Refined structure of dienelactone hydrolase at 1.8A˚

J. Mol. Riol. (1990) 214, 497-525

Refined Structure of Dienelactone

Hydrolase

at 143A

Dushyant Pathakt and David Ollis Department of Biochemistry Molecular Biology and Cell Biology Northwestern University Evanston, IL 60208, U.S.A. (Received 11 September

1989;

accepted

10 January

1990)

The structure of dienelactone hydrolase (DLH) from Pseudomonus sp. B13, after stereochemically restrained least-squares refinement at I.8 A resolution, is described. The final molecular model of DLH has a conventional R value of 0150 and includes all but the carboxyl-terminal three residues that are crystallographically disordered. The positions of 279 water molecules are included in the final model. The root-mean-square deviation from ideal bond distances for the model is 0.014 A and the error in atomic co-ordinates is estimated to be 015 A. DLH is a monomeric enzyme containing 236 amino acid residues and is a member of the /I-ketoadipate pathway found in bacteria and fungi. DLH is an cl/p protein containing seven helices and eight strands of p-pleated sheet. A single 4-turn :~,a-helix is seen. The active-site Cys123 resides at the N-terminal end of an a-helix that is peculiar in its consisting entirely of hydrophobic residues (except for a C-terminal lysine). The P-sheet is composed of parallel strands except for strand 2, which gives rise to a short antiparallel region at the N-terminal end of the central P-sheet. The active-site cysteine residue is part of a triad of residues consisting of Cysl23, His202 and Aspl71, and is reminiscent of the serinelcysteine proteases. As in papain and actinidin, the active t’hiol is partially oxidized during X-ray data collection. The positions of both the reduced and t’he oxidized sulphur are described. The active site geometry suggests that a change in the conformation of the native thiol occurs upon diffusion of substrate into the active site cleft of DLH. This enables nucleophilic attack by the y-sulphur to occur on the cyclic ester substrate through a ring-opening reaction.

1. Introduction Dienelactone hydrolase (DLHf) is an enzyme of the /I-ketoadipate pathway, a catabolic pathway found in bacteria and fungi (Schmidt & Knackmuss, 1980: Ornston & Yeh, 1982; Schliimann, 1988). DLH catalyses the hydrolysis of the cyclic ester, dienelactone ((Z)-(5.oxo-2(H)-furanylidene))acetic acid) through a ring cleavage reaction to yield maleylacetate (4-oxo-2-hexene dioic acid) (Fig. 1). The /I-ketoadipate pathway serves a vital role in the biodegradation of toxic aromatic compounds introduced into the environment both as natural products and as industrial effluent (Dickson, 1980; Ghosal et al.. 1985; Clarke & Slater, 1986; Miller & Lingens, 1986; Rojo et al., 1987). Final products of

t Present address: Ijepartment of Molecular Biophysics and Biochemistry, Yale University, Pli’ew Haven, (1T 0651 1, 1’.8.A. 1 Abbreviations used: UI,H. dienelactone hydrolasr (EC 3.1.1.45): r.m.s., root-mean-square; m.i.r.. multiple isornorphous rrplaremwt~.

the pathway are acetyl CoA and succinyl CoA. which are metabolites of the tricarboxylic acid cycle. DLH from Pseudomonas sp. B73 is a monomeric protein containing 236 amino acid residues and having a molecular weight of 25,500. Inactivation studies with the active site reagents chloroacetic acid, dibromoacetone, diethyl pyrocarbonate. N,-p-tosyl-L-lysine chloromethyl ketone (Tos-LysCI\;,HCl), N-tosyl-I,-phenylalanine chloromethyl ketone (Tos-PheCH,CI), dibromoacetone and diethylpyrocarbonate have implicated a cysteine and a histidine residue with the active site of DLH (D. Pathak & D. Ollis, unpublished results). Additionally it has been observed that’ DLH has slow esterase activity on the chromophoric ester substrate p-nitrophenyl acetate and t’he synthetic peptide substrate trans-cinnamoyl imidazole (D. Pathak & D. Ollis, unpublished results). These findings have prompted us to believe that the mechanism of DLH may be akin t’o that of the thiol proteases. An X-ray crystallographic study of DLH has recently resulted in the determination of the

498

and L). Ollis

D. Pathak

Dienelactone

Dienelacrone hydrolase

Maleylacelate

_o,!xJy

*-

Figure 1. substrate and produrt of dienelactonr hydrolasr. Uoth compounds are intermediates in thr chlorobenzoatr degradat,ive arm of t.he P-ketoadipate pathway. three-dimensional structure of DLH at a nominal resolution of 2.8 A (1 w=O.l nm) (Pathak et al.,

act’ivitg of I)LH. it was decided to refine the st ru(‘ture of the enzyme at higher resolution. The motivation for t’his work was to visualize the atjomic d&ail of DLH with a higher degree of accuracy. It. was hoped t,hat the enhanced resolution of the structurr would permit a clearer understanding of t~hr atjomi(a interactions that lead t,o the formation of a unique t,hree-dimensiona. st)ructure wit)hin the more general framework of a,n cc/,4 protein. Moreover. it, was hoped t,hat, the refined st,ructure would permit :i comparison of the active site structure of DT,H with that of the rrfined structures of rrprexentativt~ rnembers of t’he serine and thiol proteases. thrreb? yielding further insight, intJo t,he c~orrrlation of ac+ivt s&e geometry wit’h catalytic mechanism for DI,H and these prot)eases. of the In this paper M’P present the refinrrnent structure of DLH from Pseudomonas ,sp. Rlr’I itt 1.8 ,t resolut’ion. describe the detailed strucBt,ure of the molrculc. and briefly discuss the mechanistic. inferences that we draw from the strucatnrr.

2. Methods

1988).

DLH is an E/P protein composed of eight st,rands of b-pleated sheet and seven helical segment,s. An examination of the structure of DLH showed that cysteine 123 resides in a cleft in the molecule. nestled against the N-terminal end of an a-helix. In close proximity to this cysteine residue were found histidine 202 and aspartate 171, in an arrangement reminiscent’ of the catalytic triad of the serine and oysteine proteases. This finding lent further credence to our assumption of the mechanism of DLH. Since this structural study, two mutants of DLH in which cysteine 123 has been converted to a serine (C123S DLH) and an alanine (C123A DLH) have been engineered (I). Pathak et al., unpublished results). Both of these mutants show drastically reduced activity on the natural substrate dienela&one. The mutant proteins have been crystallized and found to be isomorphous with crystals of native DLH and their structures partially refined (D. Pathak et al., unpublished results). Armed with what appears to be unequivocal evidence for the association of cysteine 123 with

DLH

nas purified

and crystals

grow,) as (lrs(~ribrtl

(Pathak e:tnl.. 1988). The crystals had latticar const’xnt,s identical with those used in the multiple isomorphous replacement (1n.i.r.) structure determination of DLH (Pathak rt al.. 1988). The space group was E?,Z,?, and unit cell dirnensions were 48.90 x x 71.45 x x 7823 8. All crystals were stabilized in a buffer solution of’ 1.8 M-PO4 (Na+/K+) at pH 6.3.

High resolution data were c*olleet,ed from :i separatr crystals of DLH. All of the data were collected at ambient temperat,ure on a NicoM Area DetectSor (Durbin it ol.. 1986). The X-ray source was a R,igaku R17300 rotating anode X-ray generator providing (‘UK, radiation through a graphite monorhromator. Power sett,inps used were 200 mA at GOkV. Two or 3 scans in w of a]3proximately 100” each and with a step size of 025” were employed. C#J was varied with each scan and x was held constant at 45”. Table 1 surnmarizes the agreement statist,ics for data. Data processing, reduction. merging and scxaling were

Table 1 Summary

Nunber

Data set Crystal Crystal Crystal

1 2 3$

of

observations 67,630 121,496 176,655

JNumber of unique

reflections 18,929 24,946 26,926

of data collected Number

of

missing unique reflections

N”,,)t (Total)

5750 2111 3210

4.05 500 574

w”)t (1.8 A) IX.17 28.56 26.38

Statistics are cumulative, data from crystal 2 being merged with data from csrystal 1 and data from crystal 3 being merged with data from the 2 preceding data sets. w h ere I(i,h) is the observed intensity for the ith observation, and t R =I C lz(LhlU(hKl/U(h)), h i is the mean intensity of the reflection h, for all measurements of f(h). H( 1.8 L4) is the R-factor for the resolution shell between 1.9 A and 1.8 !I. 1 The resolution of this data set extended to 1.7 A with an equal number of reflections missing as observed in the resolution shell between 1.8 A and 1.7 A. The R-factor in the shell from I.8 19.to 1.7 .k was 46.030/,. The number of unique reflections to 1.8 .k was 24,988 whilr the number missing was 568.

499

Refined Structure of Dienelactone Hydrolase accomplished using the Xengen integrated software package (Nicolet Instrument Co., 1987). Intensities were corrected for Lorentz, polarization and detector deadtime effects. an updating algorithm used for background correction and local scaling was performed in shifts of w (Howard et al.. 1985). (c) Thr 24 d resolution

starting

model

The starting model for refinement was built into an electron density map. calculated at 28 A, using m.i.r. techniques (Pathak et al., 1988). The quality of this map permit’ted unambiguous placement of all but the carboxyterminal 3 C, atoms of DLH. Moreover, good side-chain density was observed for almost all residues. The activesite cysteine 123 side-chain appeared to have excessive electron density associated with it but at the resolution of the map it was uncertain whether this was a result of oxidation of the thiol, the presence of tightly bound water molecules or merely a ripple artifact from a nearby heavyatom site. ((1) Ikfinement Refinement was performed using the stereochemically restrained least-squares refinement method developed by Hendrickson & Konnert (Hendrickson & Konnert, 1980; Hendrickson. 1985). An updated version of the program package. PROTtN/I’ROLSQ, incorporating the FFTbased algorithm of Agarwal, was obtained from Barry Finzel (Agarwal, 1978; Finzel, 1987; Sheriff, 1987). The refinement was completed using the Ten Eyck-Tronrud refinement package. TNT (Tronrud et al., 1987). Geometry constraints were applied on bond distances, bond angles. nonbonded contacts, planarity of groups. chiral volumes, torsion angles, van der Waals’ distances and isot.ropir temperature factors. During all stages of the refinement. cycles of automated refinement were interspersed with manual model building (see below). Structure factor weighting was varied as the refinement progressed with the weighting initially favouring structure factors and later idealization of model stereochemistry. All reflections were used, corresponding initially to an inter-planar spacing less than 5 A and greater than t,he

high resolution limit of the data being refined against. After the R-factor had reached 1539/,, those reflections with structure factors less than 3 times the standard deviation were excluded from the refinement. Towards the end of refinement, after all thermal parameter restraints had been removed, all of the data were included in the refinement once more. This resulted in an R-factor increase of approximately 1 O/c.Finally thermal parameter restraints were replaced and the low resolution limit of the data was extended back to 10 A. On extending the resolution back to 10 A, it was noticed that difference map holes appeared on all solventaccessible oxygen atoms except those denot,ing solvent sites. The relative prominence of these holes was influenced by the level of restraint’ placed on B-parameters in the refinement; however, as long as data below 5 A in resolution were included in the refinement, a relatively high B and holes greater than 5cr (a approx. @08 eA’3. holes between -0.4 and -@5 eA- 3, persisted in F, - Fc difference maps. It was noted that holes did not appear on all oxygen atoms or on the sulphur atoms of methionine or cysteine residues. Below 5 A resolution the scatter contribut’ion of the disordered solvent in the crystal becomes significant and exclusion of solvent parameters in the refinement can lead to erroneous B-factor refinement for solvent-accessible atoms. Additionally, as discussed by Moews & Kretsinger (1975) when the average electron density of solvent and protein are approximately the same. low-order reflections have reduced intensities. Given the observation that the holes do not manifest themselves on every atom (even the strongly scattering sulphur atoms) it, is unlikely that this effect is due simply to a systematic reduction in low-order structure fact,ors. Nonetheless, it appeared likely that a correction for solvent scattering would alleviate the problem. TO verify this belief it was decided to incorporate a solvent continuum into the current model. The refinement program TNT, which allows the application of a crude but effective solvent correction to the model. was used for this purpose (Ten Eyck & Tronrud, 1987). Difference maps calculated with the resulting model were less noisy, with a (T value of 5.685 x lo-* eAe3. No significant systematic difference map density was observed. Table 2 summarizes the course of the refinement.

