Eur. J. Biochem. 213, 67-75 (1993) 0 FEBS 1993

Substrate interactions between trypanothione reductase and W-glutathionylspermidine disulphide at 0.28-nm resolution Sue BAILEY', Keith SMITH', Alan H. FAIRLAMB' and William N. HUNTER'

Department of Chemistry, University of Manchester, England Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, England

(Received November 13, 1992LJanuary5, 1993) - EJB 921618

The enzyme trypanothione reductase has been identified as a prime target for the rational design of inhibitors which may have clinical use in the treatment of tropical diseases caused by the genera Trypanosoma and Leishmania.To aid the design or identification of new inhibitors of this enzyme we have elucidated the structural detail of a trypanothione reductase complexed with one of the naturally occurring substrates, Ilr-glutathionylspermidine disulphide, by single-crystal X-ray diffrac- tion methods at 0.28-nm resolution. The model for the Crithidia fasciculata enzyme-substrate com- plex has an R-factor of 14.8% and root-mean-square deviations of 0.0015 nm and 3.3" on bond lengths and angles respectively. Hydrogen bonding and van der Waals interactions between the enzyme and substrate are dominated by the amino acid side chains. The substrate binds in a rigid active site such that one glutathione moiety is in a V-shape, the other in an extended conformation. One spermidine moiety binds closely to a hydrophobic patch in the active site formed by a trypto- phan and a methionine. Distances between the methionine S6 and the terminal N of this spermidine suggest that a hydrogen bond may supplement the hydrophobic interactions in this part of the active site.

Parasitic diseases continue to devastate both the physical health and material wealth of people living in many tropical countries. Current chemotherapy is frequently unsatisfactory with low efficacy, drug toxicity and drug resistance pre- senting some of the major problems (Goodwin, 1987). Basic research has recently been devoted towards the identification of suitable metabolic pathways that can potentially be ex- ploited by rational drug design (Hol, 1986; Fairlamb, 1989). In the case of trypanosomes and leishmania, causative agents of African sleeping sickness, Chagas' disease and cutaneous and visceral leishmaniasis, the discovery of trypanothione [iV,P-bis(glutathionyl)spennidine, T(SH), ; Fairlamb et al., 1985) has attracted attention. This metabolite is unique and essential to these parasites and suggests a viable target for chemotherapeutic attack. This is because trypanothione and its metabolic precursor (I" -glutathionylspermidine ; GspdSH) appear to have taken over many of the protective anti-oxidant functions that in mammalian cells are mediated by the sulphydryl group of glutathione (L-y-glutamyl-L-cys- teinylglycine; GSH). The diverse roles of the glutathionyl-

Correspondence to W. N. Hunter, Department of Chemistry, University of Manchester, Oxford Rd, Manchester, England M13 9PL

Far: 061 275 4598. Abbreviations. r.m.s., root mean square ; GSH, reduced gluta-

thione; (GS),, glutathione disulphide ; T(SH),, reduced trypano- thione; T(S),, trypanothione disulphide; GspdSH, P-glutathionyl- spermidine ; (GspdS),, W -glutathionylspermidine disulphide.

Enzymes. Glutathione reductase (EC 1.6.4.2) ; trypanothione re- ductase (EC 1.6.4.8).

Note. The crystallographic coordinates described here have been submitted to the Brookhaven Databank (Bernstein et al. 1977) and assigned the identifier 1TYP.

spermidine conjugates has been reviewed elsewhere (Fair- lamb and Cerami, 1992). In the course of these anti-oxidant functions, GSH and T(SH), are oxidised to their respective disulphides [(GS), and T(S),; Fig. 11, with the products being rapidly reduced by NADPH via their cognate flavoenzymes, glutathione reductase in the mammalian host and trypano- thione reductase in the parasite (Shames et al., 1986).

Trypanothione reductases and mammalian glutathione re- ductases share approximately 40% sequence similarity and the catalytically important residues are conserved (Aboagye- Kwarteng et al., 1993 ; Fairlamb and Cerami, 1992). The pro- teins are both active as homodimers of subunit mass around 52 kDa, contain a non-covalently bound FAD and use NADPH to provide a reducing equivalent in the enzyme mechanism (Ghisla and Massey, 1989). The critical differ- ence between trypanothione reductase and mammalian gluta- thione reductase is that the enzymes are only capable of pro- cessing their cognate substrates (Henderson et al., 1987). Thus, mammalian glutathione reductase will not catalyse the reduction of trypanothione disulphide [T(S),] to T(SH), or N-glutathionylspermidine disulphide [(GspdS),] to GspdSH at any significant rate ; neither will trypanothione reductase process (GS), to GSH. The exclusive specificity of these en- zymes suggests that it may be possible to find inhibitors of trypanothione reductase that would not affect the mammalian host glutathione reductase such that, if the trypanosomal enzyme were selectively disabled then the parasites would succumb to oxidative stress.

