Crystal structure of neutral protease from Bacillus cereus refined at 3.0A˚resolution and comparison with the homologous but more thermostable enzyme thermolysin

J. Mol. Biol. (1988) 199, 525-537

Crystal Structure of Neutral Protease from Bacillus cereus Refined at 3-OA Resolution and Comparison with the Homologous But More Thermostable Enzyme Thermolysin Richard

A. Pauptit, Rolf Karlsson, Daniel Picot, John A. Jenkins Ann-Sofie Niklaus-Reimer and Johan N. Jansonius Department of Xtructural Biology, University of Basel, Klingelbergstrasse Switzerland

The Biocentre 70, GH-4056 Base1

(Received 26 June 1987, and in revised form

16 October 1987)

Neutral protease from Bacillus cereus exhibits a 73% amino acid sequence homology to thermolysin, for which an accurate crystal structure exists. The B. cereua enzyme is, however, markedly less thermostable. The neutral protease was crystallized and diffraction data to 3.0 a resolution were recorded by oscillation photography. The crystal structure was solved by molecular replacement methods using thermolysin as a trial molecule. The solution was improved b;y rigid-body refinement and model rebuilding into electron density omit-maps. The atomic co-ordinates were refined to R = 21.7% at 3-O a resolution. Comparison of the resultant model with the thermolysin structure shows that the two enzymes are very similar with a root-mean-square deviation between equivalent C”-atoms of 048 8. The y-turn found in thermolysin is transformed into a p-turn in the neutral protease by the insertion of a glycine residue. There appear to be no contributions to the enhanced thermostability of thermolysin from additional salt bridges, whereas contributions in the form of extra hydrogen bonding interactions could be important. Other factors that may affect thermostability include the two glycine to alanine exchanges and perturbations in the environment of the double calcium site.

1. Introduction

calcium binding residues are highly conserved in NP (Sidler et al., 1986b). The two enzymes are therefore expected to have a very similar structure and mechanism of action. The amino acid sequences of NP and TLN are aligned in Figure 1. Both sequences lack cysteine residues. There is a single insertion in NP between residues 25 and 26, numbered 25a to preserve the TLN numbering scheme. The calcium ions are also numbered as for TLN (Colman et al., 1972). TLN is produced by the thermophilic bacterium Bacillus thermoproteolyticus, and must survive and function at elevated temperatures. It maintains over half its activity following a one hour incubation at SO%, whereas NP is only thermostable to 60°C. There has been considerable interest in determining the factors responsible for thermostability in proteins. Following the structural elucidation of TLN (Matthews et al., 1972a,b), it became apparent that thermostable proteins need not display any unusual structural features. As the free energy difference between the folded and

Neutral protease from BaciZZus cereus (NP)? is an extracellular metallo-endopeptidase that hydrolyses the polypeptide chain at the imino side of aromatic and hydrophobic residues (Feder et al., 1971; Sidler et al.: 1986a). The amino acid sequence for NP has been determined by Sidler et al. (19866). The enzyme shows 73% sequence identity to thermolysin (TLN), f or which the amino acid sequence refined (Titani et al., 1972) and an accurately crystal structure (Holmes & Matthews, 1982) are available. TLN consists of two domains, one mainly b-pleated sheet and the other mainly a-helical, with a zinc-containing active site cleft between them. There are four calcium ions that bind to and stabilize the TLN molecule. The active site and the t Abbreviations

used: NP, neutral protease from thermolysin; r.m.s., root mean square; m.i.r., multiple isomorphous replacement.

Bacillus

cereus; TLN,

525 0022-2836/SS/O30525-13

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NP: TLN: NP: TLN: NP: TLN: NP: TLN: NP: TLN: NP: TLN: NP: TLN:

160 NSSNLIYQNESGALN-EAISDIFGTL YTAGLIYQNESGAINEAISDIFGTL

170...-

NP: T‘N : NP: TLN: tip: TLN : NP: TLN : NP: TLN : NP: TLN :

Figure 1. The amino acid sequence of neutral protease (top) aligned with the sequence of thermolysin (bottom). Identical residues are boxed.

unfolded states of proteins is typically not greater than 16 kcal/moI (1 kcal = 4-184 kJ) (Creighton, 1983), and as the stabilization offered by a single hydrogen bond or salt bridge is of the order of 1 to 3 kcal/mol (see, for example Perutz, 1978), a significant increase in thermal stability does not require a large structural change. In fact, there are ma,ny homologous amino acid sequences available that indicate that the thermostable proteins are probably ext,remely similar to their less stable counterparts (see, for example, Argos et al., 1979). It ha,d nonetheless been noticed that proteins from thermophilic organisms are often rich in arginine content. (Frank et al., 1976) and usually lack eysteine (Hocking & Harris, 1976). It has been shown that the guanidination of surface lysyl residues (in moderation) can effect an increase in thermal stability in several proteins (Qaw & Brewer, 1986). Perutz & Raidt (1975) found that they could rationalize the increasing thermostability in a series of ferredoxins by the addition of some hydrogen bonds and surface salt bridges without modifying the protein conformation. Modelling studies on a thermophilic ferredoxin from Clostridium thermocellum suggest that salt bridges connecting the amino and carboxyl termini are responsible for the enhanced stability of this protein (Bruschi et al.,

et al.