Table 2 Course of the rejinement of DLH stage Bond length (A)

Input 0 Bond angle (“)

lnput ~7 Torsion angle (“) Input (T Planar groups (A) Input (r Had contacts (A)

Input (T Overall Rf Besolution ranged (A) Number of reflections Number of water molesmles

I

2

3

4

5

6

0.207 0.030 14.557 0.040 37dlo9 15000 0.157 0020 0567 0500 0320 50-3.0 4479 0

0.100 0030 13.585 0040 32.533 15000 0.048 0.020 0401 0500 0270 5.Gl.9 19910 0

0065 0030 7.188 0.040 24,767 15000 0.028 0.020 0477 0500 0.220 SO-l.9 19910 0

0.025 0.030 2.884 @040 22.859 15M)o 0.02 1

@021 0.030 2.647 0040 22,715 15000 0018 0020 0563 0.100 0.162 100-l% 23835 242

0,014 0.010 3,405 (3.000) 22.H57 1.%OOO 0.013 0920 0065 0.100 0150 100-l% 23835 279

The values are r.m.s. deviation from ideal stereochemistry. t Angle distance (l-3) restraint (0.040 A) applied in PROFFT restraint (30IO) applied in TNT refinement used in stage 6.

1 R= llF,l - l~cll/l~ol.

0420 0.501 0.500 0.139 5S1.8 22687 230

refinement used in stages 1 to 5. Angle

D. Pathak and D. tIllis R

L

Figure 2. F--F” Y L man1 calculated with residues 121 to 125 omitted posityoning of these residues is evident, from this Figure.

(e) Fourier

spthems

and

graphics

Maps generated with both “combined” and “model” phases were instrumental in analysis of the model. During initial refinement’ the m.i.r. map was used to verify the current model. Lat’er, phase combination, as described by Remington et al. (1982), and as implemented in t)hr PROTETlV program package (Steigemann. 1974) was used to calculate “combined” difference maps. In the final steps. phases calculated from the current model were used in the generation of difference maps. Coefficients used in these Fourier synt’heses were (1) F,- F, map 2K0- PC and map. (4 (IF01- IE‘,OexpW,) (2lE’,l -)F,l)exp(i@,). where Q, is the phase calculated from the current model. The model was inspected in maps displayed on an Evans and Sutherland PSS60 graphics work station using t,he program FRODO (Jones. 1978). Maps were also exam ined in hardcopy. These maps were especially valuable for insp&ion of new solvent, sites added to t)he model. in which portions of the model were .‘Omit”-maps. delet,ed and the rest of the model used to calculate phases for an F,-F, Fourier synthesis (Artymiuk & Blake. 1981). were employed in examining ambiguous areas of t.he structure. (f)

Addition

oj solvent

from the model. The lac*k of ambiguity

iti

or an occupancy of less than 03. it was deleted from t.he model. It was noticed that some solvent sites had been erroneously assigned to spurious pO- PC drnsit~y arising probably from series termination errors iii the difference Fourier syntheses. (g) Diwretrly

di.sordrrrd

rrsidurs

The criteria used for modelling residues as bring discretely disordered have been discussed by7 Sheriff’ rt (cl. (1987). Positive p,-FC density near positive WO-pC density that could not be resolved as being solvent or by adjustment of the model was examined for pot)ential static disorder. Proposed disordered atoms were built into t’he density and refined for a few cycles. Disappearame of difference density and reappearance on removal of the repositioned at,oms was noted for. Two residues in l)I,H were modelled as exist’ing in 2 distinct conformers. S49 was modellrd as having its 0’ atom discrrt,ely disordered with an occupancy of 6.5 for tach side-chain conformer. The active site cyst,eine. cysteine 123. was modelled as disordered conformers. 011~’ existing in 2 discretely reduced and the other oxidized (see below). When TNT was used for refinement the disc.retel? disordered residues wercl built as special groups and thrzir stereochemistries defined in t,hr standard gromet,ry (sards

to thu model

Solvent molecules were introduced in the modrl once the K-fact.or had dropped below 2000. All the solvent, sites identified were modelled as water molecules. Solvent sites were locat,ed both during the process of model inspection and with the aid of a peak picking program. A program that located all existing atoms. including symmetry mates. in t,he vicinity of any atom in the protein was helpful in ensuring that each solvent molecule was located in a structurally and chemically reasonable environment (within 3.5 A of potential hydrogen bond donors or acceptors). Solvent molecules were added to the model with a H-factor of 20.0 A2 and an occupancy of 1.6. Solvent occupancies were considered variable and allowed to refine simultaneously with their R-factors. Application of of o~cupamy shifts were alternated with application thermal parameter shifts. If the occupancy of a wat.er molecule refined to a value between I.0 and 0.9 after several C*J-&s. it was fixed at I.0 and only it,s K-facto1 refined in subsequent, cycles. Occupancy and N-factors of water molecules were followed through the refinement and solvent sit,es were inspected against 2F,- 14’,and omitmaps. If t,he modelled site did not, lie in prominent densit) and, addit,ionally. refined to a K-value greater t,han 70 A”

At t,hr end of retinrmc~nt. hydrogen atoms Mtrc’ atldrtl to the tinal model. using the program H(:EN (writt,en 11). S.E.V. Phillips. from the (‘(‘P4 program suit(>). Hydrogen a,toms for the hydroxyls of serint.. t hrrorrim and t,yrosine have torsional freedom and NY’ trot Arthur ately positioned. Additionally. the library used did not include hydrogen at,om definitions for ~*,vsteinr~ or fat, either of the histidine side-czhain nitrogen atomb.

.-Zsrefinement of DLH progressed. the ~‘XCYWtlonsit~y for the side-chain of C’ysl23. took on a dist~inctly tripart,it,r appearance. Main-rham electron density for (‘ys I23 ant1 its adjacent, residues w-as unambiguous. as tin 1)~ seen from Fig. 2. This result,ed in ambiguity only in thrs positioning of the t,hiol. Tn omit-maps where the T-sulphur atom was exc~lutled. heart-shaped densit,g appeared beloa bhe B-carbon atom. This appeared t,o be the result of the overlap of 2 prominent’ peaks with one of lesser promi nence. It was stereoc~hemically possible to posit,ion th
501

Rejined Structure of Dienelactone Hydrolase L

R

Figure 3. Stereo diagram of the cardioid densit’y for Cys123, obtained by calculating an FO- pc map with Cys123 omitted from the structural model. Visible is the nub of densit’y at the apex of the cardioid.

reside within any one of the 3 peaks. It was found, however. that no matter which of these 3 positions was chosen. a large F,-- F, hole resulted as the position of the y-sulfur. Fig. 3 shows omit-map density obtained by removing Cysl23 entirely from the model. At the apex of the heartshaped feature can be seen a smaller ball of electron density. The most reasonable fit of model to density that maintained permissible stereochemistry required that the thiol br built as occurring as 2 discretely disordered conformers, one free and the other oxidized. The free thiol was built so as to occupy the left lobe of the density, while the oxidized sulphur was modelled to lie between the right lobe and the vent’ricular apex of the cardioid density. Two oxygen atoms were modelled into the density, O-l occupying the apical density and O-2, the right, lobed portion. ,4 water molecule was positioned in the electron dense nub t,hat extends out, of the apex of the cardioid. This site refined at earlier stages of the refinement to an occupancy of approximately 0.4. The distance of this water molecule, Wat,234, from oxygen atom O-l was found to be shorter t,han commonly observed hydrogen bonding distances. In addition, the distance of Wat234 from the position of the oxidized thiol was also too small. These distances were 2.4 a and 2.9 A, respectively (see Srinivasan & Chacko. 1967). Comparison with the partially refined structures of (‘123s DLHase and C123A DLHase show that a water molecule is found in both the latter structures in the same location (see Fig. 16). These observations suggested that DLH is partially oxidized. Wat234 exists in the same fraction of crystalline DLH as the unoxidized native thiol of Cys123. Oxidation of the thiol results in a conformational perturbation of the y-sulphur so t,hat it is found in the new location represented bp SGOX in Fig. 3. The observed electron density is consist~ent with an oxidized form of cysteine carrying 2 oxygen atoms on its y-sulphur atom. most probably as the sulfinic acid moiety. The occupan,cT for the thiol sulphur was set to Q4 and that for the oxldlzed sulphur, to @6. Occupancies for O-1 and O-2 were also set to 0.6. These values were arrived at both from chonsiciering the refined occupancv value of Wat234 and by calculating difference maps with various values for thr oclcupancy for these atoms. Refinrment of (‘~~123 as having a discretely disordered thiol resulted in featureless FO-FC difference maps. During the refinement using PROFFT, the 2 oxygen atoms, O-I and O-2, were allowed to refine without any geometrical restraint being imposed on them. During refinement with TNT. residue 123 was built into the

standard geometry table as a special group. t’hr geometry of the sulphinic acid moiety of which was constrained to sp’ geometry. Difference maps indicated that the fit of model to electron density thus obt’ained, was good.

3. Refinement Results (a) The initial

and refined models of DLH

Refinement resulted in an overall improvement in the model structure. The r.m.s. deviation from ideal stereochemistry was improved considerably with a resulting overall R-factor of 15 y. (see Table 2). The greatest difference between the initial and final refined co-ordinates resulted from t’he identification of the active site cysteine as being partially oxidized in the crystal. Refinemen.t of the structure also permitted a more precise demarkation of’ secondary structural elements in DLH (Fig. 4). At the end of refinement 279 bound water molecules were identified; 42 refined to unit occupancy, while 19 refined to occupancy values between 0.9 and 1.0.

I

APLQSIUP

Figure 4. Single-letter coded amino acid sequence of DLH showing the position of p-strands 1 t,o 8 and helices A to G. Unbroken arrows and rectangles represent strands Broken lines represent the and helices, respectively. demarkation of secondary structure prior t,o refinement of the structure. Every 20th residue is marked with a star.