Schulz and co-workers have dissected, in considerable detail, the structurelactivity relationships of human gluta- thione reductase (Janes and Schulz, 1991; Karplus et al., 1989; Karplus and Schulz 1987, 1989). The glutathione re-

68

coo'

0

(a) N'-glutathionylspermidine disulfide, [GspdS]~

coo'

I NH;

\ I

(b) Trypanothione disulfide, T[SI2

Fig. 1. Molecular formulae for trypanothione reductase sub- strates. The physiologically important substrates are (a) W-gluta- thionylspennidine disulphide. The secondary amine of the spenni- dine component is labelled N4, the primary amine is N8. (b) Trypan- othione disulphide [IV,IP-bis(glutathionyl)spermidine disulphide].

ductase structure has subsequently been utilised by a number of groups as the template onto which a model of trypano- thione reductase has been constructed using computer graph- ics methods (Murgolo et al., 1991 ; Smith et al., 1991 ; Hor- jales et al., 1993). Some of these models have been used in the design of mutant enzymes created by site-directed mu- tagenesis in an attempt to define the substrate specificity of trypanothione reductase (Bradley et al., 1991 ; Henderson et al., 1991; Sullivan et al., 1991; Walsh et al., 1991). Model- ling has also been used to identify phenothiazines and related uicyclic compounds as lead structures for selective inhibitor design (Benson et al., 1992).

Our crystallographic studies are designed to complement these approaches to inhibitor design. The structure of trypan- othione reductase isolated from the insect parasite Crithidiu fusciculutu has been determined in two crystal forms (Kur- yan et al. 1991 ; Hunter et al., 1990, 1992). We now report the structure of this enzyme in the tetragonal form complexed with one of the naturally occurring substrates, M-glutathion- ylspermidine disulphide.

MATERIALS AND METHODS

Crystallisation, data collection and processing Crithidiu fusciculutu (wild-type clone HS6) was cultured

as described previously (Shim and Fairlamb, 1988) and try- panothione reductase isolated and purified according to Shames et al. (1986). Single crystals of the enzyme were grown from an 18-mdml solution of the protein using am-

Table 1. Statistics relevant to the data collection. Ray,,, = Elf@)- <I> I/ZZ(k) where Z(k) and Z represent the diffraction intensity va- lues of individual measurements and the corresponding mean values. 0 represents the standard deviation.

Resolution Independent R,,, >3af reflections (all data)

nm 0.885 0.626 0.511 0.443 0.396 0.361 0.335 0.313 0.295 0.280

Total

783 2125 2776 3290 3735 4057 4449 4630 4927 5076

35848

0.06 0.07 0.08 0.08 0.07 0.09 0.11 0.14 0.19 0.24 0.096

%

89.8 87.8 85.4 89.3 88.2 82.6 75.4 62.4 49.5 36.9 70.0

Hunter et al. (1990). The crystals belong to space group P4, (a = b = 12.89 nm, c = 9.28 nm) and contain the enzyme dimer in the asymmetric unit. A single crystal (dimensions 0.12 X 0.12 X 0.8 mm) was soaked for 2 days in a solution of 10 mM W-glutathionylspermidine disulphide (custom-syn- thesised by Bachem) and 10 mM NADPH (Sigma) in 0.1 M sodium potassium phosphate, pH 7.0 plus 70% saturated am- monium sulphate. No attempt was made to exclude oxygen from the sample. The high concentrations of phosphate and sulphate utilised in the mother liqour and the length of the soak serve to provide an abortive complex of enzyme and substrates. We observed only a small change (0.2% change in c) in the unit cell parameters of the soaked crystal com- pared with the native crystals. Although the crystals are rela- tively sensitive to the intense radiation beam provided at the synchrotron, by using a 0.2-mm collimator we were able to collect the data over a period of 10 h by translating along the length of the rod-shaped crystal as decomposition became evident.

The data were collected on station PX9.6 at the Synchro- tron radiation source, Daresbury Laboratory using an Enraf- Nonius FAST area detector and a wavelength of 0.089 nm. The experimental configuration of this station has been de- scribed by Helliwell et al. (1986). The ring current ranged over 215-14OmA at 2 GeV. The crystal was aligned with the c axis offset by about 45" to the rotation axis and 100" of data were measured in 0.1" frames using exposure times of 20 dframe. Data were processed with the MADNES software (Messerschmidt and Pflugrath, 1987) using the option EVAL 6 (P. Brick, unpublished) and the CCP4 program suite (SERC Daresbury Laboratory, 1986). A total of 59390 measure- ments yielded 35 848 independent reflections, 95.3 % of the data, to a resolution of 0.28 nm. The data merged with an overall Rsym of 0.096. Statistics relevant to data collection are presented in Table 1.