1986), in agreement with the observations of Perutz (Perutz & Raidt, 1975; Perut,z; 1978). Argos et al. (1979) suggested that certain amino acid exchanges might be beneficial to thermostabilit,y, and that the preferred exchanges that they observed in a series of ferredoxins, lactate dehydrogenases and glyceraldehyde-3-phosphate dehydrogenases might, also be found in other systems. The general consensus at present is that the thermostabilitg of a protein is due to small and subtle stabilizations (by hydrogen bonding, salt bridging, van der Waals’ strengthening of secondary structure contacts, elements, etc.) that, occur distributed throughout the molecule (Colman et u,l., 1972; Singleton & Amelunxen, 1973; Zuber, 1976). In a system where bwo homologous proteins differ in thermostabilit’y, the st,abilization must occur as a result of the changes in the amino acid sequence. These may be numerous, and locating those that contribute to thermostability remains a difficult task, especially as it has been demonstrated that only one single exchange (Arg to His) in pha,ge TB, lysozyme is capable of effecting a sizeable decrease (14°C) in melting temperature (Griitter et al., 1979). Using the guidelines of Argos et al. (I979), an increase in t’he thermostability of the neutral prot’ease from Bacillus stearothermophilus has been successfully engineered through a single Gly to Ala replacement (Imanaka et al., 1986). Two individual mutations that increase thermostability in kanamycin nucleotidyltransferase have been shown to have a cumulative effect when t,hey occur eoncurrently (Matsumura et al., 1986). ,4 cumulative effect is also inherent in the results of Imana,ka e:t ai. (1986), where a single thermostability-decreasing point mutation in the neutral protease from B. stearothe~moph~~~s can only be compensated by two thermostability-enhancing point mutations when they occur concurrently. In order to understand the variation of prot,ein thermostability in nature, structural differences between relat,ed mesophilie and thermophilic proteins should be carefully examined. Such especially at the three-dimensional compa,risons, structure level, are still sparse in the literature. An analysis of the differences in prima,ry structure between TLN and KP has been given by Sidier et al. (19863). Earlier comparisons of the neutral protease from a different mesophilic bacterium, Badus subtilis, to TLN (Levy et al., 1975; Pangburn et al.: 1976; Grandi et al., 1980) were complicat,ed by incomplete sequence informat,ion. The DNA sequence of the B. subtilis neutral protease is now available, but it shows only 45% homology to TLN and the calcium binding ligands for Ca-3 and Ca-4 are not conserved (‘Yang et al., 1984). Presumably one or both of these celcium ions are absent in the B. subtilis neutral protease; which would explain its extreme sensitiviby to autodigestion. Because fewer varia,bles are involved, WI? is a better candidate for comparison with TLN than is the B. szcbtilis neutral protease. In order to be able to make such a comparison at the tertiary

Crystal

Xtructure

of B. cereus Neutral

structure level, the X-ray crystal structure of NP was determined and inspected in the hope that the structural differences to TLN might allow some correlation with the difference in thermostability. It should be realized that matters are greatly complicated by the fact that these enzymes are proteases which are capable of autodigestion. This means that reversible local unfolding may be rapidly followed by irreversible degradation. Hence, strictly speaking, differences in amino acid sequence can alter thermostability not only by affecting the structural stability but also by affecting enzymic activity. This study deals with structural aspects only.

2. Materials

and Methods

(a) Crystallization

The enzyme was isolated and purified by Sidler et al. (1986a), who kindly supplied it deep-frozen in Tris buffer (0.05 iv-Tris-(hydroxymethyl)aminomethane, 0.01 Msodium chloride, 0.01 M-calcium chloride (pH 6.0)). The buffer was exchanged by dialysis against 0.05 Mpotassium succinate containing 0.01 M-calcium chloride at pH 6.0. Autodigestion of the protease in solution was minimized by handling it at temperatures as little above 0°C as possible. Crystallization was carried out by the hanging-drop vapour-diffusion method (Davies & Segal, 1971). The reservoir solutions contained 18 o/o (w/v) polyethylene glycol (PEG 6000) in 0.05 M-potassium succinate buffer (pH 6.0) and 0.01 M-calcium chloride (this calcium ion concentration should ensure full occupancy of the 4 calcium sites necessary for protein stability). Drops of 16 ~1 were formed by mixing equal amounts of reservoir and 20 mg protein/ml solutions. Hexagonal bipyramidal crystals grew to a size suitable for X-ray analysis (although rarely larger than 0.3 mm x 0.3 mm x 0.3 mm) in about 10 days. While the presence of calcium chloride is essential, small variations in PEG concentration, protein concentration, drop size and pH have little effect on the crystallization. The crystals belong to the hexagonal space group P6,22 and have unit cell edges of a = b = 76.5 A and c = 201 A. There is one 35,000 dalton monomeric molecule per asymmetric unit and 12 molecules in a unit cell volume of 952,000 ip3, i.e. V, = 2.27 d3/dalton. This corresponds to a solvent content of 46%, which is normal for proteins (Matthews, 1968) (1 a ==0.1 nm). (b) Data

collection

and processing

The diffraction data were recorded at room temperature on Kodak no-screen (NSST) film using an Enraf-Nonius Amdt-Wonacott rotation camera and nickel-filtered CuKcl radiation from an Elliott GX-20 rotating anode generator operated at 38 kV and 38 mA with a 0.2 mm focusing cup. Crystals with the largest dimension 0.25 to 0.30 mm were mounted in siliconized Debye-Scherrer glass capillaries which were sealed with epoxy resin to avoid the warming inherent in waxsealing. The crystallographic c-axis was oriented parallel to the rotation axis. As crystal slippage can especially be a hazard with bipyramidal morphology, the solvent plugs in the capillaries were given B calcium chloride concentration 20 mM greater than that in the mother liquid at the crystal in an attempt to prevent the crystal