D. Pathale and D. Ollis

502

data are included in t,he Luzzati error analysis, the assumption that all disagreement between F, and I’, values are due to co-ordinate errors. does not, hold. The reason for the low resolution departure is because only a linear scale factor was used to minimize the difference in F0 and FC. The function used by TNT to compensate for the lack of a solvent continuum in the F, values involves an exponent,ial term that includes a solvent R-value. Furthermore. t’he correction for bulk solvent, scattering is not’ very sophisticated. From the Luzzati plot we estimate a mean co-ordinate error of 0.15 A for t’he refined model of DLH. (d) C‘v?formational I 0.0

0.2

I

I

I

5. Plot

I

0.4 Inverse

Figure

I

of R-value

1

I

I

0.6

1

I

I

I.0

0.6

resolution

against

inverse

resolution

A-‘). (+) R-factor; (0) R-factor (bin scaled); ( x ) R-factor (F > 3~7); ( + ) R-factor (F > 3a, bin scaled). Also plotted (broken lines) are theoretical error lines (Luzzati, 1952): (0) 0.10 A; (0) @2OA; (0) 0.15 A. (2sinO/1,

(b) Quality

of the electron

density

map

The absence of significant features in the final difference map calculated for the refined structure of DLH, attests to the quality of the electron density distribution and the final molecular model. An F,-F, difference Fourier synthesis resulted in a map with a standard deviation of5.685 x 10s2 eAM3, and a maximum and minimum of 2.786 x lo- ’ eAP3 and -2.827 x 10-i eA3, respectively. (c) Bccuracy

of the model

Figure 5 depicts a Luzzati plot for the refined structure of DLH. The error lines were calculated using Table 1 and equation (51) of Luzzati (Luzzati, 1952; Derewenda et al., 1982). The two curves for refined data were obtained by sort,ing reflections in the range 10 A to 1.8 A into 20 shells containing approximately an equal number of reflections and calculating individual linear scale factors for each shell so as to obtain a minimum R-value. Curves are drawn representing all the data used in refinement , and data for which Fobs was less than 30. Both curves follow the Luzzati error line corresponding to a mean error in atomic positions of 015 A, except for bins representing higher resolution data and the bin between 5.0 A and 10.0 A. Here a clear departure from the error line is evident especially for the curve that represents the complete data. Since the best R-values correspond to data with the largest intensities, and the highest resolution data correspond to the lowest intensities, the increased R-factors seen at the high resolution limit of the data used in refinement are a result of the inclusion of poorly determined data. When these

arqles

Main-chain torsion angles (4; II/) are represented on a Ramachandran plot, shown superimposed on a steric contour map for an L-alanyl polypeptide chain, in Figure 6. The u/B nature of DLH is apparent from the two clusters of points in the diagram. The greatest density of points in the dist,ribution is seen around (-60”, -4O”), reflectring the smaller permissible region of Ramachandran conformational space available to r-helices than t,o p-sheet. Ser203 and Tyr145 are the only uon-glycine residues in the left-handed helical region of t,he Ramachandran map and both are associated with turns in the polypeptide chain and will be discussed later. Only two non-glycine residues lie well outside the allowed regions. These are Met1 and Cys123. The N-terminal Met1 lies in poor density and has higher than average atomic R-factors, indicat,ing disorder and perhaps, consequently, an erroneous 4 value. For Cys123, both 4 and 9 torsion angles (52” and - 105”, respectively) have sterically unfavourable

Figure regions (1965). stars.

6. Ramachandran plot for DLH. “Allowed” are according to Ramakrishnan & Ramachandran All residues, other than glycine. are denoted bJ

503

Re$ned Structure of Dienelactone Hydrolase values. This is the active site cysteine, and it lies at the tight, junction between /?-strand 5 and a-helix D. The sharp nature of this turn and the interactions that stabilize it result in the unusual main-chain conformation for Cysl23. This turn is diagrammed in Figure 2 and discussed further in the text. Similar torsion angles are found for Ser123 and Ala123 in the partially refined structures of C123S DLH and C123A DLH respectively. These angles are (59”, - 114”) for Ser123 and (70”, - 116”) for Ala123. This confirms that the unusual conformation angles are specific to this particular polypeptide turn rather than a result of a local perturbation of the polypeptide backbone due to the oxidation of the active-site thiol.

4. Description of the Structure of DLH The overall structure of DLH remains as described earlier (Pathak et al., 1988). DLH is an IX//?protein consisting of an eight stranded p-pleated sheet with seven parallel strands and one antiparallel strand (strand 2). This central sheet structure is surrounded by seven a-helices. All but the Cterminal three of its 236 residues have been refined against data between 1.8 A and 10 A. Also incorporated into the structure are 279 molecules of ordered solvent. all identified as water. Stereoscopic a-carbon views of DLH are shown in Figure 7. Visible is the active site cleft, thrust into the centre of which lies a-helix D. Ensconced at the

base of this helix is the active site cysteine 123. Also clear is the a/b nature of DLH with its characteristic P-pleated sheet fringed with helices. (a) Secondary structure The pattern of hydrogen bonding, taken together with dihedral angles, was used to define secondary structural elements in DLH. Main-chain hydrogen bonds were considered to exist if the C=O.. N distance was less than 3.2 A and the C=O H and N-H . . 0 distances were less than or equal to 2.5 A. The minimum associated angles permitted were 90”. All distances and angles were scrutinized for possible bifurcated hydrogen bonds. In cases of doubt, the distances and calculated with angles co-ordinates that included main-chain hydrogen atoms were considered more reliable than the corresponding C=O . . N distances and angles. Less stringent guidelines were used in defining side-chain and water-mediated hydrogen bonds (discussed later). Table 3 lists the average parameters for main-chain hydrogen bonds within secondary structural elements while individual values for each hydrogen bond are listed elsewhere (Pat>hak, 1989). (i) He&es DLH contains seven helices, all but one of which lie on the periphery of the protein. Helices were identified by the main-chain hydrogen bonding pattern and dihedral angles of the residues they

Table 3 Mean hydrogen bond parameters N..

O(A)

H...O(A)

in helicex and sheet C=6..

N(“)

C=8

.H(“)

N-fi.

O(“)

A. H&es (A) Residues 42-54 2.95(0.10)

2.02(0.13)

155(4)

148(5)

157(11)

302(0~10)

2.07(010)

158(8)

152(S)

158(2)

2.97(0.12) Residues 124-135 3.01(0.15) Residue 126 included 3~10(022) (E) Residues 148-159 2.94(010) (F) Residues 17.5-187 2.97(014) (G) Residues 213-228 2.95(@11)

2.03(0.11)

152(12)

148(6)

155(7)

2.09(@16)

155(9)

148(11)

153(12)

2.22(029)

147(21)

139(24)

149(15)

1.99(@09)

125(4)

119(5)

159(9)

2.03(0.16)

149(20)

149(7)

158(12)

2~00(0~11)

156(6)

150(7)

159(Q)

2.03(013)

155(11)

149(8)

157(10)

(1%) Residues 77-87 (C:) Residues W-108 (D)

Overall mean 2.97(@13) excluding helix 15 and water molecules

B. fi-s/wet Anti-parallel

strands 1, 2, 3 and 4: Residues X-10. 13-21, 29-34 and 57-62. 2%3(0.14) 1%6(@14) 152(11) 149( 10) Parallel strands 4. 5, 6, 7, 8; Residues 57-62, lltS-122, 13%145, 163-167 and 191-197. 1.97(013) 2.91(@13) 161(6) 155(6)

157(7)

Overall mean

160(7)

2%8(0.13)

1.93(0.14)

157(9)

153(8)

164(4)

II. Pathak and 11. Ollis

Figure 7. (a) Stereoscopic plot of a-czarbon at,om positions for DLH. Hrlices and st,rands are labrllrd. \.isihlr is thcb active sitr cleft with helix D jutt,ina out within it. (b) Ribbon schematic> of DLH prrpare:d using the program R!l KHOK written by Priestle (1988). comprise. From this examination it) was found that six of the seven helices display typical cc-helical geomrt,ry while one, helix E, adopts an entirely 3,, conformation. Table 4 lists the values of dihedral angles for secondary structural elements in DI,H. The average dihedral angles for the m-helical residues in DI,H are -63” (6) and -41” (6) for C#Jand I//. respectively. These values are in good agreement with values obtained for other well-refined structures (e.g. act’inidin, - 63”. - 40” (Baker, 1980); myohemerytherin. -65”) -42” (Sheriff et al., 1987); crambin, -63”, -440” (Hendrickson & Teeter, 1981)) though they differ from values for an ideal helix ( -57”: -47”) (Arnott & Dover, 1967). The mean hydrogen bond lengt,h for cr-helices. defined by the distance (‘=O .x, is 2.97 a (013) and the associated angle, (y=i...N, 155” (11). The ends of helices were demarcated by the first and last residues participating in hydrogen bonds t,hat follow t,he helical pattern. In several instances. at) the ends of helices, the hydrogen bonding pattern

‘ helix. These residues havca follows that for a Jlo been included in the a-helices. but, not in t,hr c~alcalation of mean C$ and $ angtes. The ext.remities of helices are often distorted and frequently (sonlain bifurcated hydrogen bonds. Tn t>hesr cask t,hch observed hydrogen bonding pattern may c~onforrn to both 3,, and a-helical motifs. Helices K and (’ are the onlv helices in l)l,H without an int)rrvening stretch ;f fl-shecbf Helix t< turns into helix (’ ,r;in a connecting 3,, turn. Helix 13 contains t,hree bifurcat,ed hydrogen bonds and helix C contains one. At’ the N-terminal end of helix I) is found t hch active site cysteinr 123. C:vs123 has not been included in the helix due t,o its abnormal dihedral angles, (ZY, - 105”) a,nd it appears to btt rcsiducb i+ 1 of a y-turn. Figure X(a) depicts residues present in helix I), while (b) shows 1he main-chain h.ydrogetr bonding pattern within the helix. The initial turn in helix T) has both a-helical a,ntl 310 hydrogen bonding, with Leu 124 0 f’orming a 3.6,, hydrogen bond with I,culBX N and a 3,0

Refined Structure of Dienelactone Hydrolase

505

Figure 8. (a) Stereo plot of helix D. Main-chain bonds are filled while side-chain bonds are open. (b) Stereo plot of helix D showing the pattern of main-chain hydrogen bonding within it and with neighbouring water molecules. All hydrogen bonds including possible bifurcated bonds, are included. Hydrogen bonds are depicted by dotted lines.

Table 4 Summary of dihedral angles in secondary structure of DLH Structural element

Mean d, 0

Mean + (7

Helix Helix Helix Helix Helix Helix Helix

-62(5) -62(5) -63(4) -65(5) -65(13) - 64(5) - 64(5)

-41(7) --41(Z) - 40(6) -37(6) -24(14) -41(4) -41(4)

Gln54 Leul7 Asp92 Lys135 Gly148. Gln152 and Va1159 Pro175, Phe186 and Gly187 Va1213. Phe227 and Leu228

-63(6)

-41(6)

Residues above and helix E excluded

136(15)

Gly13, Gly17, Asp62, Aspl39. Tyr145 and Leul91

A B C D E F G

Ovwall P-Sheet

mean:

- 117(20)