Refinement proceedures The mean fractional isomorphous difference (Cl Fs-FN I/

E I F N l , where Fs is the experimentally observed structure amplitudes for the enzyme-substrate complex and FN those for the enzyme by itself) between this data set and the native data. which was measured under similar exDerimenta1 con-

Y

monium sulphate as &e precipitating agent as detailed by

69

ditions (unpublished), is 18.2 %. A difference map calculated using coefficients F,-F,, acdo revealed strong and continu- ous density at the substrate binding sites and at the NADPH binding site.

We decided to refine the structure completely and so, prior to interpretation of the density, the current native model was refined against the data from the substrate-bound crystal using the X-PLOR package (Briinger, 1990). A heatinglslow cooling step, up to 15OO0C, was first carried out followed by several cycles of conventional positional refinement applying restraints on non-crystallographic symmetry. An overall tem- perature factor of 0.2 nm2 was applied. The R-factor, defined as ZIF,-F,IIZIF,I, was 20.6% for all data between 0.8- 0.28 nm. Density maps were then calculated using the coef- ficients 2F0-Fc, acdc and F,-F,, acalc. The electron density was consistent with two W-glutathionylspermidine disul- phide molecules located in each active site of the enzyme.

X-PLOR was again used for further cycles of refinement interspersed with graphics fitting using FRODO (Jones, 1978; P. R. Evans, personal communication). The substrates were included in the model and restraints on non-crystallo- graphic symmetry released. Solvent molecules, all treated as oxygen atoms of water, were progressively added at each stage of map inspection and included in subsequent refine- ment calculations. Several solvent molecules displayed iso- tropic thermal parameters greater than 0.5 nm’. These were in general deleted but we noted 16 which, despite such a high B value, were in well defined density and on this basis they were retained. Individual atomic temperature factors were also included in the refinement but not allowed to drop below 0.03 nm2. The refinement was terminated with a crys- tallographic R-factor of 14.8% for the 32393 reflections with F, > OF, between the limits of 0.8-0.28 nm resolution.

RESULTS AND DISCUSSION Crystallography

The crystal structure is isomorphous with the native structure (Hunter et al., 1992) and has been fully refined (see Materials and Methods). The final model comprises a protein dimer (residues 1A-487A for the first subunit, 2B-487B for the second, 7418 non-hydrogen atoms with an average isotropic thermal parameter, B,, = 0.13 nm2) plus two FAD molecules (106 atoms, B,, = 0.05 nm’), four W-glutathionyl- spermidine molecules (116 atoms, B., = 0.47 nm2), two NADP molecules (96 atoms, B,, = 0.48 nm2) and 400 solvent molecules treated as oxygen atoms. The B values for solvent positions ranged over 0.03-0.64 nm2 with B , = 0.28 nm’. The final model has root-mean-square (r.m.s.) values on bond lengths and angles of 0.0015 nm and 3.3” respectively. For dihedral angles the r.m.s. is 25.8’. The presence of a dimer in the asymmetric unit provides an internal check on the structure and we observe very similar results, commensurate with the level of accuracy of this structure, for each active site interacting with the substrate. A least-squares fit of all the enzyme Ca atoms, with the exception of MetlA, results in an r.m.s. deviation of 0.035 nm from non-crystallographic symmetry. A Luzatti plot (not shown; Luzatti, 1952) suggests an average positional error of approximately 0.025 nm. The first residue of the B subunit, a methionine, and the last four residues at the C-terminus of both subunits have no electron density and are not included in the model. The structure is well ordered, as represented by the relatively low thermal parameters (see above), and the fit to electron density is ex-

cellent. It is worthy of note that the residues comprising the disulphide substrate binding site of trypanothione reductase are some of the most ordered in the structure (see below). In the final 2F,,-F,, electron density map (where F, and F, represent the observed and calculated structure factors re- spectively, aCdc the phase calculated from the molecular mo- del) there is continuous density at the la level for all main chain atoms. Fig. 2 shows the disulphide substrate in one of the binding sites with the associated electron density of an omit map.

The model contains one difference from the amino acid sequence deduced from the gene sequence (Aboagye-Kwar- teng et al., 1993). This is located at position 126 where a tryptophan replaces a phenylalanine. The decision to place a tryptophan in this position was based on the evidence pre- sented by the electron and difference density maps. A Rama- chandran plot (Ramachandran and Sasisekharan, 1968) indi- cates 13 violations. Three of these are at either the N or C- terminal regions. Particular residues of note are Tyr4.5, Ala47, Ser315, Arg331 and Ser433 which display the same strained conformations in both subunits. These latter residues are well defined, in tight loops and their conformations are stabilised by hydrogen bonds to residues also in the loop.