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527

accumulating moisture by vapour diffusion. The I&ray beam was collimated such that the crystals were entirely irradiated. Although the crystals diffracted well, the films had to be placed 100 mm away from the crystal in order to resolve the reflections in the direction of the long c-axis, thereby recording data to only 2.9 a. An oscillation range of 2” per film pack (2 films/pack) was used, and 24 film packs with overlapping data from 6 good crystals completed a total oscillation range of 35”. Each 2 deg. oscillation required an exposure of 11 to 12 h. The crystals were stable for around 45 h of Xray exposure, enabling 6 to 10” of oscillation data to be collected per crystal. No attempt was made to collect the blind region. Pairs of still photographs (at 4 = 0” and 4 = 90” around the spindle axis, 1.5 h exposure/still) were taken at regular intervals to monitor crystal orientabtion. Data from the 24 film packs were digitized on an Optronics Photoscan System P-1000 rotating-drum scanning densitometer with a raster size of 50 pm and a 3.0 optical density scale. The optical densities were corrected for non-linearity in film response b,y an empirically obtained correction curve. Typically around 300 reflections on the front film and 30 on the back film were overexposed. The data were processed using a version of the Cambridge oscillation program system (Nyborg & Wonacott, 1977) which incorporates the profile analysis algorithm of Rossmann (1979) as modified by Wilson et al. (1983). The final Rmergevalue of 3.0 A resolution (where R merge= ~ij(Fi)-F,I/~iF,, and (Fi) is the average of 4 over all of its symmetry equivalents) was 5.5% for 6865 unique reflections (constituting 91 o/o of the unique data) obtained from 35,938 measurements. Data beyond 3.0 ,/I gave significantly worse Rmerge values and were not used.

(c) Structure

determination

Early attempts at structure solution by multiple isomorphous replacement (m.i.r.) included an extensive search for heavy-atom derivatives, covering derivatives of mercury, silver, gold; lead, barium, uranyl, palladium, cadmium, rhenium, ruthenium, platinum, iridium, tungsten, iodine and several lanthanides, using 01.5 to 2.0 mlvr-heavy-atom concentrations and soaking times of 1 to 7 days. Due to lack of success (see Results), the m.i.r. approach was eventually abandoned in favour of structure solution by molecular replacement, using TLN as a trial structure. Co-ordinates of TLN refined at 1.6 ,!% resolution (Holmes & Matthews, 1982) are available from the Brookhaven Protein Data Bank (Bernstein et al., 11977). The rotation search was carried out using the computer program ALMN written by Eleanor Dodson (1York University). This program is a version of the fast rotation function of Crowther (1972). A single TLN moleculle was positioned in a PI unit cell with arbitrarily chosen cell axes of 100 a and cell angles of 90”. Structure factors were then calculated within the resolution range 3 to 15 a assuming an overall temperature factor, B, of 25 8’. The step size (in the Eulerian angle system CI, /I, y) for the b rotation was initially 5” and was decreased to 0.5” in the vicinity of the solution. The step size for the tl and y rotations was 2.5”. The search was carried out using: data in various resolution shells between 3.5 and 8.0 A. The volume of Patterson space used was that within the hollow sphere of inner and outer radii of 5 and 30 A, which produced acceptable results. The correctly oriented molecule was submitted to a translation search using the T2 translation fun&on of

R. A. Pauptit

528 Neutral proteose

Thermolysm

L Figure 2. Schematic diagram showing the orientation of the TLN and NP molecules in their unit cells. The TLN molecules must be rotated approximately 70” if they are t,o pack into the NP unit cell.

et al. were omitted. The resultant maps are sometimes referred to as “omit-maps” (see, for example, Artymiuk & Blake, 1981). Two electron density maps were compiled from such omit,-maps. In the first, the polypeptide chain was divided into 11 segments of approximately 35 amino acid residues each (the segments overlapping by 5 residues), and omit-maps were calculated and examined for each of the segments in turn. In the second: the molecule was divided into 10 sections with almost equal at,om populations. Omit-maps for the 10 sections (which overlapped by 1 grid plane) were combined to give a map covering the whole molecule. This second type of map has the advantage that interactions between different stretches of the polypeptide chain can be modelled, but it involves combining the sections into a single map. The maps were examined and the NP model was rebuilt using the FRODO soft,ware described above. Refinement was carried out using the “TNT” program of Tronrud et al. (1987). This program is a restrained least-squares refinement program that makes use of fast Fourier transforms. The refinement’ was interrupted at’ several stages to allow inspection and correction of the model on a. graphics display system using electron dens&y maps that were calculated with 3F,-2F, Fourier coefficients.

(a) Structure Crowther & Blow (1967), as implemented in the computer program TFSGEh’ written by Dr Ian Tickle (Birkbeck College, London). All the observed data were included. The translation function was carried out in both the enantiomeric space groups PC?,22 and P6,22 in order to resolve the space group ambiguity. (d) Model Improvement The TLK molecule, placed and oriented as indicated by the solution of the molecular replacement method, was subjected to rigid-body refinement using the program CORELS (Sussmann et al., 1977). The model was defined as a 2-domain entity consisting of residues 1 to 156 and 157 to 316. Three cycles of scale factor refinement, 4 cycles of refinement using data between 10 and 8 a resolution, and 3 cycles of refinement including data between 10 and 7 a resolution were performed. It was conceivable that the rigid-body refinement could be adversely affected by contacts involving those TLN sidechains that are not conserved in NP. For this reason, the refinement was repeated using a ‘Xrimmed” TLN model in which the side-chains that were not identical with those in R’P were replaced by alanine or glycine; whichever was more appropriate. The correct KP side-chains were built into the model on an Evans and Sutherland PS-300 eolour graphics display system hosted by a VAX 11/730 computer using FRODO soft’ware (Jones, 1978) developed at Rice University, Texas (Pflugrath et a.l., 1984). The side-chains were positioned manually using only stereochemical criteria as no electron density bad yet been calculated. The conformations of those side-chains that are identical in the 2 proteins were not altered at this stage. For the earliest electron density maps, phase information derived from the modified TLN model had to be used. In order to reduce bias towards t,he TLPU’ model, the maps were limited to a sma.11portion of the molecule (about 10%) for which the contributions to the phasing