Residues excluded in calculating averages

506

D. Pathak arLd D. Ollis

hydrogen bond with Ala127 N. The hydrogen bond parameters are 3.13 d. 157” and 3.16 A, 119”, respect,ively. J,eu124 N also participates in a bifurcated hydrogen bond and is the donor to both Wat236 and Tyr122 0, t,he first residue of t’he y-turn that leads strand 5 into helix II. The next turn, similarly, cont,ains a bifurcated hydrogen bond. A hydrogen bond between Gly125 E and Tyr122 0 is complement,ed by a hydrogen bond between the glycine amide nitrogen and the carbon)71 oxygen of Gln35. The C=O S distance and the associated angle are 2.88 a and 115” for the former, and 2.82 A and 156” for the latter. respectively. A dubious bifurcated hydrogen hod is seen between the carbonyl oxygen of My126 and t’he amide nitrogen atoms of Ala129 and Phe130. Hydrogen bonding with the forrner donor is of 3,,. while with t,he latter. of 3.6,, r-helical character. Both distances are very long, 3~37A for the first and 343,4 for the second. The C=O ?1’a,ngles are 96” Other bifurcations arc’ and 141”, respectively. evident in helix I). The last a-helical hydrogen bond is formed by the main-chain oxygen of I,cu131 and main-chain nitrogen of LysJ35. Helix 1) turns into st’rand 6 through a 3,, bend that links Ala133 0 a,nd Glgl36 S. Six wat>er molecules contact helix I). Thescb contacts are formed by hydrogen bonds with t,hc main-cha,in oxygen atoms of Ala127. LeuJ 3 I , Ala,J33, Ala134 and LysJ35. and the main-chain nitrogen of Leu124. The hydrogen bond between Ala127 0 and Wat483 is tridentate, while that between Leu131 0 and Wat36X is bifurcated, as is the hydrogen bond between Leu 124 N and Wat236. Ala127 0 participates weakly in bot,h a-helical and 31,,-helical hydrogen bonds with l,eu131 N and Phe130 I$, respectively. LVater molecules Wat376. Wat346 and Wat275 hydrogen bond wit,h Ala1 33 0. Ala134 0 and l,ysJ35 0. Helix I) is composed entirely of non-polar residues. except for llys135, the tinal residue in the helix. and its environment within the active sitcl cleft of J>LH is consistent with its hydrophobic nat’ure. The positively charged Lysl35, situated at t,he carboxy-terminal of helix II, will tend to stablizr the virtual negative charge associated with t’hc helix dipole. Hydrophobic helices are extremely rare in globular proteins. being seen mainly in membrane insert,ed regions of membrane bound proteins. Blundell et al. (1983). in investigating solventinduced dist,ortions in a-helices, were unable to find examples of refined structures of complet,ely hydrophobic helices. However, using the a,verage c= 0. S angle and related hydrogen bond distance obtained from the hydrophobic “regions” of amphipathic helices, these workers generated a hypethetical hydrophobic a-helix. They found an unusually long average C=O . N bond distance of 3.04 19. The associated average angle they observed was 153.2”. From these results they concluded that the “hydrophobic helix” would be unstable and

therefore not seen in globular prot,eins. Sincatsthen. the structures of membrane-associated proteins have confirmed the existence of hydrophobic hclices and helix 1) of JILH provides a refined structure for a hydrophobic helix within a non-membranr~-associated protein. It should. of course. bt‘ remembered that helix D is not’ in an entirely hydrophobic* environment since it lies in a solvent,-ac~c,essible calef’t of the protein. However. the hydrophobic> nature of the lining of this cleft (see section (d), Mow) mtl the hydrophobic nature of the residues withit helix D have the effect of ordering the wat,er molecules within t’his region. These water molecules form an extended ordered network (see Figs IX and 22) but only six molecules actually contjact helix 1) (see above). Helix I) also has the lowest ac~cessiblr surface area per residue of all t,he helic*es (see Table 8). The average (‘=O N distancir in hc,lis I>. 11’tjhe long hydrogen bond between (ilyl26 0 and Phel30 N is included. is 310 A. with an associa,ted angle of 137”. If, howclver. this hydrogfxn bond is ignored. irnplying a missing hydrogen bond within the helix (no other atoms lie within hydrogen bonding range of (ilyl26 0). t,hen t,hr average C’=O . N distance and associated angle arr 301 x and 155’. resp&ively (see Table 4). Thrsc values appear to be consistent, with the model presented I)> Blundell d al. (1983) but t)he associated anglr of’ 141” is far smaller. Tf one were to c~orrsider 1htksct average hydrogen bonding parameters as the sole determinant of helix stal%lit)y. helix 1) would htl prediczted to br unstable. This does not. howc~vvr appear to be so. An indicat)ion of this is t,hat t.hch rnean R-fact*ors for both main-chain and side-chailr atoms of the helix are noi high. These values arc’ 15.9 8’ and 20.0 A*. respectively. It should t)(a pointed out t,hat Lys135 has the highest atomic, H-factors and exclusion of this residur results in &factors ot mean main-chain and side-chain 15.2 x2 and 16.9 A*. respectively (see Table 8). The unfavourable hydrogen bonding parametr~rs for helix I) arts c*ompensated for J))- bifurcated hydrogen bonds formed by main-chain at,oms within it. Additional stabilization is surely J)rovidrd by the hydrophobic na.ture of its amino acid side~chains and thr hydrophobic lining of the ac+ivf*-sitcb c+lrft. It is likely. therefore, that, as rnorl’ rt+inetl strucf ures of hydrophobic helices bec~ornc~availablc~. it will t)(h found t,hat, just as ionic interactions amongst thy side-chains of hydrophilic* httlicsrs arc’ important stabilizing forces iu addition to main -(-hain hytlrogen bonding (Sundaralingam pt al., 1987. Matqust~t~ B Raldwin, 1987). SO are side-cbhain interac‘t ions. both intra-helical and with the onvironmc~lrt’. import,ant st,ahilizing fact)ors for hydrophobic~ h~~licr~s. Helix IC (Fig. 9) is the only isolaM 3,,-Mix occsurring in DJ,H and it extends from (:l,v148 tjo Glul59. The first helical hydrogen boncl links t hrk carbonyl ox,vgen of (:lyl48 with the amide nitrogen of Lysl51. and is followed by another 3,,) iurn linked J))- Leu149 0 and Clnlh2 S. ?r pc~ssil~l~~ hydrogen bond bet.wcrn the carhonyl oxygtbn of

507

Refined Structure of Dienelactone Hydrolase n

L

cu1i5a PRO157

PRO&

0

Q

UAT250

fAT250

‘. VALISB ‘.I\

s

‘.

..

‘. ‘I\

5

VALl56

Figure 9. Stereo plot of the main-chain atoms of helix E showing ink-a-helical made with neighbouring water molecules.

Glu150 and the amide nitrogen of Leu153 is replaced by a hydrogen bond to Wat350, which in turn hydrogen bonds with Leu153 N. This distorts the 3,,-helix so as to prevent hydrogen bonding between Lyslril 0 and Asn154 N, but allows the orientation of Gln152 such that two further 3,0-helical turns are formed by its hydrogen bonding through its carbonyl oxygen to Lys155 N and Leu153 0 hydrogen bonding with Va1156 N. Asn154 0 hydrogen bonds with Wat250 but does not complete a link with Pro157 N. The last two hydrogen bonds in helix E are extant and connect 1~~~15.5 0 and Va1156 0 to Glu158 N and Va1159 N, respectively. Wat350 is similar to the inserted water molecules that Sundaralingam & Sekharudu (1989) find to result in directional changes in a-hehces. This water molecule, by inserting into the 3,,-helix, accent’uates its overall curvature by making it kinked. This allows helix E to reverse the course of the polypeptide chain. Helix E bends around the hydrophobic core of the protein and is reminiscent in this respect to the distortions found in amphipathic helices by Blundell et al. (1983), (see above). The average C=O . . N hydrogen bond length for the four 3,c-helical turns in helix E (excluding the turn stabilized by Wat350) is 2.94 A (610) with a

hydrogen bonding :md hydrogen bonds

mean C=O . N angle of 125” (4). Six hydrogen bonds were used in this calculation (Pathak, 1989). As observed for 3r, and a-helices by Baker & Hubbard (1984), the hydrogen bonding parameter that differs significantly between helix E and the cc-helices in DLH is the angle C=G H. The mean C=O . H angle observed for 3r0-helices is 114” (10) and for a-helices is 147” (7). For helix E. this value is 119” (5), while for the a-helices in DLH, the corresponding value is 149” (8). Interestingly, however, unlike the observation by Baker & Hubbard of long C=O.. H distances for 3,,-helices, the mean C=O . . . H distance for helix E (I.99 A (909)) is smaller than the overall average for cr-helices in DLH: 2.03 A (0.13). The values found by Baker & Hubbard are 2.17 A (0.16) and 2.05 A (0.15) for 3,, and ol-helices, respectively. Furthermore, while Baker & Hubbard found that the angle subtended at the hydrogen atom, N-H.. 0, is only slightly less linear for 3,c-helices, as compared to a-helices (153” (lO)_and 157” (9), respectively), in DLH the mean N-H. . 0 angle is slightly greater for helix E: than for the a-helices (159” (9) and 157” (lo), respectively). Thus, it appears that the hydrogen bonding geometry for helix E is not remarkably unfavourable, and this is likely the reason for its occurrence in DLH.

508

D. Pathak

and D. Ollis

L STR-3

STR-4

STR-2

82

62

(bl

Figure 10. (a) View of the central p-pleated and main-chain

sheet of DLH.

Only

main-chain

hydrogen bonds are shown. (b) Stereo view of the antiparallel

Mean r$ and $ angles are -65” (13) and -24” (14); not including Gly148. Gln152 and Vall59. a,nd lie in the 3,0-helical region of the Ramachandran map. These values show a substantial difference from those for an ideal 3,0-helix -Sri”), only in 4 (Ramachandran & (-49”. Sasisekharan, 1968). This value of r$ places helix E: more squarely in an allowed region of the Ramachandran map, implying less steric strain on the residues within the helix. Helix F contains a bifurcated hydrogen bond at its amino end. Pro175 forms a 3%r3 a-helical hydrogen bond through its carbonyl oxygen atom with the amide nitrogen of Arg179. The bond distance is 3.14 A. while the associated angle is 160”. The shorter distance, however, occurs through a 31a-helical turn that results in a hydrogen bond between Pro175 0 and Ser178 N. The distance of 2.88 A is associated with a less favourable angle of 139”. Similar, possibly bifurcated, hydrogen bonds are found in all the helices except helices R and E.

(ii)

at,oms are displayed.

Strands

are lahrlled

domain of the b-sheet of IILH.

/3-Sheet atructurc

DLH contains an eight-stranded central b-sheet. that comprises approximately 21”/;, of the protein’s residues. Two views of the sheet are shown in Figure 10. The characteristic twist associat,ed wit,h

1

2

4

3

5678

Figure 11. Diagram illustrating the topolopic~;tl arrangement of the strands of the p-sheet of I)T,H. (Year arrows represent strands and their dirrct,ion.

509

Refined Structure of Dienelactone Hydrolase

Figure 12. St)ereo diagram side-chain bonds are open.

of the single isolated stretch of b-ribbon

P-sheet, can be clearly seen in Figure 10(a). The P-sheet is predominantly parallel, with a single strand, strand 2, contributing to a small N-terminal stretch of antiparallel P-sheet. Reference to Figure 11, which outlines the topological connectivit,y of the sheet, shows that strands 2 and 3 are not adjacent, but are separated by the interposition of strand 4 between them. Thus, rather than the single antiparallel strand 2 giving rise to a P-meander, it results in a three-stranded nonadjacent antiparallel sheet separated from the parallel sheet domain at strand 4. Strand 4 exhibits different hydrogen bonding patterns OQ either edge as typified by the bending of the C=O . N angle either towards or away from C”. This may be seen in Figure 10(b) (see also Baker & Hubbard, 1984). It may be noted that, with nine residues, strand 2 is the longest strand in the P-sheet structure. It is through hydrogen bonding with the N-terminal half of strand 2 that strand 1 is contiguous with the central /&sheet of DLH. Strand 1 is short and contains just three residues. Ile8 and SerlO both make two hydrogen bonds with strand 1 (see Fig. 10(b)), while Gln9 is bereft of any hydrogen bonding with strand 2. However, this residue participates in a hydrogen bond through its NE2 nitrogen atom with Wat389 which, in turn, hydrogen bonds with the carbonyl oxygen of Gly13, thus connecting the two strands. The carbonyl oxygen of Gln89 hydrogen bonds with a water molecule, Wat423. Strand 2 bulges at two points along its length. The first bulge, which may be classified as a Gl-type P-bulge (Richardson et al., 1978), is formed by residues Gly13, His14 and the opposing SerlO. The glycine at position 1 of this bulge is concomitantly the glycine at position 4 of a type I reverse turn. It should be noted that most Gl-type bulges share a glycine at position 3 with type II reverse turns (Richardson et al., 1978). The second bulge is a wide-type bulge and allows strand 2 to transfer its hydrogen bonding from

in DLH.