Overall structure of trypanothione reductase and location of the disulphide substrate binding site

A diagram of the trypanothione reductase dimer showing the domain organisation and location of the substrates in the two active sites is presented in Fig. 3. The fold and domain structures are similar to that of human glutathione reductase at a level commensurate with the sequence similarity of ap- proximately 40% (Kuriyan et al., 1991 ; Hunter et al., 1992). Each trypanothione reductase monomer contains four do- mains. Domains I and 11 bind FAD and NADPH respectively and display ‘Rossmann’ folds (Rao and Rossmann, 1973). Domain I11 has been referred to previously as the central domain (Karplus and Schulz, 1987) and domain IV forms the interface with the partner subunit. The secondary structure of the enzyme comprises approximately 30% a-helices, 30% P-sheet and 40% in a variety of other conformations. The functional dimer contains two disulphide substrate binding sites. Each is located in a cleft constructed by contributions from domains I and I11 from one subunit and domain IV from the other subunit (Fig. 4). The cleft is approximately 2.0 nm long by 1.5 nm wide and 1.5 nm deep. It is lined on one side by three helices and on the other side by a small section of residues in an extended conformation, two short helical segments and, at the periphery, the edge of a section of j3- sheet. The floor of the active site is formed by a helical segment on which is located the redox active disulphide formed by Cys52 and Cys57.

W-Glutathionylspermidine disulphide binding The amino acid segments that construct the trypanothione

reductase active site comprise residues 14-21, 52-58, 106-113,335-339 from one subunit and 396’-399’, 461’- 470’ from the partner subunit (the prime, ’, is used to signify residues from the other subunit). We have designated the two active sites as site A and site B, dependent upon which sub- unit contributes the FAD and the redox-active disulphide pair Cys52-Cys57. The nomenclature of the substrate is as fol- lows: [GspdS], comprises yGlu, Cys, Gly and Spd with the

j suffix I or I1 to identify each of the two W-glutathionylsper-

70

Fig. 2. The electron density for the substrate. The density shown corresponds to an F,-F,, aCacalc map in which all atoms of the Substrate were omitted from the structure factor calculation. For clarity, the substrate has been divided into two, only one glutathionylspermidine component is shown with associated density and different views have been selected. (a) Gspd-IB with 20 density, (b) Gspd-IIB with 1.2a density. A certain amount of positive density may represent solvent positions that have not been included in the model.

Fig. 3. A stereodrawing of trypanothione reductase and sub- strate. Arrows depict P-strands indicating the C to N direction of the polypeptide chain, helices are shown by spirals. The secondary structure assignments are according to Kabsch and Sander (1983). The different colors are used to identify the different domains. Yel- low and green represents domains I and 11, pale blue and red show domains I11 and IV. The active site is located in a V-shaped cleft. The substrate molecules are shown as van der Waals spheres colored according to atom type; C black, N blue, 0 red, S yellow. The view is approximately into the non-crystallographic twofold axis. Figs 3 and 4 were prepared with MolScript (Kraulis, 1991)

midine segments and chain identifier A or B according to which active site they bind.

The 23 amino acid residues that have been identified as playing a role in directly binding the disulphide substrate, either by hydrogen bonding or van der Waals interactions or both, are listed in Tables 2 and 3. We note that there are no major rearrangements of residues when comparing the structures of the native enzyme refined at 0.26-nm resolution with the enzyme-[GspdS], complex. The presence of the

Fig. 4. The secondary structure that forms the active site of try- panothione reductase. A stereodrawing showing the elements of secondary structure that contribute to the active site of trypanothione reductase and the substrate depicted in a ball-and-stick fashion. The same color scheme as applied in Fig. 3 is used. Note that the red sections belong to the partner subunit.

acidic residues, Glul8, Glu466’ and Glu467’; help to provide an overall negative charge in the active site. This serves to compensate for the positively charged substrates utilised by trypanothione reductase (Fig. 1).