determination

Most of the heavy-atom derivatives that produced measurable changes in the X-ray intensities also produced changes in the unit’ cell dimensions, especially in the c direction, and were therefore useless. Only a samarium and a mercury derivative were isomorphous to the native protein, and for these diffractometer data were collected to 6-O a and 8-O -4 resolution, respectively. Unfortunat,ely, the difference Patterson maps were uninterpretable. The molecular replacement was straightforward. The initial orientation search produced a correct peak at the Eulerian angles a = SO”, j3 = 70” and y = 2.5”. The best results were obtained when data. in the resolution range 3.5 a to 8.0 A were used, giving a peak height of 4.5 standard deviations above the mean for the correct solution with the highest spurious peak occurring at 3.7 sta,ndard deviations. The correct peak could a,lso be readily located when data in the resolut,ion ranges 3.5 to 5-O 8, 4.0 to 6.0 A, 5.0 to 7.0 A and 6.0 t,o 8.0 -4 were used, but in the latter two ranges it was not the highest peak. A subsequent search in t,he vicinity of this solution using a 0.5” grid for ,B refined the P-angle to 68.5”. A comparison of the TLX and NP unit cell angles dimensions shows that‘ the above rotation are very reasonable. TLN crystallizes in P6,22 with a = b = 94.2 8, c = 131.4 8, and y = 120”, and the elongated molecules (64 a x 38 A x 37 8) lie wit,h the long molecular dimension 20” out of the a-b plane. In t,he unit cell of NP, t,he c axis is elongated at the expense of t,he a and Fi axes (a = b = 76.5 8, c = 201 d: y = P20”). The situat,ion is drawn schematically in Figure 2. In order to pack the TLN molecules in the NP unit cell, it is

Crystal

Structure

of B. cereus Neutral

necessary to rotate the molecule (roughly 70”) so that its long dimension becomes nearly parallel instead of nearly perpendicular to the long c axis. The results of the rotation function indicate that exactly this happens: the only rotation that effects a change of orientation with respect to the c axis is the p rotation (y is very small and a is a rotation about the c axis), and this rotation has the appropriate value of b = 68.5”. The largest peak in the translation function map for the space group P6,22 (7.3 standard deviations above the mean; highest spurious peak was at 5.5 standard deviations) corresponded to the correct solution. Structure factors calculated for the molecule translated to the position indicated by this solution scaled against the observed data with an R-factor of 50.8%. The highest three independent peaks in the translation function map for space group P6,22 corresponded to positions of the molecule that gave R-factors of 58% (random agreement) and were taken to be incorrect. Thus, the space group in which NP crystallizes was established as P6,22. Further support for the molecular replacement solution was obtained through an intermolecular contact search. If the molecules generated by space group symmetry would interpenetrate, giving rise to too many short intermolecular contacts, the solution would have to be rejected as incorrect. A search for all amino acid residues involved in intermolecular contacts of less than 1.5 a revealed only two such cases. An additional search showed that only 29 out of 317 residues were involved in intermolecular contacts of less than 3.0 8. All of these residues were on the surface of the molecule, nine of them were residues that have shorter sidechains in NP and many of the contacts were in fact potential hydrogen bonds. As an additional check, symmetry-related molecules of TLN in the NP unit cell were generated and the crystal packing was inspected on the graphics display system. The molecules packed beautifully with excellent interdigitation of domains.

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529

to 7 8) was lower than that for the whole molecule refinement (R = 0.42, 10 to 7 d) and so the “trimmed” model was used for further work. On the graphics display system, it could clearly be seen that the two refined models differed considerably more from the starting model than they did from each other. The trimmed TLN model was converted to that containing the correct NP side-chains, which involved changing and orienting the 85 difierent amino acid residues on the graphics display using the original TLN model and general stereochelmical constraints as guidelines. This resulted in a dr’op in R-factor of 5%, to 44%, and revealed some interesting effects of the substitutions (e.g. Leu275 in TLN to Phe275 in NP, see Discussion). The model was subsequently rebuilt into an omitmap consisting of segments covering 30 to 35 residues. The calculated omit-map clearly revealed inaccuracies of the model, especially in side-chain orientation. Figure 3 shows portions of this ma,p. In Figure 3(a) the reorientation required for Thr129 is obvious. Figure 3(b) shows Thr38 and Ile39 after they were moved into the electron density. The clear side-chain density for this threonine, a glycine in TLN, shows that the omit-map is success&l at reducing model bias. The fact that the omit-maps indication produce interpretable density is another that the solution is correct. The quality of the omitmap electron density deteriorated somewhat near the surface of the molecule, but overall it was good

(b) Model improvement The CORELS rigid-body refinements of the whole and of the “trimmed” TLN molecule in the NP unit cell moved the domain centres about 0.4, 0.2 and 0.1 a in the x, y, and z directions, respectively. The rotations around the domain centres showed values of up to 2”, and the two domains did not behave in the same way. The relative orientations of the two domains following CORELS refinement were inspected on the graphics display system. In both refinements, the result was that the two domains had rotated slightly apart such as to widen the active site cleft. Following the CORELS refinement, the R-factor calculated for the new model using all the observed data had dropped to 49 %. The final R-factor for the “trimmed” TLN CORELS refinement (R = O-40, 10

