a

similar shape for each coil to that of the extra-cellular lyases and also has 8 coils together with an

extra PB3 strand. However, the sequence is significantly shorter than that of the other pectinases

and there is no N-terminal helix, there is only one notable T3 loop (residues 2742) and there is noC-terminal extension. The small size is not associated with an unusually repetitive structure. There

are left-handed a-helical turns and some stacking of aliphatic groups. However, the only

asparagines stack is external and there are no obvious aromatic stacks. Alignment with Bacillus

subtilis pectate lyase gives an RMSD of 1.94 (A for 158 Cas (of 197), showing that this enzyme is

another member of the pectinase superfamily. The active site contains several striking similarities

to the other lyases and some surprises, including the site of calcium binding. The calcium position

is different from that of the single calcium seen in BsPel and PelL. This site also does not

correspond to any of the sites reported by Scavetta et al. (1999) for PelC but is near aspartates in

both PelE and BsPel. A full description of this interesting structure is eagerly awaited and

hopefully it will be possible to model or observe substrate binding and thus understand in detailthe role of calcium, which is also necessary for the pectate lyase activity of this family.

2.2. The P22 phage tailspike endorhamnosidase

The P22 phage tailspike endorhamnosidase (TSP) has been for many years one of the principal

model systems used to study protein folding and misfolding both in vivo and in vitro (King et al.,

1996; Betts et al., 1997; Seckler, 1998; Betts and King, 1999). The final rate-limiting trimer

maturation reaction, which has the same rate in vivo (Goldenberg and King, 1982) and in vitro

(Danner et al., 1993), produces a folded trimeric enzyme which is unusually stable both to high

temperatures and even more usefully in presence of denaturants including SDS. Thus this system

has allowed a clear separation of issues relating to folding from those relating to the stability ofthe folded protein. Many temperature sensitive folding (tsf) mutants have been found which are

stable once folded but which will not fold above some non-permissive temperature. Several

supressor mutations (su) that restore folding to tpf mutants at the non-permissive temperature

have also been identified. The determination of the TSP structure depended on the fragmentation

of the enzyme by recombinant expression and crystallisation of the two fragments, residues 1124

and 109666 (Miller, 1995; Miller et al., 1998a). The structures of these fragments of TSP

(Steinbacher et al., 1994, 1997) revealed that the major part of the protein forms a parallel b-helix

while the N-terminal (residues 1108) and both C-terminal (residues 541666) domains have anti-

parallel b-folds. This information opened the way for the genetic and in vitro folding studies to be

interpreted in terms of atomic interactions.The nomenclature describing the structure of TSP was developed independently from that of

the pectinases. The articles describing the structure of TSP describe the three stranded parallel

b-helix in terms of sheets A, B and C corresponding to PB2, PB3 and PB1 of the pectinases. The

overall shape of the monomer resembles a fish with the parallel b-helix domain taken as the body

and the extreme C-terminal domain taken as the Caudal fin. The long loops off the parallel

b-helix are then named Dorsal and Ventral fins, corresponding to T3 and T1 loops,

respectively. There are 13 complete coils in the parallel b-helix domain of TSP and these coils have

the characteristic L or kidney shape also seen in the pectinases and the N-terminal region of

pertactin with a stack ofaR residues at the start of sheet C (PB1). Like the pectinases but unlike



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pertactin, the parallel b-helix has an N-terminal a-helix instead of a PB1 in its first coil followed by

the first strand of sheet A (PB2). The detailed shape of the parallel b-helix is more similar to the

pectic lyases than pectin methylesterase or pertactin, mostly because the aR residue at the start of

sheet C (PB1) and the second inward pointing residue of sheet B (PB3) tends to be larger, forcingsheets B and C (PB3 and PB1) apart. One possible alignment of the parallel b-helix with BsPel

gives an RMSD of 2.02 (A for 174 Ca atoms. There is one long T3 loop from residues 197 to 260

forming the Dorsal fin. There are some long T1 loops, forming the Ventral fin, one of which is

poorly ordered in the X-ray structure. Although TSP is a hydrolyase, the T1 region is more similar

to the pectic lyases than the hydrolyases and there is only a very small region where the hydrogen

bonds of a sheet PB1a are present between residues 267268 and 287288. However, the T1 region

has an interesting example of internal stacked cysteines. The turn at T2 is generally similar to that

seen in the pectic lyases and has similar geometry. The interior of the parallel b-helix is almost

exclusively hydrophobic, without polar stacks such as the asparagine ladders of the pectic lyases,

and with few buried waters, mostly associated with the amides of the T2 turn. There are severalaliphatic stacks especially at the C-terminal end of PB3 and an aromatic stack on PB1 (residues

Phe 284, Phe 308 and Phe 336). However, there are also aligned rather than stacked interactions

and edge to face aromatic interactions.