Given the similarities between (GS), and (GspdS), with their conserved y-glutamylcysteinyl moieties and the simi- larities in certain sections of the active sites of glutathione reductase and trypanothione reductase, it is not surprising that we observe similar conformations in the two systems. In an analogous fashion to (GS), (Karplus and Schulz, 1989), one IV -glutathionylspermidine (Gspd is used to signify the glutathionylspermidine component of the dimeric substrate) binds with the yGlu-Cys-Gly moiety in a V-shape. yGlu-I interacts mainly with domain I11 and IV residues whereas the Cys-I, Gly-I and Spd-I components interact mainly with domain I residues. The spermidine component fits into a

71

T R SUBSTRRTE RCTlVE SITE B TR SUBSTRRTE ACTIVE SITE B I R I

Fig. 5. A stereoview of N’-glutathionylspermidine disulphide in site B. Specific amino acid side chains on a Ca plot of five amino acid segments lining the active site. The view is similar to that used in Fig. 4. The one-letter code for identifying enzyme residues is used. Atoms are depicted as spheres of increasing radius in the order C < N < 0 < S. This figure was obtained with PLUTO (SERC Daresbury Laboratory, 1986)

hydrophobic niche formed at Trp21 and Metll3. The second Gspd binds in an extended mode. The yGlu-I1 segment inter- acts with domain IV residues whereas the Cys-11, Gly-11, Spd-I1 portions only contact residues belonging to domain 1. The spermidine moiety flails out across one of the a-helical sections of this FAD binding domain (Figs 4 and 5).

Specific contacts that may represent hydrogen bonds formed between the substrate and the enzyme are detailed in Table 1. The two binding sites have been refined indepen- dently and for each a best fit, between substrate and density, has been achieved. This has resulted in minor differences between the active sites, particularly in the solvent structure, which are commensurate with the resolution of our data.

There are 12 enzyme residues implicated in hydrogen bond interactions when both site A and B are considered. Of particular interest are the residues Glu18, Serl09 and Metll3. Glu18 is held in position by a hydrogen bond from Asn22 and able to accept another hydrogen bond from the amide linking Gly-I and Spd-I. Serl09, in site B, appears to form a hydrogen bond with N4 of Spd-11.

The occurrence of hydrogen bonds in proteins that in- volve a sulphur atom has recently been surveyed (Gregoret et al., 1991). It has been noted that hydrogen bonding to the Sy of methionines is not particularly common. In our struc- ture the two distances observed between N8 of Spd-I and Sy of Met113 are 0.29 nm and 0.36 nm in sites A and B, respectively. These distances are, given the accuracy of this structure, comparable to the distance of 0.34 nm observed for N-H-S hydrogen bonding interactions on the basis of small molecule crystal structures (Hamilton and Ibers, 1968). Met113 also partakes in van der Waals interactions with the substrate (see below).

Solvent-mediated linkages formed between substrate and enzyme also contribute significantly to stabilising the com- plex. This is particularly evident at the Glu-I binding sites (Table 2). The N4 atom of Spd-I is directed away from the hydrophobic cleft formed at Trp21 and Met113 and points out toward bulk solvent. In site B, N4 Spd-I is 0.37 nm from So1331 which in turn is 0.37 nm and 0.34 nm from 061 of Asn22 and Oe1 of Glul8 respectively. The terminal amino group of Spd-I1 is free to interact with solvent and in the B active site such an association is indeed observed.

In terms of van der Waals interactions, there are 21 resi- dues that have contacts of 0.4 nm or less with the substrate

that do not represent hydrogen bonds (Table 3). Of these, 18 make single or multiple contacts in the A site and 19 in the B site. Residues of note with respect to van der Waals interactions with the substrate are the aromatics Trp21, Phe396’ and His461’. In particular, Trp21 stacks over the Spd-I segment of the substrate helping to produce the most contacts between enzyme and substrate in one corner of the active site (Fig. 5). Most of the contacts involve the amino acid side chains although some of the main-chain atoms of Serl4, Leul7, Serl09, Gly112, Pro398’, Pro462’, and Ser464’ interact in this manner.

The conformations of the NADP molecules that bind to trypanothione reductase are similar to those observed in other oxidoreductase systems (Karplus and Schulz, 1989; Mattevi et al., 1992). This detail will be presented elsewhere.

A rigid active site, a flexible substrate The disulphide substrate binding site residues are

amongst the most ordered in the enzyme structure with an average thermal parameter of 0.11 nm2 for all atoms of those 23 residues in the unliganded enzyme that we have identified as being involved in the binding. In the complex with (GspdS), the average thermal parameter is 0.10 nm’. Also, there are no major alterations in side-chain conformations, when comparing native and liganded enzyme. Taken to- gether, these two observations suggest that the active site is fairly rigid. The trypanothione reductase active site is con- structed from a scaffold of secondary structure, mostly a- helices, which must help to promote rigidity. The constitu- ents of the active site involve bulky aromatic residues and a number of short side-chain residues. The residues with longer side chains are observed to interact with other residues to stabilise specific orientations. So, Glu18 is held by a hydro- gen bond with Asn22, Trp21 N E ~ hydrogen bonds with Oyl of Thrll7, Glu466’ has hydrogen bonds with His461’ N61 and the amide of Thr463‘ and Glu477‘ has a hydrogen bond- ing contact with Oy of Ser394‘. In active site B, the latter is a direct hydrogen bond, in active site A it is solvent-mediated. Hydrophobic interactions also stabilise certain side-chain conformations. Met113 is stacked below Trp21 and has the methyl group directed into a hydrophobic core formed by Leul7, Leu33 and Phell4. This helps to orient the S6 atom for interaction with the N8 primary amine of Spd-I. The ali-

72

Table 2. A listing of possible hydrogen bonding contacts formed by W-glutathionylspermidine disulphide with trypanothione re- ductase and solvent molecules.