Figure 3. (a) An example of the clarity

with which the after CORELS refinement. The reorientation required of Thr129 (thin bonds) into its position in the electron density (thick bonds) is obvious. (b) An example of the lack of model bias in the omit-map. Thr38 and Ile39 are depicted after they were moved into the electron density. The clear density for the threonine side-chain is very satisfying as this residue is a glycine in TLN. This and all other molecular illustrations in this paper were prepared with the plotting program PIPPIN written by Dr E. L. Mehler (personal communication). omit-map

suggested

model

improvement

530

R. A. Pauptit et al.

for a 3.0 A map and allowed complete rebuilding of the model. This was accompanied by a lowering of the R-factor to 42%. As the model improved on rebuilding into the omit-map, the improved quality of the derived phases should lead to improved quality of a subsequent omit-map, which should in turn allow further improvement of t’he model. Since there was interest in examining salt bridge interactions between different portions of the polypeptide chain, the second omit-map was compiled from sections perpendicular to the c axis rather than from segments of the polypeptide chain. This map presented information not seen in the previous map, and further rebuilding resulted in a lowering of the R-factor to 39.8%. A least-squares fit of the TLN molecule to the NP model provided a useful reference during rebuilding. The model was now deemed sufficiently accurate to start least-squares refinement of the atomic positional parameters. This was initiated at the Institute of Molecular Biology, University of Oregon, Eugene, U.S.A. (by J.N.J., during a sabbatical leave) with the expert guidance of Dr D. Tronrud. The atomic temperature factors and were fixed at 20 A2 and 1.0, occupancies The optimal relative weighting respectively. between the X-ray contributions to the normal equations and the stereochemical contributions was established by trial and error. Suitable results were obtained when the following relative weights were entered into the TNT refinement program: R-factor (i.e. the X-ray weights) 0.001, bond lengths 3: bond angles 2, torsion angles 0, trigonal planes 5, general planes 15, and non-bonded contacts 10. This starting set of weights also proved suitable for the refinement of TLN (D. E. Tronrud, personal communication), which certainly is not intended to suggest that it is generally applicable. The progress of the refinement is illustrated in Figure 4. The data between 15 A and 3 A were used. After four cycles (R = 30.1%) the bond lengths were behaving well but other stereochemical parameters started to degrade. For this reason the R-factor weight was relaxed to 0.0002 and the bond angle weight was doubled. Over the next ten cycles the R-factor gradually increased to 32.2% with a improvement of stereochemistry. concomitant 3F,- 2F, and F,- Fc elect,ron density maps were calcula,ted and used to update the model on the graphics display system in Eugene. This resulted in a 0.4% improvement of the R-factor, to 31.8%. Fifteen cycles of refinement with the R-factor weight set to the original value 0.001 lowered the R-factor to 23.1%. To improve the stereochemistry, four refinement cycles with the R-factor weight relaxed to 0.0005 and an additional four cycles with an R-factor weight of 0.0002 were carried out. This increased the R-factor to 23.9% and the total number of refinement cycles to 37. The refinement was continued in Base1 upon implementing the TNT program on a VAX 8800 computer. The model was once more rebuilt into a 3F,-2F, electron

density map on the graphics display system. Following this, 12 cycles (10 min central processing unit time ea.ch) with an R-factor weight of 0*001 plus six cycles with an R-factor weight of 0.0005 completed the refinement’ to produce an R-factor of 21.7% for a model with good stereochemistry. The weighted r.m.s. deviations from “ideal” geometry are 0.015 A for bond lengths, 3.3” for bond angles, 0.019 A for trigonal planes and 0.018 A for other planes. The resultant model was used to calculate an k;b- Fc difference elect’ron density map that was inspected to locate the first shell of solvent molecules. Peaks greater than three standard deviations were sorted by magnitude, reduced to a single asymmetric unit, and fed into a compmer program written by Dr J. P. Priestle (persona,1 communication), which examines the plausibility of peaks as solvent molecule sites in terms of van der Waals’ and hydrogen bonding contacts to the protein and to other pot,ential solvent molecules. The procedure is similar to that described by Smith et al. (1986) for locating structural heterogeneity. The potential solvent, sites were inspect,ed on the graphics display system using the F. - Fc as well as a 3F,-2Fc electron density map. The eleetron density for the solvent molecules was well-defined in both maps. The solvent molecules that were at! hydrogen bonding distance from the protein, with good stereochemistry, were included in the model. This amounted to 66 water molecules, two of which have 50% occupancy as they a.re located on a Z-fold axis, being hydrogen-bonded to symmetry-related carbonyl groups. Many water molecules are involved in more than one hydrogen bond and several bridge neighbouring molecules The model now includes water hgands to the zinc and calcium(4) ions. With data to only 3.0 A resolution, refinement of the solvent molecules hitherto located, as well as the search for additiona, solvent and not structure, was considered unwarranted carried out. The FO- F, map just mentioned also showed positive difference electron density along some

Figure 4. Progress in the refinement: the R-factor (01;) is plotted against, the refinement cycle number. After cycles 14 and 37 the model was inspected and corrected manually on the graphics display system.