Main-chain

bonds are filled while

strand 1 to strand 4. This bulge is associated with a break in the main-chain hydrogen bonding pattern between strand 2 and strand 4 so that Pro61 and Ala18 do not contribute hydrogen bonds to the sheet structure. Ala18 hydrogen bonds through its carbonyl oxygen with the main-chain nitrogen of Thr3. The carbonyl oxygen of Pro61 is involved in a hydrogen bond with Leu63 N. Va120 does not’ contribute to the P-sheet hydrogen bonding since its main-chain hydrogen bond participants are both on the distal edge of the sheet. Its carbonyl oxygen bonds with a water molecule, Wat449, while its main-chain nitrogen bonds with Thr3 Oyl. t’hus stabilizing the secondary structure. Table 4 summarizes the b-sheet dihedral angles and Table 3 summarizes the average hydrogen bond parameters. The mean C=O N hydrogen bond length is 2.88 A (0.13), while the mean C=c^ . . h’ angle is 157” (9). The mean hydrogen bond length for just strands 1, 2 and 4 (antiparallel sheet) is 2.83 w (0.14) with a mean C=O . . . N angle of 152” (ll), while for the parallel sheet these values are 2.91 A (0.13) and 161” (6), respectively. A few long hydrogen bonds have been omitted from this calculation because they are found at the ends of strands but are considered part of the sheet because their geometry is otherwise consistent, with their inclusion. As has been observed for other structures, the hydrogen bonds in the sheet are on average more linear than those in the a-helices and are shorter by approximately 0.1 a (see Tables 4 and 5 of Baker & Hubbard, 1984). Besides the /I-strand structure found in the sheet domain there also exists a short solitary stretch of /?-ribbon extending between Pro69 and Leu73 (Fig. 12). The mean 4 and I/J values for this piece of strand (excluding Gly70 and Pro75) are -75” (48) and 105” (55), respectively. This structure is stabilized by several hydrogen bonds, mostly between it and neighbouring secondary structural elements. Three hydrogen bonds are made with water molecules, two of these being bifurcated bonds from the

510

D. Pathak and D. Ollis

(b 1 Figure 13. (a) Stereo view corresponding to the view shown in Fig. 2 of’ residues I21 to 125 of Cl238 DLH. ElectrolI density was calculated with an F,-F, synthesis. the residues shown being omitted from the model. (b) View corresponding to that in (a). of C123A DLH.

main-chain Wat381.

oxygen

and nitrogen

atoms of Ala72

to

(iii) Turns The geometry of hydrogen-bonded reverse turns in DLH is summarized in Table 5 (Chou & Fasman, 1977; Smith & Pease, 1980; Milner-White & Poet. 1987). Of the 11 reverse p-turns identified on the basis of 4-1 hydrogen bonding, six are type I turns. Turn 1 (SerlO-Gly13) contains glycine 13 which participates in the B-bulge in strand 2. The mean C=O . N hydrogen bond length for these turns is 2.99 A (0.15) and the mean C=O.. N angle is 123” (6). There are three clear type III (or 3,,) reverse turns in DLH (other than those in helix E) and the mean C=O . N bond length and associated angle for these is 2-82 A and 129”, respectively. A single hydrogen-bonded type TT turn leads strand 4 into the solitary stretch of p-ribbon. Strand 5 leads into helix D via a y-turn (Nemethy & Printz, 1972; Matthews, 1972), resulting in an extremely sharp reversal of the polypeptide chain direction. The main-chain oxygen of Tyr122 hydrogen bonds with the amide nitrogen of Leu124. The observed dihedral angles for Cys123 are (52”. - 105”). which place it in a disallowed region of the Ramachandran map (see Fig. 6). These angles are

indicative of o-amino acid stereoisomerism. a .feature that has been observed for residue i+ 1 of y-turns in general (Smith & Pease. 1980). Figure 2 shows this region of the polypeptide chain within omit-map density, illustrat’ing the good lit of model to density. Also shown for cornparison is the same portion of the chain for the C123S DLH and C123A DLH engineered proteins in Figure 13. The dihedral angles of Ser123 and Ala1 23 are similar to those of Cys123: (59”. -~ 114”) and (70”. - 116”). respectively. In addition to these defined examples. there exist several non-classic chain reversals in DLH. The residues Gly146 to Leu149 have the characteristics of a t’ype II turn, but do not possess a 4-l hydrogen bond. There does exist, however, a pair of water (Wat249))mediated hydrogen bonds that’ link the carbonyl oxygen of Gly146 to the main-chain nitrogen of L&149. cis-proline is residue 4 in the open reverse turn that takes strand 2 into stra,nd 3 of the P-sheet. Interestingly. turn 9 and the residues leading into turn 6. by their promixity stabilize each other through main-chain hydrogen bonding (Fig. 14). The main-chain oxygen and nitrogen a.toms of Tyr197 and Glyl69, respectively. are hydrogen bonded as are the main-chain oxygen and nitrogen atoms of Ala200 and Gln170. respectively.

512

I). Pathak

and II. Ollis

L

1

R

I

I

Figure 14. Main-chain atoms of turn 9 and the residues leading into turn B illustrating the main-chain hydrogen bonds between these 2 stretches of polypeptide chain Two residues associated with turns have dihedral angles that, place them in the left-handed region of the Ramachandran map. These residues are Tyr145 and Ser203, with dihedral angles (40”, 59”) and (62”. 23”). respectively. Tyr145 lies at the C-terminal end of st’rand 6 of the P-sheet and allows t’he strand to turn into helix E. Neither dihedral angles for the residues in this turn nor the lack of hydrogen bonding is suggest,ive of a classic t,urn. Ser203 is residue 1 of the type 1 turn that leads through two more such t’urns &to the final helix of IILH! helix C. This residue is hydrogen-bonded through its carbonyl oxygen to the amide nitrogen of Arg206. Both residues lie close t,o the active site triad of DLH and are likely to be involved in t,he catalytic mechanism of DLH.

(b) Side-chain

hydrogen

bonding

The criteria used for locating side-chainassociated hydrogen bonds, while less restrict’ive

than those used in some ot’her highl!. refined strucature studies, were chosen to allow comparison with the values cited in t’he comprehensive and detailed review of hydrogen bonding within proteins. 1)~ Baker Kr Hubbard (1984). For N-H 0 bonds t’hra largest allowed distance was taken to be 2.5 a. while for C=O N distances 3.5 A was caonsidered the largest allowed distance. The associabed angles for both these bonds were required to be greater than 90”. Distances involving water molecules were also permitted a maximum of 3.5 A. For side-chain N to water 0 hydrogen bonding distances, only a 11-H A (1). donor: A, acceptor) angle wa,s cal(u lated,_ while for side-chain 0 to water 0 only a 11, A-C’ angle was calculated. Roth angles were required to be greater than 90”. Mainchain hydrogen bonds involved in stabilizing secsondary strmtural elements in DLH have been described in t,hr previous section. Table 6 summarizes hydrogenbonding parameters for va,rious c1assc.sof hydrogen bond in DLH. These values are in a,greement wjth those observed by Baker & Hubbard (1984).

Table 6

Maiwc-hain side-chain Side-chain main-chain Side-chain side-chain Main-chain water 0 Main-chain water 0 Side-chain water 0 Side-water watw 0

Hydrogen

bond averages for some classes o;f flydrogvlz

N to 0 N to 0 N t,o 0 N to

3~03(031)

201(023)

3~10(0~27)

?04(021

3~00(026)

2~03(0524)

309(0723)

2. 04(0.16)

0 to

3m(o%)

0 to

1+S3(096)

iu IILN

127(16)

IN(17)

128(ifi)

138(1X)

l.il(14)

143(12)

Ili(l6)

ltiO(

Ii!)

lr,4( I31 13O(lti)

3~00(096)

N to

)

ho&

I:i’(

d~08(0%) 125(15)

1X)

I li(17)

Refined Structure of Dienelactone Hydrolase By far the largest number of side-chain hydrogen bonds are formed with water molecules, reflecting the exposed nature of most of the polar side-chains in the protein. A consequence of this fact, as also observed for other proteins, is the finding that there are far fewer side-chain to side-chain hydrogen bonds than main-chain to main-chain hydrogen bonds. Fewer hydrogen bonds with water as the acceptor than with water as the donor are found in DLH, both for hydrogen bonds with main-chain atoms and for hydrogen bonds with side-chain at,oms. This is as observed for other proteins. As observed for other proteins, in general the H ‘^.A--Cl angle is smaller than the associated D-H A angle. An abnormally low value (117”) is seen for hydrogen bonds involving side-chain to side-chain interactions, and this may be considered a result of higher B-values and consequent positional inaccuracy associated with some of these sidechain atoms. On excluding all hydrogen bonds with H A--C angles less than 120”, the number of hydrogen bonds thus defined was reduced by half and the mean associated angle found to be 133” (9). Interestingly though, the corresponding change in the mean hydrogen bond distance is very small, 2.07 A (0.25) as compared with 2.06 A (927) for the less restrictive bond angle definition. As noted by Baker & Hubbard (1984), a more stringent and perhaps more accurate, minimum angle to be used might indeed be 120” in the definition of’ hydrogen bonds. However, as observed in their review, and as observed for DLH (side-chain to side-chain interactions excepted), the combination of distance and angle constraints screens out most angles smaller than 120” in identified hydrogen bonds. Only three side-chain oxygen-oxygen hydrogen bonds are found in DLH. While this is consistent with the lack of acid-group participation due to the pH in the crystals (pH 63), it is unusual considering the number of serine (12), threonine (7) and tyrosine (12) residues present in the protein. Only one example of side-chain hydrogen bonding with tyrosine acting as a hydrogen bond donor is seen, as opposed to four instances of tyrosine acting as a hydrogen bond acceptor. The hydrogen bond donors in these instances are the NV’ and NV2 atoms of arginine, the Ivg2 atom of asparagine and, potentially, an Nsl atom from histidine. One other example of hydrogen bonds involving the hydroxyl group of tyrosine is the interaction of Tyr144 OH with t,he Oy’ oxygen atom of Thr224. Since both residues can function either as donor or acceptor, the hydrogen bonding role of tyrosine in this case is moot A single example of a tyrosine donor sidechain is seen in the hydrogen bond formed by GlulOl 0” and the phenolic hydrogen atom of Tyr137. Thr224, mentioned above, and SerlO appear to be the only hydroxyl side-chains situated so as to be capable of serving as hydrogen bond donors to side-chain oxygen atoms. A number of hydrogen bonds are formed by hydroxyl groups and water molecules, and it is

513

likely that the hydrogen bond donor potential of these side-chain groups are satisfied in many of these interactions.