Residue Atom Protein and solvents Distances Comments

residue (A, B) atom A B

yGlu-I 01 So168, So1330 0 0.30 0.32 solvent H-bonds to Ser 470‘ Oy, So1272/329 01 So1272, So1329 0 0.30 0.34 solvent H-bonds to So168/330 01 None, So1275 0 0.35 solvent H-bonds to Glu18 0 ~ 1 , So1332 0 2 So168, So1330 0 0.35 0.36 0 2 Ser470‘ OY 0.39 0.34

0.33 0.32 N Glu467‘ 0&1 0.30 0.43 OE So1273, So1328 0 0.31 0.28 solvent H-bonds to 081 of Glu466‘

0 Serl4 OY 0.35 0.32 0 So1264, none 0 0.27 SY His461’ N E ~ 0.33 0.35 Ne2 is 0.32 nm (A) and 0.36 nm (B) from Sy of Cys 52

Gly-I N So1262, So1332 0 0.36 0.42 0 Tyrl 1 0 01 0.29 0.29

Spd-I N1 Glu18 0&2 0.33 0.32 Glu18 is held in position by Asn22 N8 Met113 S6 0.39 0.36 Met1 13 held in hydrophobic pocket

LGlu-I1 01 Ser464’ OY 0.37 0.37 01 none, So1353 0 0.29 So1353 H-bonds to Lys61 NC 0 2 Lys61 N5 0.35 N Thr463‘ 0 0.29 0.35 main-chain carbonyl N Glu466‘ 0&2 0.35 N GIu467‘ 0.28 GIu467’ has different conformation in A, B

Glu467’ has different conformation in A, B N Ser470‘ OY

CYS-I N So1262, So1332 0 0.36 0.37 So1332 H-bonds to So1275

Cys-I1 N none, So1376 0 0.33 So1376 H-bonds to Thr397’ carbonyl Gly-11 0 TyrllO ov 0.35 0.31 Spd-I1 N4 Serl09 0 0.32 two possible positions for Spd-I1 exist

N8 none, So1390 0 0.30 a fit to the best density was made in each site

Table 3. A tabulation of the number of contacts, not involved in hydrogen bonding, between (GspdS), and trypanothione reductase. Only contacts equal to or less than 0.4 nm have been considered. The specific enzyme residues involved are listed and the residues that are different in active site A and B, i.e. not within the 0.4-nm criteria, are detailed.

Residue Number of contacts Trypanothione reductase residues

A site B site

Residues that do not contact within the 0.4-nm limit

yGlu-I 17 16 Thr33.5, Ile339, His461’, Ser470‘, Glu466’ Glu466’ in B cys-I 12 12 Serl4, Va153, Vd58, Tyrll0, Ile339, His461’ GlyI 7 10 Serl4, Glu18, Tyr110, Ile339 Spd-I 33 28 Leul7, Glu18, Trp21, Met113 yGh-I1 22 16 Phe396‘, Pro398’, Pro462’, Thr463’, Ser464’ FVo398’ in B, Ser464’ in A cys-I1 1 1 Ile106 Gly-I1 4 6 Ile106, TyrllO Spd-I1 12 22 Tyr110, Serl09, Gly112 Tyr110, Gly112 in A

phatic components of the Glu466’ side chain have van der Waals interactions with Phe396’ and Va1413‘.

NMR studies on the cyclic peptide trypanothione (Hen- derson et al., 1990) and glutathione disulphide (Rabenstein and Keire, 1989) indicate a considerable degree of flexibility. This conformational freedom is aIso likely to apply to the acyclic N-glutathionylspennidine disulphide. Once in the ac- tive site, however, the conformational freedom will be re- stricted by interactions with the enzyme. This enzyme-sub- strate complex is clearly not an example of the lock-and-key

mechanism of binding (Fersht, 1985). The term ‘induced fit’ is in general used to denote conformational change on the part of the enzyme. In the present study it is the substrate that undergoes induced fit. Although similar to the ‘zipper’ scenario presented by Burgen et al. (1975), the trypanothione reductase substrate interactions may thus be classed as what we term mould-and-melt fit. The rigid active site presents a cavity or mould of defined shape, with chemical properties varying according to the locality on the mould. The flexible or molten substrate molecule diffuses into the mould and is

73

Table 4. Solvent-accessible areas for the residues comprising both (GspdS), molecules and averages when treated in isolation from the enzyme and when complexed. A solvent probe of radius of 0.14 nm was used with the algorithm of Lee and Richards, (1971).