G-y&al

Structure

of B. cereus Neutral

stretches of the polypeptide chain backbone, indicating local overestimation of temperature factor. Refinement of individual atomic temperature factors, which might have produced a slightly lower R-factor, was also not carried out as it was considered unjustified given only 3-O A data. 4. Discussion The NP model is based on the analysis of 3-O A resolution data only, preventing a very exact comparison to TLN (refined at 1.6 A). There are, however, many aspects of the 3 A structure that warrant discussion. the NP structure closely Not surprisingly, resembles that of TLN. The r.m.s. deviation between the superimposed main-chain atoms (N,Ca,C,O) of the two proteins is 0.88 A. This fit improves considerably when the two domains are considered separately, the r.m.s. deviation is 0.69 d for the first domain and 0.54 A for the second domain. Figure 5 presents a superposition of the C”tracings for the two enzymes. In the NP model, the orientation of the two domains results in an active site cleft which is slightly wider than that in TLN. This suggests that the NP crystals may be able to accommodate different, and perhaps larger, inhibitors than those that can be soaked into the TLN crystals. (a) The y-turn

A structural difference results from the insertion of a glycine residue after residue 25, where TLN has a y-turn. The most satisfactory geometry with which the insertion could be modelled into the NP

531

Protease

electron density was with that of a type II’ b-turn (Richardson, 1981), with the inserted glycine at the “i+ 1” or “2” position. The amino acid sequence of the B. subtilis neutral protease contains a deletion rather than an insertion at residue 26 (Yang et al., 1984). The sequence of the neutral protease from the thermophilic B. stearothermophilus (Takagi et al., 1985) has been aligned with the TLN sequence with a single tripeptide insertion (Gly-Tyr-Tyr) after TLN residue 28 (Imanaka et al., 1986). This place,s the insertion in a strand of B-sheet and is unlikely to be correct, especially as the sequences can equally well be aligned with an insertion following residue 25 or 26, i.e. at the y-turn. Thus, it appears that the TLN y-turn is avoided in at least three homologous proteins. (b) Calcium

sites

The metal ion environments are very similar to those in TLN. The electron density for the outer calcium, Ca-2, in the double calcium site has been elusive. Not until the second omit-map could isome density be found, about 1 A further away from Ca-1 than the expected Ca-2 position. In the ‘TLN structure determination, the density for Ca-2 was also weaker than that for Ca-1 (Colman et al., 1,972). In that study the site was confirmed by substitution with the more electron dense strontium and barium ions. However, these metal ions were located O-5 A further away from the Ca-1 position. In NP, upon placing Ca-2 in the observed density, an alternate ligand binding arrangement becomes apparent. The calcium ion still binds to Glu177, Glu190 and to the carbonyl moiety of Asn183,, but

Figure 5. A superposition of the Ctracings of TLN (thin lines) and NP (thick lines) after refinement. The filled circles represent the metal ions. The amino and carboxyl termini, as well as the zinc ion, are labelled. This view shows tha,t the active site cleft is slightly wider in NP. The insertion that transforms the y-turn in TLN to a p-turn in NP appeau-s at the extreme top of the Figure.

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et al

182

Figure 6. The double calcium site. (a) A superposition of the TLN (thin bonds) and K-P (thick bonds) double calcium sites. The calcium hgands Asp138 and Glu177 have been omitted for ciarity. Ca-2 in the NP model (open circle) is closer to Asp191 than is Ca-2 in TLN (filled circle). This suggests that whereas in TLN Asp191 forms a salt bridge with residue 182, in h’P it could be involved in calcium binding. (b) The KP double calcium site in electron density. The density was calculated with 3Fo-2F, Fourier coefficients upon completion of the refinement, and is contoured at one standard deviation. Ca-2 could be bound direct,ly to Asp191 instead of through a water molecule as in TLE.

now Asp191 is also in a position to bind it, in competition with AspI85. The situation is illustrated in Figure 6. If indeed Ca-2 is bound directly to Asp191 rather than through a water molecule as in the TLK model, further credence is lent to the co-operative dissociation of Ca-2 and Ca-4 as proposed by Weaver et al. (1976). The low electron density observed (before refinement) for Ca-2 might be explained if the calcium ion is shared between two ligand binding sites, one closer to Asp185 and one closer to Aspl91. Near the end of the refinement, a difference electron density map was calculated in which the contributions from the calcium ions were omitted from the phasing. In this map, Ca-2 appeared as a single peak of equal strength (10 standard deviations) t’o the Ca-1 peak, indicating that Ca-2 occupies a single site only. The density for Ca-2 was now located approximately 0.5 a closer to Asp191 (i.e. further away from Asp185) than in TLN. In this position, it is not clear at t’he current resolution whet,her Ca-2 binds

AspI91, Asp185 or both. As calcium binding has long been known to affect stability (Endo, 1962), the exact nature of binding at, the double calcium site and any differences to TLK should influence the relative thermostabilities of the two enzymes. Reinvestigation of this region at higher resolution is desirable. (c) Salt bridges Salt bridges, especially those that link amino acids far apart in the sequence, can offer an important contribution to the stabilization of protein folding and hence to thermostability. The I2 salt bridges in the refined structure of TLh’ are listed in Table 1. This list is different from the one published for the unrefined structure (Colman et al., 1972). The first ten salt bridges in Table I; five in each domain, are conserved in NP. Salt bridge I I in TLN is replaced by salt bridge 16 in NP (vi& infra). The 12th salt bridge, Arg285-Asp31I: stabilizes the

Crystal

Structure

of B. cerew

Table 1 The salt bridges in TLN Separation in TLN (ip) 1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16