(c) Thermal motion Atomic B-factors, or thermal parameters, are a measure of the degree of thermal motion experienced by the atoms in a crystal. B is related to the mean square amplitude (,k2) of atomic vibration by B= (8n2/3)G2. The final model was restrained to reflect the correlation of isotropic thermal parameters of bonded atoms., using a function that sums the square of the difference in B-factors between all pairs of bonded atoms (Tronrud et al., 1987). The mean B-value for all protein atoms in DLH is 21.7 A’, compared with a mean main-chain B-value of 180 A2 and a mean side-chain B-value of 258 A’. The mean B-value for all atoms, including water molecules, is 23.2 8’. Figure 15 highlights regions of DLH for which main-chain atoms have lower than average B-factors. The main-chain B-values for secondary structural elements other than turns, are lower than the average for the protein (see Fig. 15). This is as expected and is a consequence of the lower conformational entropy for a polypept’ide chain stabilized in a regular conformation. There is also a correlation between the level of atomic mobility and the environment of residues in the protein. As expected, residues in DLH with greater solvent accessibility generally exhibit a greater degree of mobility, especially in their sidechains. In keeping with this is the observation that lower average B-factors are seen for the p-sheet than the helices. The /?-sheet occupies t’he central core of DLH, which is predominantly hydrophobic. while the helices (with the except’ion of helix D) lie on the periphery of the protein and are consequently more solvent exposed. Table 7 lists the average B-values and accessible surface area for helices and j-sheet in DLH. Accessible surface area was calculated for a sphere of radius 1.4 A, using the algorithm of Lee & Richards (1971). Helices E and F are the only helical or p-sheet elements that display higher t’han average mainchain B-factors throughout their extent (see Fig. 15). The main-chain atoms of these helices also have the largest accessible surface areas of the helices in DLH (99.4 A2 and 141.0 A%‘,respectively) and considerably larger accessible surface area per residue ratios t)han the rest (8.3 and 10.8 respect’ively, compared with an average for all 7 helices of 54).

(d) Side-chain environment Figure 16 is a histogram of residue t)ype in DLH while Figure 17 is a plot of accessible surface area for residue type in DLH. There is a preponderance of non-polar residues over polar residues: 148

Figure 15. Stereo diagram of the main-chain atoms of DLH with those regions of the polgpeptidr~ vhaiu con~prisinp atoms wit)h lowrr than average main chain H-values. highlighted. The view wrresponds to that shown in Fig. 10.

compared with 86 of the latt’er. As expected. most’ non-polar residues have low accessible surface area. Polar residues. on the other hand. are found both in the interior of the protein and on the surface. Charged residues lying in bhe interior of the protein are ion paired. The single cis-proline. Pro27. occurs in t,he open t,urn t,hat brings strand 2 into st’rand 3. sidr-chains (i) ,Von-polar A hydrophobic core is considered import)ant for globular proteins, both in the energetics of folding and in stabilizing the folded structure (Pace, 1975: Matheson $ Scheraga. 19790.6: Tanford. 1980: GhClis 8r Yen. 1982). The central P-sheet of DLH is hydrophobic, containing 31 non-polar residues out of a tot,al of 50 that contribute to the sheet struct)ure. Six of the remaining 19 residues are glycine. Of these non-polar residues, only seven a,re aromatics. Except for strands 1 and 8, and part of &and 2, which form t,he edges of the sheet and t,hus have. each. one solvent,-exposed side. t,he accessible

surf’ace area of strands 3 to 7 is X4 .\‘. ~~sclutliny t,hree residues that lie at the ends of strands (se? also Table 8). The hydrophobic core of IILH is ac.c.entuatrtl I)! an aromatic kernel consisting of a clust.er of art)mat’ic and histidine side-chains. As has been noted. from a survey of 31 high-resolution J)rot,eitl struca tures. aromatic side-chains do not stack J)araIJel with each other as observed for IISA bases (Burle) & Petsko. 1985: Singh & Thornton. 1985). Ratjher. networks of aromatic interactions arv formed in which the phrnyl rings are oriented aJ)J)roximat.el~ perpendicular to each other. R’hile J)arallel sbwkiny of aromatic rings is not sew within thv h?-drophohic4 core of l)J,H and edge-to-face ring interact ions are found. a clear I)rePOr~(lrrellc~eof t,hese perpendicular ring plant interactions over all ot,hw forms of’ interaction is not wident. As has hecn noted by others. not all hydrophobic~ residues are found in solvent -inacwssiblc regions (Rose et CL/.. 198.5). Aianirw and prolinc (lo no1 display a marked J)ropensit,J- for being found either

515

Refined Structure of Dienelactone Hydrolasr

Ala

Vol

Lou Ilr

Phe Tyr

Trp

Pro Met

Cys

Sar

Thr

Asn Gin

Asp.Glu

His

Lys Arg

Residue

Figure

16. Frequency

of occurrence of amino acid type in I)LH

in the int,erior or on the surface of DLH (see Fig. 17). Helix 11, as noted earlier, is composed entirely of residues and glycine, other than hydrophobic Lys135, the last residue in the helix. Figure 18 displays helix D and the side-chains of residues in its vicinity. Also included in the Figure are, ordered water molecules in proximity to the helix. From this Figure it is clear that the side-chain environment of helix 11, in keeping with its low total accessible surface area, of 38.8 A2 (excluding Lys135, which by itself has an acressihle area of 120.5 ,A2), is very

Figure 17. Plot of’ side-chain-accessible surface area for residue typ’ in I>LH. Pun&ate squares represent accessible surface area calculated as for Fig. 20. Filled circles represent the acbcessible surface areas for the residue X in the model triprptides Ala-X-Ala (Lee 8: Richards. 1971).

hydrophobic. The only residues. other than Lysl35. Gith an accessible surface area greater than 2 A2 are Ala127 (13% A2), I >eulBl (151 X2) and Ala134 (6.6 A2).

(ii) Polfrr sidwhaina The polar residues in DLH. while showing a clear tendency towards high solvent accessibility, are also found buried in the interior of thcb prot>cm. Buried polar residues, bot*h charged and neutral, make hydrogen bonds through their sidcx-chains with other internal polar residues. Asp99 is the only charged residue in DLH with no ac,cessible surface area and it forms a salt bridge with Arg66 Nq2. through its 6% oxygen atom. Similar charged-pair interactions. as well as hydrogen bonds wit’h water. are seen for the other charged side-chains in Dl,H. SerlO also has zero accessible side-chain surface area and it hydrogen bonds with Arg66 iV2. Gln35 is completely solvent-inac~essil)lr and is oriented such that its side-chain is in hq’dropen bonding position with the carbonyl oxygen of (:lu36. Thr224, similarly solvent-inaccessiblr. forms a hydrogen bond t,hrough its hydroxyl group with His166 N”‘. The hydroxyl group on the phenolic ring of Tyr197 is situat,ed close to His1 66 so as to 1)~.able to taktl part in a possible hydrogen bond with the 61 nitrogen atom of Hisl66. Sincr thcl phr~nolic* sidrchain of tyrosine and the hydroxyl group of threonine are capable of serving bot,h as hydrogen bond donors and acceptors. it is not c+ar whicah nitrogen atom of His166 oarries a protoll. Exposed charged residur>s hytfrogt~~ bond both with ordered water molecuks and with bulk solvent,

516

Il. Path&

and D. Ollis T P

Figure 18. Stereo diagram of helix 11and the ordered wa,ter molecules and side-chain atoms in it,s vicitlity. Oxy,ut*n atoms of water molecules are depicted by %caoncentric(air&s and lahrlled with a wave (- ).

(iii) Tryptophan, and tyrosinP All three tryptophan residues in DLH have low accessible suriace areas. Nevertheless, both Trp50 and Trp88 hydrogen bond with wat.er molecules through their cl nitrogen atoms: Trp50 with Wat356 and Trp88 with Wat357. Trp196 makes a weak hydrogen bond with Thr183 0”‘. All but one of the tyrosine residues, despite their hydrophobic environment take part in hydrogen bonding interactions with other side-chains, mainchain oxygen atoms and water molecules. Tyrll does not appear to be involved in llydrogen bonding with any modelled atoms, but is capable of interaction wit,h the bulk solvent. (iv) Discretely disordered sidr-chains Only Ser49 and Cys123 were finally built into the model as existing in more than one distinct conformer within the crystal. The hydroxyl group of Ser49 is found in two distinct) locations. One conformer h,ydrogen bonds with Wat3ld whose occupancy refined to a value of 0.5658. The occupancy of this conformer was set to 0.6. The other 40% of the time, the hydroxyl oxygen swings

around and is stabilized by a hydrogen Arg45 N ql. (‘~~123 is discussed below.

(e) Therzctioe-xitr The major change in the activy site struct,urc of’ 1)LH frorn its original description arises from the identification of cysteine 123 as existing in two distinct conformers within tht3 c+rystal. One conformer is in t’hr native, reduced state of t’he t,hiol. while t,hr other is oxidized and bears two oxygen atoms on t>hr y-sulphur. The identificat’ion and building of the act,ive-sit,c t,hiol as existing in reduced and oxidized, discret)el> disordered conformers has been discussed in the t,ext and illustrated in Figure 3. Figure 19 displays thr active sit,e residues Asp1 7 1. His202 and (lys123 with both its conformers. in 2J’,,--PC difference density. These residues are similar to the catalyt’ic t)riad seen in the thiol and srrine proteases (see Frrsht, 1985). The similarit’ies and differences in the catalytic roles of these residues in DLH, with those in thtx proteases will be discussed later (see Discussion). At the centre of thr tria,d is His202 which is oriented such that itsL Val I atom is poised at hydro-

Table 8 k\‘wmmczry

qf inter-molecular

bond with

hydrogen

tend kztrractionn

517

Refined Structure of Dienelactone Hydrolase

u

d Figure 19. Stereo view of’the active site residues Cys123, Asp171 and His202 within are

bot#hthe native reduced and the oxidised

conformers

gen bonding distance from the 061 atom of Aspl71. This distance is found to be 2.61 8. Asp171 061 can also form hydrogen bonds with Wat310 and WaMOO, which are at distances of 2.64 A and 3.11 8, respectively, from the 61 oxygen atom. Figure 20 displays residues in the vicinity of Cys123 and the putative catalytic triad. A hydrogen bonding interaction between Asp171 0” would stabilize the tautomer of His202 with a proton on N”l. This would permit the NE2 atom to participate in a hydrogen bonding, or proton abstracting, interaction with the thiol of Cys123. The thiol group appears to be tethered in place by three weak

Figure 20. Stereo view of the active Hydrogen vicinity.

bonds formed

of Cys123.

Also included

2F0- F, electron is Wat234.

density.

Sho wn

hydrogen bonds, and a fourth shorter one. The NE2 atom of His202 is at a distance of 3.58 a from the thiol sulphur, while Glu36 OC2 is positioned at a distance of 3.44 A and Wat234 at a distance of 3.52 8. from the y-sulphur. The shortest hydrogen bond is formed by Wat253 which is found at a distance of 3.08 w from Cys123 Sy. Wat253 was modelled to lie in a well-ordered solvent, site (R= 30.42 A2, Q=O+3). It is situated so as to potentially take part in a network of hydrogen bonds (see Fig. 20). Ser203 Oy and Glu36 OE2 make hydrogen bonds of length 2.68 a and 2.54 A, respectively, with Wat253, while hydrogen bonds of length 2.61 a

site triad: Cys123, His202 and Asp171. Both conformers of Cysl23 are included. by these residue are depicted by dotted lines. Also included in the Figure are residues in the

518

Il. Pathak a,nd D. Ollis P

Figure 21. Stereo diagram tiepi&lg the acative site triad anti the network of ortlrrrd water rnolt~wlrs in t,ho regioll. molerules funnel in from t,hr lip of t,he active-sit,e-containing cleft wit’h the conicsal ciistrihution rtafircting thcb narrowing of t,hr czleft at it,s base where Cys123 lirs