Residue Area molecule in isolation Area when complexed AI-C

A B average (I) A B average (C)

yGlu-I

Gly-I Spd-I yGlu-I1

Gly-I1 Spd-I1

cys-I

cys-I1

Totals

nm2

2.19 1 .oo 0.60 2.67 2.10 1.03 0.68 2.68

12.95

2.17 0.96 0.58 2.58 2.15 1 .oo 0.69 2.88

13.01

2.18 0.98 0.59 2.63 2.13 1.02 0.69 2.78

12.98

0.78 0.06 0.08 0.93 0.28 0.38 0.33 1.82 4.06

0.79 0.03 0.08 1.14 0.43 0.47 0.28 1.63 4.85

0.79 0.05 0.08 1.04 0.36 0.43 0.31 1.73 4.46

13.9 0.93 0.51 1.59 1.77 0.59 0.38 1.05 8.52

pressured by non-covalent interactions into the correct orien- tation and specific conformation for binding to occur and subsequently catalysis. Only a small quantity of energy would be expended to alter the substrate conformation given the flexibility of the spermidine adducts involved.

We have calculated solvent-accessible areas, using a sol- vent probe radius of 0.14nm, for the substrate in isolation and when complexed with trypanothione reductase (Table 4). Although we do not know the precise conformation of the uncomplexed (GspdS),, by treating it in isolation from the enzyme we gain some insight into what area of the substrate is utilised in the interactions with trypanothione reductase. The results show that approximately 66% of the substrate area becomes buried when the enzyme-(GspdS), complex is formed. This suggests an important role of the hydrophobic part of the active site in stabilising the complex. In energy terms the results presented in Table 4 suggest that the hydro- phobicity component of binding approximates to 84 kJ mol-' (20 kcal mol-'; Chothia and Janin, 1975). The proportion of buried surface area on complex formation is not uniform over the substrate molecule but observed to vary from residue to residue. In general, Gspd-I is more buried than Gspd-11. The largest decrease in surface accessibility (95 %) occurs at Cys- I, the smallest decrease at Spd-11. Indeed, the Spd-I portion of the substrate is significantly more buried than is the Spd- I1 component. The stacking of Trp21 over Spd-I serves to enhance the protection of aliphatic methylene groups from solvent.

Of the 23 residues listed in Tables 2 and 3 which interact directly with the substrate, only Gly112 is not conserved in the other trypanothione reductases (Aboagye-Kwarteng et al., 1993). In trypanothione reductase from T. cruzi this position contains an aspartate (Sullivan and Walsh, 1991), in T. con- golense trypanothione reductase a glutamate (Shames et al., 1988). The C. fusciculutu trypanothione-reductase-(GspdS), complex structure implicates the Glyll2 Ca in contacting the spermidine component (Spd-11) of the substrate in binding site B (Table 3). The orientation of the peptide main chain suggests that a side chain would be directed away from the substrate. It is, however, possible that slight rearrangements of either the aspartate or glutamate side chains and the Spd- I1 section of (GspdS), could facilitate a direct interaction be- tween the oppositely charged groups in other trypanothione reductase molecules. In molecular modelling studies on T. congolense trypanothione reductase such an interaction in-

Fig.6. The location of charged residues on the surface of trypanothione reductase and glutathione reductase near to a di- sulphide substrate binding site. Atoms comprising the three do- mains that form the active sites of (a) trypanothione reductase and (b) human glutathione reductase have been depicted as space-filled spheres. Atoms comprising aspartates and glutamates are colored red, basic arginines, histidines and lysines are colored dark blue and all other enzyme atoms are white. The cognate substrates for trypanothione reductase and glutathione reductase are shown in light blue and yellow with the latter color being used to identify the Cys- I and Cys-II residues. The direction of viewing is similar to that employed in Figs 3-5.

74

volving a glutamic acid interacting with the spermidine por- tion of (TS), was postulated by Murgolo et al. (1991). We now have access to X-ray-grade crystals of recombinant try- panothione reductase and plan to investigate this further.

Comparison with (GS)2 binding to glutathione reductase Based on the structure of the active site of native trypano-

thione reductase, we and others (Hunter et al., 1992; Kuriyan et al., 1991) have put forward explanations for the differing substrate specificities of human glutathione reductase and trypanothione reductase. Our study of the complex with N1- glutathionylspennidine disulphide now provides direct evi- dence concerning trypanothione reductase enzyme speci- ficity.