2-8 2-l 2-7 2-8 3.0 2-8 3-4 2-7 2.6 2.4 3.0 2.7 (5.0) (4.4)

and NP

Base Acid Lys&AsplB Lys18-Asp72 Arg35-Asp32 Arg35-Asp82 HislO&Asp124 Arg203-Asp170 His25GAspZ 15 His231-Asp226 Lvs262%Glu302 A&269-Asp294 [Arg]Lysl82-Asp191 Arg285-Asp311[Ser] [Arg]Lys260-Asp261 Lys2WAsp215 [Val]Lys7%Asp180[Ala] [Lys]Arg182-Asp180[Ala]

Separation in NP (ip) 2.8 2.4 3.2 2.4 2.9 2.5 3.1 2.3 3.3 2.9 2.6 3.1 3.0 2.4

Sequence differences are indicated in brackets.

position of the carboxyl-terminal helix in TLN, but is not present in NP where residue 311 is a serine (see Fig. 7(a)). A single bond rotation would allow Arg285 in NP to interact with the negatively charged carboxyl terminus, but this does not seem to occur, The positioning of the carboxyl-terminal helix could be one of the final stages in protein folding (and one of the first in unfolding) and hence could affect the relative stabilities of the two enzymes. While TLN has two salt bridges not found in NP, the latter contains four salt bridges (13 to 16 in Table 1) that are absent in TL,N (see Fig. 7). In TLN, Asp261 is located 5.0 A from Arg260 and 5.4 A from Lys265, i.e. about midway between two positive charges. In NP one of these positive charges is lost as Lys265 becomes an alanine, and Asp261 forms a well-defined salt bridge with the remaining positively charged side-chain of Lys260 (salt bridge 13 in Table 1; see Fig. 7(b)). Salt bridges between adjacent residues, however, presumably contribute little towards the stability of the overall folding. Salt bridge 14, Asp215-Lys219, does not occur in the TLN crystal structure even though the residues involved are conserved (see Fig. 7(c)). In TLN Asp215 and Lys219 are separated by Tyr251 (both form hydrogen bonds with the Tyr251 hydroxyl group). This is not the case in NP, where Tyr251 only interacts with Lys219, allowing the Asp215 Lys219 salt bridge to be made. The reason this charge interaction is not exploited in the TLN crystal structure may be found in the different crystal packing arrangements of the two enzymes. The .Asp215 and Lys219 residues are both involved in salt bridges to a neighbouring molecule in the TLN structure: Asp215 forms a 2.6 A salt bridge to the neighbouring Lys45, and Lys219 forms a 2-9 A salt bridge to the neighbouring Asp43. It can reasonably be assumed that in solution, the

Neutral

Protease

533

Asp215Lys219 salt bridge observed in the NP structure can be formed in both enzymes. The Lys79-Asp180 salt bridge found in NP (salt bridge 15 in Table 1) cannot be present in ‘TLN where residues 79 and 180 are valine and ala:nine, respectively. This salt bridge is of special interest to protein stability as it connects the two domains, on the side of the protein opposite the active site cleft. It is unlikely that this salt bridge is responsible for the small differences in domain orientation and the widening of the active site cleft in NP mentioned earlier, since the main-chain separation between residues 79 and 180 does not differ significantly from that in TLN. Asp180 is not only involved in the NP interdomain salt bridge, but also in a salt bridge with Arg182. The side-chain of residue 182 has a completely different orientation in TLN, where the salt bridge Lys182-Asp191 is observed i.e. salt bridge 11 (Table 1) in TLN is replaced by salt bridge 16 in NP. This means that in NP, Asp 191 is free to bind Ca-2 at the expense of the Ca-2Asp185 interaction! The situation is depicted in Figure 7(d). Thus, the double calcium site is affected by a salt bridge involving the other domain. Even if it is argued that the NP salt bridges 13 and 14 (see Table 1) do not offer llarge stabilizations, and that NP salt bridge 16 is only a replacement for the TLN salt bridge 11, the missing carboxyl helix positioning salt bridge 12 foun.d in TLN is presumably still adequately compensated in NP by the interdomain salt bridge 15 alone. Thus, it appears that, in contrast to the ferredoxin system (Perutz & Raidt, 1975), the enhanced thermostability of TLN cannot be attributed to the presence of additional salt bridges. This is consistent with the lack of influence of ionic strength on the thermal inactivation temperature observed by Pangburn et al. (1976). (d) Hydrogen

bonding

Hydrogen bonding interactions are less powlerful but much more numerous than salt bridges and also play an indispensable role in the stabilization of protein folding. In the refined TLN model, 275 hydrogen bonds can be counted in which the participating non-hydrogen atoms are no further than 3.2 A apart. In the NP model, 226 hydrogen bonds are found that obey the same cutoff criterion. Of the 226 interactions counted in NP, 182 also occur in TLN, 15 cannot occur in TLN because of amino acid sequence differences, 17 do not occur in TLN because of different side-chain orientations, and 12 main-chain hydrogen bonds are not seen in TLN. Of the 275 interactions counted in TLN, 182 are common to NP, 18 cannot occur in h’P because of sequence differences, 27 are missing in NP because of different side-chain orientations, and 48 main-chain hydrogen bonding interactions are not observed in NP. The last number is too high as the main chain folding is almost identical for the two proteins. If we increase the hydrogen bond length