These water

and 2.96 A are formed with \Vat234 and Wat427, respectively. In it,s oxidized conformer. the y-sulphur is held at a separation from His202 SE2 and Lru124 Iv that is observed for slight,ly short’er than t,hat usually sulphur~-nitrogen hydrogen bonding dist,ances (see Srinivasan B Chacko. 1967). The former distance refines t,o a value of 3.43 At while t,he la,tter refines to a value of 3.22 4. Wat236 is found at a distance of 3.24 Aq from thr oxidized sulphur atom. The t,wo oxygen atoms, O-l and O-2. of t,hr oxidized sulphur form hydrogen bonds t’hat t)ether t)hr sulphur atom in this position. 0-I hydrogen bonds weakly wit)h I,eul%l N. yielding a hydrogen bond length of 3.57 a. O-2 forms a 2.76 A hydrogen bond with the ~2 nitrogen atom of His202 and a 2%5 A hydrogen bond with W&236. molecdt~. water disordered discretely Thr \Z’at%34, refines to distances of 2.95 ,q and 2.24 is, from t hr oxidized sulphur and O- I rrspc~c+ively. These distances arp rather small compared to observed distances for interactions between such atoms and are further confirmation that Wat234 exists in that fraction of t)hc c*rvst,al that contains

unoxidized thiol. Thch onI>. hydrogen bonds t’h;lt b’at234 makes are with Wat,253 and Wat427: thrsc> distances are %%I ip and 2.53 ‘4. rrspt~c+ivcly. It can c+arly hc seen that oxidat’ion of thr t~hiol would nccessitatc a c.oriforrnational acljustment 01. the side-chain of (‘ysl%3, in order t)o ;l,c~c~ommodatt, the two oxygen atoms. This adjustment is wcc’omplished w&h minimal pert~urbation of thr act.ivci sitta structure. with the t,hiol rotat.iny ahont the (V”lr bond. to its new locat’ion wht’rc it is stabilized by. ;I new set of hydrogen bonds. Movement of the C’” atom in the ado@on of thr.‘9 net\- c~ontormat~ion iz vrry slight). Figure 21 shows the side-chains of rfGlurs of I trot ac%ive site triad and the network of ortkrrtl watrtr molecules in the region; while Pigurc 22. which includes at1 side-chains with atoms within X AA 01’ C,‘y~l23, illustrates the hydrophophic nat’urts of t IW hmng of’ t)he actJive site cl&. The great nurnhcAr 01’ sidc>bcatiains that c*nc~losr t ht. c~ai,iLlVt ic. ;Womittic~ residues arc’ complemented t)J- ot,her nonpolar s&l chains. Only three polar side-chains. in addition to the catalvtic triad. are found in this region of DLH. These residues are Clu3li. Ser203 and ~A’rg206. and it

Refined Structure of Dienelactone

Hydrolase

519

Figure 22. Stereo diagram showing the side-chains of the active site triad and of residues with atoms nit,hin 8 A of the side-ihain atoms of Cys123.

is very likely that these residues play an important role in the catalytic function of DLH. Roth side-chain oxygen atoms of Glu36 are within hydrogen bonding range of the side-chain nitrogen atoms of Arg206. The NV’ atom of Arg206 is also hydrogen-bonded with Ser208 Oy. Both the mainchain nitrogen atom of Ser203, and its hydroxyl oxygen, form hydrogen bonds with the carbonyl oxygen of His202. The carbonyl oxygen atom of Ser203 is hydrogen-bonded to the amide nitrogen of Arg206. Ser203 and Arg206, along with His202 of the catalytic triad, lie in a flap-like domain of the protein. This region had previously been considered a region of random coil in the structure, and inasmuch the only long region in DLH lacking any secondary structure (Pathak et al., 1988). It is now apparent, that the flap comprises three consecutive type 1 turns, stabilized by bifurcated hydrogen bonds (see Table 5). As was noted in the previous 2.8 -4 study, this flap constitutes one of the walls of the active site fissure. Given the fact that the abovementioned polar residues reside in this flap, in addition to forming a bulk-solvent inaccessible crevice in the protein. it is possible that this flap-like domain has catalytic significance. Tts looped out nature could confer flexibility which can be exploited by allowing movement’ of the residues it comprises in order to accommodate the substrate and facilitate release of enzymatic product.

(f) Water structure All solvent sites in DLH were built as wat’er with the final model containing 279 water molecules. Of these, 42 refined t,o unit occupancy and 19 to occupancy values between @9 and 1.0. The mean B-factor for all the water sites is 33.14 8’. Water molecules were considered internal to the protein if they had zero accessible surface area and

lay no less than 4 A away from any other modelled water molecule. This yielded a single internal solvent site, Wat299. This water molecule is very snugly enveloped by the surrounding residues and Figure 23 displays those residues most int’imately in contact with it, within 2F,-FC difference density. Three hydrogen bonds, one each from Tle33 0. Gln35 N and Pro61 0 hold Wat299 in what is otherwise a fairly hydrophobic environment. The distances associated with the hydrogen bonds are 2.82 A, 2.89 w and 2.82 8, respectively. Wat299 has unit occupancy and a low R-factor of’ 10.3 8. The y-sulphur of Cys60 can be seen in the Figure, existing in the free reduced state. The distance between Wat299 and Cys60 8” is 3.79 w. rather too long for electronic interactions. The restricted environment, of Cys60 and the relatively immobile Wat299 t’hat acts as a plug for the cavity, suggest that it is the atomic environment’ that results in the oxidation of Cys123, but t,he protection of Cys60 from the same fate. Only three water molecules in DLH are situtated so as to be capable of hydrogen bonding with more than four prot,ein atoms. Wat291 is strategically located so as to stabilize the type III turn that takes strand 4 of the central P-sheet into the isolated B-ribbon that extends from Pro69 to (iln76. This turn (turn 2) is also stabilized bv a hydrogen bond between Tyr64 0 and Gln67 N. himilarly, all of the other water molecules with hydrogen bonds to more than three protein atoms are involved in interactions with turns in the protein. Whether this is an indication that the turns involved require t,hese water molecules for the added stability provided by the additional hydrogen bonds, or serve as hydrogen bonding nuclei for the formation of t’he polypeptide t’urn, or simply, through the orientation of protein atoms made possible by the polypeptide chain reversal, stabilize solvent molecules in t#he vicinity so as to partially order them in the crystal, is unclear.

520

11. f’athak and D. Ollis

Figure 23. dFo- Fc electron

density

for M:at%HS, thr single internal

As is the rule for hydrogen bonding in proteins in general (Baker & Hubbard, 1984), water molecules in DLH form far more hydrogen bonds with oxygen atoms than with nitrogen at,oms: 170 of the former as compared with 80 of the latter. Nineteen hydrogen bonds are formed by the hydroxyl groups of serine. threonine and tyrosine residues wit,h water molecules.

(g) (‘rystal packirq The packing of molecules in crystals of DLH is illustrated in Figure 24. The closest approaches between atoms from different molecules are those that are associated with hydrogen-bonded interactions, and these are listed in Table 8. The bulk of the close approaches are mediated through complex networks of water bridges between molecules. Trp50. Tyrl12 and Phe173 are the only aromatics found at the surface of DLH that are involved in close intermolecular approaches. but do not form hydrogen bonds. The histidine ring and the indole ring of the tryptophan are approximately parallel and this interaction does not appear to be stabilized by hydrogen bonds with either molecule or with ordered water. The act’ive site residues of DLH are far removed from protein atoms from any other molecule in the crystal and it is extremely unlikely that the modest,

Figure 24. Stereo diagram

depicting

water

site in DLH

and thr residues

surroumhtlg

it.

number of intermolecular hydrogen bonds within crystals of DLH impose any structural influence OII DLH. It is t,herefore not, likely that lattice cont)acts result in a crystal structure that. for DLH, wonld differ to any significantj degree from a solution structure.

The structure of DLH was originally determined using multiple isomorphous replacement with fout heavy-at’om derivatives. Table 9 lists t,he heavyatom sites and their proximity to protein atoms that might represent possible ligands for the heavy atoms. Heavy-atom site numbers refer back to the original structure determination. Only the unique sites are listed. Site 2 of derivative PTCN (same as site 3 of PTCl) and site 3 of derivative PTCl (same> as site 2 of derivative AUCN) are rather far from protein atoms. considered chemically likely to serve as binding sites. Other than these. t’he binding sites identified for KAuBr, and KAu(CN), are not unusual (SW Blundell & ,Johnson, 1976). K,Pt(CN), would bc expected to yield the stable anion (Pt(CN)~ and bind electrostatically t’o positively charged sidechains. However, PTCR: 1, PTCN 3 and PTCI 3 at-r found to lie close t,o glutamate side-chains (see Table 9).

crystal

packing

in DLHasr

R.efined Structure

qf Dienelactone Hydrolase

5. Discussion A wealth of biochemical knowledge exists for the cysteine proteases and the related serine proteases (Bender & Kt%dy. 1965; Glazer & Smith, 1971; Lowe, 1976; Polgar, 1977; Kraut, 1977; Neurath, 1984; Fersht,, 1985; Baker & Drenth, 1987; Sprang ef al., 1988: Schowen, 1988). This information includes well-refined, high resolution structures of several representative members of these families of proteases (actinidin, Baker, 1980; papain, Kamphuis et al., 1984; subtilisin, Neidhart & Petsko, 1988: chymotrypsin, Tsukuda & Blow, 1985; trypsin, Read & James, 1988). In spite of this abundance of fact, many aspects of the molecular mechanisms that underlie the catalytic function of these enzymes is still unclear. In particular, the sulphydryl proteases are considerably less well understood than the serine proteases. Dienelactone hydrolase (DLH) bears a compelling resemblance t)o these enzymes in that it possesses the very similar catalytic triad of amino acids at its active site: cysteine 123, histidine 202 and aspartate 171. The finding of these familiar catalytic residues in a novel locale, provides a great, opportunity to characteristics of these residues probe the functional related to their structural configurations in a different system. It may be noted here that the catalytic t,riad present in DLH is a hybrid of the triads seen in the papain class of thiol proteases and the trypsin and subtilisin classes of serine proteases. The former contain cysteine, histidine and asparagine residues, while both of the latter two contain serine, histidine and aspartic acid at their active sites. Tn this respect DLH bears greater similarity t,o the viral cysteine proteases which contain the

Table 9 IIeavy-atom Derivative wmpound KAUlb,

Heavy-atom site

Nearest protein atom(s)

AllI3K

C60 sy C60 , 0 K86 NC Cl23 RY Cl23 W(OX) H202 NE2 H202 N” E460 OE2 E46 0”’ T207 W’ K86 Nr E460 0”’ El84 0 Plll 0 El84 o”* Y130 N E94 0” R206 N” Fl73 N E94 0” E94 0” N41 NdZ Cl23 W(OX)

1

AITBR 2 AITBR 3

K&((V),

ITCN

1

PTCS 2 PTCN 3 I’TVl 3

KAu((‘lJ),

positions

PTCI 4 AUCN 4 AIJCN

5

AllCN

6

AV:cS 7 AUCN 8

Thtancr (4 2.76 2.58 491 1.88 1.92 2.21 383 2.62 2.7 1 3.19 433 348 3.88 325 5.83 404 2.77 2.97 3.87 2.07 3.99 3.96 2.99