The fragment 109666 had the same oligosaccharide binding and endoglycosidase activity as

the full length protein (Miller et al., 1998a) which acts both as an adhesion factor in phage binding

and as an enzyme. Steinbacher et al. (1996) determined the structure of three complexes of TSP

with receptor lipopolysaccharide fragments comprising two O-antigenic repeating units from

three Salmonella species, giving the first direct evidence for the substrate binding site of a parallel

b-helix enzyme. The substrate bound to the same cleft created by sheet C and the loops on either

side (i.e. T3PB1T1) and was bound almost parallel to the helix axis as was later seen in pectate

lyase C (Scavetta et al., 1999) and chrondroitinase B (Huang et al., 1999). The binding site showswobble with alternative binding sites for fragments from different strains but the terminal

rhamnose is in almost the same position in the three complexes. The binding of the O-antigen

involves mostly small hydrophilic side-chains and only the side chain of Lys 302 shows significant

displacement on forming the complex. However, the binding does bury a considerable surface

area, showing the excellent complimentarity of the protein and the polysaccharide. The reducing

end of the polysaccharide was bound towards the C-terminus of the parallel b-helix. Aspartates

392 and 395 and glutamate 359 were identified as likely to be involved in catalysis. Mutation of

any of these residues to the amide, either asparagine or glutamine, caused a dramatic loss of

activity (Baxa et al., 1996) but all three mutants continued to bind substrate and product with

wild-type affinity. An inverting endorhamnosidase mechanism was suggested (Steinbacher et al.,1996) in which a water molecule observed to bind in contact with Glu 359 and Asp 395 acts as the

nucleophile, while Asp 392 protonates the glycosidic oxygen.

The structural data revealed that all the 62 tsf mutants were in the parallel b-helix region of

TSP (Haase-Pettingell and King, 1997). Miller et al. (1998a) had reported that the removal of the

N-terminal domain to give the fragment 109666 unmasked the effects of two tsf mutants, G244R

and D238S, and four suppressor mutants, V331G, V331 A, A334 V and A3341, on the kinetics of

folding and unfolding. Subsequently Miller et al. (1998b) reported the properties of a tailspike

fragment corresponding to the isolated parallel b-helix domain consisting of residues 109544

(bhx). This fragment has low but measurable endorhamnosidase activity (0.2% of the wild-type)



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and is a mixture of monomers and trimers. Unlike the larger species, it folds and unfolds

reversibly. Further truncation at the C-terminus of the parallel b-helix produced increasingly

unstable proteins which could not be purified showing the importance of capping the parallel b-

helix. Schuler and Seckler (1998) conclude that the P22 tailspike folding mutants act by globallystabilising or destabilising thermolabile monomeric folding intermediates in which the central

parallel b-helix is topologically similar to the native structure but less tightly packed. Betts and

King (1999) point out the alternative that the rate of the self aggregation process is reduced. An

obvious question might be why are the su mutant sequences not selected by evolution? Val 331 is

close to the substrate and the V331G or V331A mutants are less active than wildtype. The case of

A334 V and A3341 is more complex in that the intermediate and bhx are stabilised by these

mutations, which produce a classical aliphatic stack, but the final trimer is destabilised (Beissinger

et al., 1995) although it has a similar structure to the wild type enzyme (Baxa et al., 1999).

Recently Schuler et al. (2000) have described the rational design of mutants aimed at producing

the tsfand su phenotypes. A tsfmutant was produced by the mutation T326F, which introduced amuch larger residue into the parallel b-helix. The most surprising feature was how little the

structure was distorted with very small shifts of the main chain and only a slight but significant

increase in the dissociation constant for an octasaccharide (no change was seen with T326S and

T326V). The steric strain was mostly absorbed in an unusual conformation of Phe 352 although

strain was transmitted as far as Val 362 on the opposite side of the helix. The rigidity of the

parallel b-helix (or the plasticity of its internal residues) may suggest the mechanism which has

preserved the shape of parallel b-helices while erasing all trace of sequence similarity. Potential su

mutants were designed by mutation of residues Glu 359 and Trp 391 at the active site following

the hypothesis that these mutants had not been observed because of the loss of activity. These

residues conformation lies on the edge of the allowed region of the Ramachandran plot.

However, E359A was much less stable than wild-type and E359G was still less stable than wild-type, suggesting that the side chain of Glu 359 is important for stability as well as for activity.

W391A and W391G were also less stable than wild type. The binding of octasaccharide was also

seriously affected but the crystal structures of E359G and W391A were similar to wild type except

for some small changes near the side chain of Trp 391.

After describing the role of the parallel b-helix in folding, it is necessary for balance to note that

Robinson and King (1997) showed that the formation and breaking of disulphides involving Cys

613 and Cys 635 in the C-terminal Caudal fin domain was critical for the folding to the native

conformation. This domain is formed by chains from each monomer of the trimer and has been

called a triple b-helix by Seckler (1998). This domain contains anti-parallel b-sheet but Kreisburg

et al. (2000) note some similarity in the packing compared to the parallel b-helix and suggest thatthis packing might be a model for amyloid.

Misfolding of TSP has been studied by King and colleagues (King et al., 1996; Speed et al.,

1996, 1997) with the surprising result that aggregation is specific rather than random and that

folding intermediates do not coaggregate with each other but only with themselves. It was possible

to directly identify by native gel electrophoresis sequential multimers as the earliest intermediates

along the in vitro aggregation pathway. Schuler et al. (1999) have similarly shown that bhx

aggregates via a linear polymerisation mechanism observing monomers, dimers, trimers and

tetramers. The secondary structure was relatively unchanged but tryptophan fluorescence was

quenched. Fibrils were observed which bound Congo Red and gave the green birefringence



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363 and Arg 384), while His 116, Arg 184 and Glu 333 might interact with a substrate rather than

a disaccharide. The only side chains moving significantly on forming the complex belong to Asn

213 and Arg 363 (4 and 1 (A, respectively). Of the residues near the active site, conservation

between the three sequences suggests that Lys 250, Arg 271 and Glu 333 may have important rolesin catalysis while Arg 318 and Arg 363 may be significant in defining the specificity.

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Architecture of B-Hellices Parallel)