Of particular note with respect to the differing substrate specificities of human glutathione reductase and trypano- thione reductase are the size and charge of the active-site cleft and specific interactions involving residues on one side of the active site. It has been shown previously that the try- panothione reductase active site is more open than the gluta- thione reductase active site and that the charges were differ- ent (Kuriyan et al., 1991; Hunter et al., 1992). The more open trypanothione reductase active site is required for the larger substrate. We now confirm that in trypanothione re- ductase the charge of all residues interacting with (GspdS), is overall negative, whilst in glutathione reductase it is posi- tive. This difference in charge is explained by the electro- static requirements of the cognate substrates. A positive charge accompanies the trypanothione reductase substrates, a negative charge for (GS), engendering charge comp- lementarity between enzymes and cognate substrates.

A comparison between our trypanothione reductase and human glutathione reductase shows that there are six non- conservative amino acid differences in the residues involved in the interaction with substrate. In the format trypanothione reductase residue/glutathione reductase residue these are Glul8/Ala34, Trp211Arg37, Asn22/Arg38, Ser109/Ile113, Metll3/Asnl17 and Ala343/Arg347. The first five differ- ences all occur on one side of the active site (Fig. 5) and are key to the question of the differential specificity of trypano- thione reductase and human glutathione reductase. Trypano- thione reductase uses Asn22 to stabilise a conformation of Glu18 that then, in turn, accepts a hydrogen bond from the amide linking Gly-I and Spd-I. Asn22 is also positioned to interact with the substrate through a solvent-mediated link- age as observed in site B. Glu18 places a negative charge in the active site. Trp21 caps this portion of the active site and, together with Metll3, forms a hydrophobic patch to bind the Spd-I residue. Met113 offers a hydrogen bond accepter and polarizable sulphur atom together with van der Waals interac- tions to produce a significant component to the binding of Spd-I. The carbonyl group of Serl09 is able to accept a hy- drogen bond of length 0.32 nm from N4 of Spd-I1 in site B.

Human glutathione reductase presents the positive Arg37 and Arg38 to repulse the positively charged trypanothione reductase substrates. In addition, the presence of Asnll7 with the arginines produces a hydrophilic patch in the active site. Asnll7 would not provide a mixture of hydrophobic and hydrophilic properties, as does a methionine (Gellman, 1991), to interact with Spd-I. The Ala343/Arg347 substi- tution merely removes another large positively charged am- ino acid residue from the active site of trypanothione re- ductase, in accordance with the structural requirements of a large positively charged substrate. Our structural results are

in broad agreement with the mutational analyses carried out on recombinant glutathione reductase and trypanothione re- ductase (Bradley et al., 1991 ; Henderson et al., 1991 ; Sulli- van et al., 1991 ; Walsh et al., 1991).

The distribution of charged residues on the surface of trypanothione reductase and human glutathione reductase is quite different (Fig. 6). Trypanothione reductase has a ring of acidic residues on the exterior of one side of the disulphide substrate binding site, the side which binds the positive Spd components. This may serve to attract the respective sub- strates, perhaps also to pre-orient them before they slip into the cleft. In human glutathione reductase basic arginine and lysine residues are located in this area of the enzyme presum- ably to carry out the same function of interacting with the negatively charged glycine carboxylates of (GS),.

The trypanothione-reductase-(GspdS), complex gives clues to the mode of binding of the other physiological sub- strate T(S),. The Spd-I component of (GspdS), appears to approximately mimic the position of the spermidine bridge of trypanothione. However, although there are clear similari- ties, we think that the precise detail of the trypanothione re- ductase/T(S), interactions would be difficult to predict given the flexibility of the substrate and the asymmetry of the sper- midine bridge. This detail would be better determined exper- imentally.

In terms of inhibitor design, we now have access to an accurate model of the trypanothione reductase active site and details about which residues are involved in substrate binding and their mode of interaction. We judge it to be useful that the targeted active site is fairly rigid and will now proceed to the study of enzyme-inhibitor complexes with the hope that we can improve on the encouraging results of previous inhibitor studies (Benson et al., 1992; Henderson et al., 1988; Jockers-Scherubl et al., 1989).

We thank our colleagues and the staff at Daresbury Laboratory, in particular M. Papiz and P. Rizkallah, and A. Mattevi and W. Hol for useful discussions and comments. This program of research is funded by the Wellcome Trust, The National Institutes of Health (USA), the Royal Society and the Science and Engineering Research Council Daresbury Synchrotron Laboratory. S. B. acknowledges receipt of a short-term European Molecular Biology Organisation fellowship.

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