521

same catalytic triad of residues as DLH (Bazan &Z Fletterick, 1988). The determination of a refined, high-resolution structure for DLH is a vital step towards the elucidation of its enzymat,ic mechanism. (a) Oxidation

of the active thiol

While the coup de grace in understanding the structural basis for enzymatic activity is certainly a combined structure of enzyme complexed with the transition-state between substrate and product, it is clearly important to understand the native structure of the active site. In this end crystallographers have been thwarted in their study of the thiol proteases by the extreme propensity towards oxidation of the active site sulphydryl group by X-rays (Jocelyn, 1972; Packer, 1974). All the well-refined high-resolution structures of these enzymes contain oxidized active site thiols. Unfortunately, DLH is not an exception and is found t’o be partially oxidized during data collection. Nevertheless, we have been able to deduce the three-dimensional orientation of the active site thiol in that proportion of the crystal in which it is not oxidized. In the original structure descriptions of papain and actinidin, it was suggested that, the oxidation of the active site thiol resulted in the addition of two atoms of oxygen to the y-sulphur atom, yielding the sulfinic acid moiety (Drenth et al., 1975; Baker. 1980). In the carefully refined structure ofactinidin. Baker observed a weak extension of electron density, suggesting that one oxygen atom was less well ordered than the other. In a more recent resolution extension and refinement study of papain by Kamphuis et al. (1984), these workers now contend that the active site thiol of papain is oxidized with three oxygen atoms, as the sulphonic acid moiety. It should be stressed that in the present study, in building the active site of DLH. we have attempted not to be biased by these previous results, oi* by mechanistic considerations. Rat)hrr, we have allowed solely the experimental data, namely the electron density maps. to guide the c=onstru&ion of the final model. In this we were aided hy the arailability of the two mutant enzymes (11231’: DLH and Cl 23A DT,H. Details of this work along with kinetic: characterization of the mut,ant enzymes will b(> presented elsewhere. Examinat.ion of the elect’ron density for the sidechain of Cysl23 in 2F,, - F, and omit-maps indicated a clear excess of density, the most likely explanation for which being X-radiation-induced oxidation of the thiol. The most reasonable (i.e. in keeping with accepted steric and geometrical constraints) and refineable (i.e. yielding good refinement statistics and featureless difference maps) modelling of the electron density suggests that the active site t’hiol of DLH is oxidized with two oxygen at)oms attached to the y-sulphur atom. The oxlda.tion is partial and causes the active site thiol to exist, as t,wo discretely disordered conformers within crystal. the A partially ordered water molecule. Wat234, is found in t,he vicinity of the nat’ivc, reduced thiol and

522

D. Pathak and D. O&s

Figure 25. The c>atalytic triads of DLH. papain and subtilisin. Atoms of I)LH ~IIV joint~tl 1)~ till~cl buntis. atoms of’ papain by oprn bonds and atoms of subtilisin by broken bonds. The 5 ring atoms of the side-chain of eat+ histidinta UPW superposrd and the transformat,ion applied to each t,riad. For pq)ain. thca hist~idinr ring was orirntpd so as to suyq~s~~ its P’ atom with thr (“62 atom of the histidine ring of DLH. This reflects thr 180 rotation of’thrb histidine ring of papa.irI about t’hr C8P(‘y I~ond. relative to thr rings of I)LH and suhtilisin.

is displac~ed by the oxygen at,oms of the oxidized conformer. The refined occupancy of Wat234 was used to r&mate the percentage oxidation of the thiol wit,hin the cqc&al. The occupancies of the reduced and oxidized conformers of the thiot were fixed to reflect a GO’& oxidation. The fact, that Wat234 is indeed a water molecule and not a spurious side-effect from the oxidation ot the active sit’e sulphur is confirmed by the fact that, a water molecule is found at the same location in t’hr structures of the two mutant) enzymes, Cl238 DLH and C‘123A DLH. in which t’he active site sulphur has been converted to the non-radiation-labile residues. serine and alanine. respect’ivety (Pathak el rxl., unpublished results). Figure 13 illustrates the engineered active sit,es of both of those proteins within omit)-map density. and t,he location of the corresponding water molecule (Fig. 2). The reduced t’hiol ties at the base of the active site cleft. and is held in position by four hydrogen bonds. I’pon oxidation. the thin1 swings around its (?c’” bond in order to accommodat!e t,he t,wo oxygen atoms. This results from t.hr fact, that the location of the reduced sulphur places it very snugly amongst it,s neighbouring atoms and the addition of two atoms. at,tached to t,he sulphur, necessitates a movt’mcnt of the sulphur. A rotation about t,he C’“Ys bond allows minimal perturbation of the rest of the st,ruct,ure in t)hr micro-environment of the thiol. Given the similar nature of t,he actjive sites of‘ L)LH, actinidin and papain, it is possible that a similar oxidized state may exist for t.he active sit,es of these tatt)er enzymes. This would resolve the discrepancy in the interpretation of t)hr electSron density for thr active site cyst,eine residues of actiand papain, with a third oxygen atom nidin suggested to exist in papain being prrhaps a water tnoteculc~.

Having fixt~i t I~(. position of’ c.ystrirrcs 1%~ III t tits native form of t’he cnz~mc. it is int,t~rt~si.ing to cornpart’ the activr sitcb t’rlatl of I)LH with those ofa representative cystrine and a representative serinr protease. Papain (Kamphuis et ~1.. 1984) and sub tilisin (Neidhart B Prtsko, 1988) trarc, been chostbn for t,his purpose. (‘o-ordinates for papain and sut)Gtisin wery obtained from t’he Rrookhavrn Prot.rin Data Bank. The act ivr sitrs of Dt,H, papain atltl subtilisln arr all twatfd ilt t hf, N-terminal rnd of an a-hrlis. In addition to this l)I,H bcsars superfic*iwt similarit> with subtilisin in its topology. Both enzj.mes art’ r//l proteins and both havta thrGr activr sitrs loc*ateti in a cleft that lies at t.he carboxy ends of t hcs fourth and CRh strands from the (’ trrmini of i h(> f)rotc,ins. Figurfs 25 illustrates tht, relat ivr oric~ntat~ion of’ residurs of the, catatyt,ica triads of lItAH, l)apaitl all(l subtilisin. This arrangetnf>nt rrsult’s f’rom aligning only the ring at,oms of the> I hrrt, hist itlint rAduf+. It ‘is apparent from this Figurt~ that whitt, t trc spatial arrangement of the t hrrr triads is tlifferrrrl the orient)ation of the donor and ncc*f,ptor atorrlh involved in hydrogen bonding bf~twrr~n t hth Asf)i.+\\sn residues and the histidinr residues is cIuitt% similar Tn all t hretl c+ases thr ptanr formed by the Co and atoms Of’ ASp/ASIl tll~~t
Refined Structure of Dienelactone Hydrolase solution between the hydroxyl group and the imidazole ring of serine proteases (Smith et al., 1989)). It should also be not#ed that the orientation of the histidine is different in papain (and actinidin) from subtilisin and DLH. Frorn this comparison it is clear that the catalytic triads of these three crystalline enzymes are very similar in their hydrogen bonding characteristics. The differences in relative orientation of these residues probably reflects the difference in structure of the respective substrates of the enzymes and the location of the target sites for nucleophilic at’tack. (c) :I// echanistic

inferences

Given the similarity of the active site residues in DLH with those of the proteases, the inhibition studies with various active site reagents, and the drastic1 reduction in activity of t’he C123S DLH and C123A DLH mutant enzymes? it is extremely likely that DLH functions in an analogous manner with regard to the active site nucleophile. The thiol of Cys123 is probably activated for nucleophilic attack by His202, which is stabilized in the appropriate tautomer by Asp171. This is suggestsed by the short hydrogen bond that exists between Asp171 0” and His202 N”‘. Cys123 Sy is within hydrogen bonding range of His202 N”‘. though the hydrogen bonding angle associated with this distance IS not optimal and the sulphur atom is not coplanar with the imidazole ring (Cs-Sy . . NE2= 94”). Hydrogen bonds with sulphur as donor are ext,remely rare, both in proteins and in small molecule structures, and it is unlikely that a hydrogen bond exists between Cys123 Sy and His202 NE2. When substrat)e diffuses into the active site a conformational switch is induced which results in t’he active site thiol swinging around to a position more nearly coplanar with the imidazole ring and close to t,hat of t,he oxidized thiol (Pathak. 1989). At this juncture, proton abstraction can occur yielding a mercapt#ide-imidazolium ion pair with the sulphur poised for nucleophilic attack. In this section we discuss a series of possibilities regarding the mode of enzymatic act)ion followed by DLH. While speculative for the most part, this discussion is relevant’ in conjunction with the atomic detail afforded by the high-resolution struct’ure of the enzyme. Unlike a peptide substrate, the dienelact’one cyclic> ester contains a network of conjugated double bonds. As a result,. nucleophilic addition can conceivably occur not only at the acyl carbon but also across one of the doudle bonds in the manner of a Michael addition (see Massey-Westropp & Price, 1981). Tt is not conclusively established yet’ that) nucleophilic attack by DLH occurs at t’he acyl carbon of dirnelact’one. The conformational switch that results in the y-sulphur at,om swinging out to a more exposed position. coplanar with the histidine ring. is probably t,riggered by the entry of substrate intro the active-site cleft,, causing the cleft to widen slightly. The widening is induced both by steric conditions

523

and by the destabilization energy due to the introduction of the negatively charged carboxylate group of the substrate into the hydrophobic environment, of the crevice. This widening would disrupt the hydrogen bonds that’ t’ether the sulphydryl in place, freeing it to swing out. In this respect it should be remembered that’ His202. Ser203 and Arg206 all lie in the flap-like domain t’hat forms one of the walls of the active-site cleft. The loosely looping nature of this flap could confer upon it a flexibility that would permit’ movement of residues wit)hin it in response to the diffusion of substrate into the active site. Tt should also be recalled that Ser203 is held in a strained conformation, as suggested by it’s unusual dihedral angles. Tt, is likely that upon entry of subst’rat’e. t’he flap moves slightly, allowing the active-site crevice to widen. One of the events mediated by this t’ransient distension might involve destabilization of the hydrogen bond holding Ser203 in place allowing both it and Arg206 t’o take part in the catalytic events t’hat follow. It is also possible that there exists an equilibrium in the native protein between the two positions of the active thiol. This would imply t’hat the thiol may be switching back and forth between a neutral non-hydrogen-bonded conformation and a charged species hydrogen bonding wit’h its abstracted proton on His202. Experimental observations (Lewis et d., 1976; Creight’on et al.. 1980: Roberts et al.. 1986) and a recent simulation study (Rullmann et a,Z., 1989) suggest that the active site of papain may be oscillating between zwitterionic and neutral stat’es. The active sites of DLH, papain and subtilisin: all lie at the N terminus of a helix. It has been suggested that the virtual positive charge associated with the a-helix dipole would serve to stabilize negatively charged substrates, and in the case of the proteases, tJoenhance the proton donating abilit’y of the nucleophile and stabilize the negatively charged tetrahedral intermediate (Ho1 c?t al.. 1978). It is likely that’ t’hese mechanism are also at work in DLH. To this end the unusually hydrophobic nature of helix D of DLH, and its hydrophobic environment’, would serve to accemuate the effect of the helix dipole. Tn terms of substrate recognit’ion it, is likely that the hydrophobic environment is important in stabilizing the ring of dienelactone. while the helix dipole serves to stabilize the negatively charged carboxglate. While residues responsible for substrate specificity have not been delineated, it, is likely that t’he polar side-chains in the active site are important in this regard. While the mechanism of DLH remains speculative at this time. the refined structure at, a resolution of 1.8 A provides a sound basis for biochemical studies with the enzyme. The st’ructure provides a reliable model against which biochemical results can be interpreted. In addition, t’he structure points out dire&ions along which further biochemical investigations may be pursued. Such studies are under way.

ll. Pathak and D. Ollis

524

The authors thank A. Mondragbn fbr many extremei? helpful discussions throughout the later stages of this work. We also thank him for reading the manuscript and providing useful comments. We thank E. Westbrook and M. Deacon for making additional computer time available to us. We acknowledge the assistance of T. Puri during an earlg stage of model rebuilding. D.P. is an Abbott Laboratories Fellow. and acknowledges this support,. The research described here was supported by a grant from the Pu’ational Science Foundation (grant no. DMB-X904341).

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Edited by W. A. Hendrickson,