534

R. A. Pauptit et al.

535

Crystal Structure of B. cerem Neutral Protease cutoff first to 3.3 a and then to’ 3.5 a, the number of missing main-chain hydrogen bonds in NP decreases from 48 to 37 and 18, respectively, suggesting that the discrepancy is due to model inaccuracy resulting from the limited resolution. Thus, a final conclusion concerning the total number of hydrogen bonds in the two proteins and the effects on thermostability would be premature. It is still encouraging, however, that as a result of sequence differences and different side-chain orientations, the more thermostable TLN appears to be able to form a greater number of hydrogen bonds than NP. An analysis at higher resolution will be necessary to confirm this. The enhanced stability of a thermostable variant of subtilisin, for which the crystal structure was determined at 1.8 A resolution, has also been attributed to slight improvements in hydrogen bonding parameters (Bryan et al., 1986). (e) Preferred exchanges Argos et al. (1979) have shown that in the systems they studied the five most frequent amino acid exchanges between related mesophilic and thermophilic proteins are, in decreasing order of frequency: (1) Gly to Ala; (2) Ser to Ala; (3) Ser to Thr; (4) Lys to Arg; and (5) Asp to Glu. The direction of exchange here (and throughout this paper) is from the less to the more thermostable protein. Sidler et al. (19866) reported that these exchanges only partially preferred “cold-hot” agreed with those observed for TLN and related proteins. Such exchanges can now be more carefully examined in the NP/TLN enzyme pair. It should be emphasized that these five exchanges do not occur with a statistically meaningful frequency in a single pair of proteins. Nevertheless, since they could be responsible for enhanced thermostability it is worth examining their effects on the structures. The most frequent amino acid exchange in proteins observed by Argos et al. (1979) on going from a mesophile to a thermophile is that from Gly to Ala. There are two such exchanges between NP and TLN (at residues 141 and 287), and none in the opposite direction. The rationale for increased thermostability arising from this exchange is that the alanine residue affords greater a-helical strength and hydrophobieity. Indeed, both exchanges occur in helices in the hydrophobic core. This exchange could also enhance the stability of the folded protein through entropic considerations, since a glycine residue sacrifices more conformational freedom upon folding than does an amino acid residue with a @atom (Matthews et al., 1987).

Residue

141 is located

between

two

helices,

at the

where

closest contact

a small

side-chain

is

required.

The sequence of B. stearothermophilus neutral protease also contains a glycine at residue 141, and it has been demonstrated that exchanging this for an alanine increases the thermostability (Imanaka et al., 1986). Ala287 is adjacent to Leu275 in the TLN hydrophobic core. It is interesting to note that as Leu275 is exchanged for Phe275 in NP, its neighbours, Leu284 and Ala287, are exchanged for Ala284 and Gly287 such that the overall sidechain volume and main-chain separation are conserved.

Only two of the nine Ser/Ala exchanges between NP and TLN (at residues 153 and 309) are in the direction that Argos et al. (1979) interpreted as enhancing thermostability. Ala153 is located at the start

of a helix

and Ala309

within

a helix

in TLN,

so the argument that alanine increases helix stability may apply here. However, in both cases hydrogen bonding interactions in NP are lost in TLN. Of the seven exchanges in the opposite direction, six result in hydrogen bond formati.on in TLN. The seventh serine extends into the solvent, where the more hydrophilic residue is likely to be favoured.

Only

two

of these seven serines,

Ser291

and Ser305 are found in helices in TLN, and only the hydrogen bonding of @er291 interferes wit’h the helical

hydrogen

bonding

network.

Although

these

seven exchanges are in the direction opposite to that expected by Argos et al. (1979) it is not difficult to see how they may benefit thermostability. Ser to Thr exchanges are believed to affect thermostability by increasing bulkiness and hydrophobicity, so if they are to be effective they would be expected to occur in the interior of the molecule. Only a single one of the nine Ser/Thr exchanges (Ser103 in TLN) between the two proteases occurs in the interior of the molecule at the edge of the hydrophobic core (where it is hydrogen-bonded to a glutamate residue), and this is one of the four exchanges that are not in the predicted direction. The remaining exchanges occur at the surface of the molecule. The contribution towards thermostability of a lysine to arginine exchange may be rationaliz,ed in terms of the greater potential for solvation and hydrogen bonding of an arginine side-chain. Three lysine

residues

in NP,

Lysll,

LyslOl

and Lys260

become arginine residues in TLN (exchanges in the predicted residue,

direction), while only one arginine Arg182, becomes a lysine. Argll and

Arg260 in TLN contribute

guanidinated

extend ‘&to the solvent and may

to thermostability

lysines

(Qaw

as demonstrated

&

Brewer,

for

11986).

Figure 7. Salt bridges that are different in TLN (th in bonds) and NP (thick bonds). (a) Arg28&Asp311: this salt bridge only occurs in TLN as residue 311 is a serine in NP. (b) Lys260-Asp261: in TLN this salt bridge does tiot exist, perhaps due to a weak attraction of Asp261 by Lys265. (c) Lys219-Asp215: crystal packing effects are responsi’ble for disrupting this salt bridge in TLN, see the text. (d) The interdomain salt bridge Lys79-Asp180 in NP is not folund in TLN where residues 79 and 180 are Vai- and Ala. In NP, Asp180 also forms a salt bridge with Argl82, replacing the Asplgl-Lys182 salt bridge found in TLN. Thus, in NP Asp191 is free to interact with Ca-2. Then Cai2 fn the NP model (open circle) is indeed closer to Asp191 than is the Ca-2 in TLN (filled circle).

R. A. Pauptit et al.

536

Residue 101 forms a hydrogen bond to Tyr42 in both proteins. Residue 182 is involved in different salt bridges in NP and TLN and indirectly affects the double calcium site, complicating the situation here. There is only one Asp/Glu exchange (at residue 150) between TLN and NP, and it is not in the direction predicted by Argos et al. (1979). However, Asp150 in TLN forms a hydrogen bond with Trpll5, and this is prevented in NP by the greater length of the glutamate side-chain. Hence this exchange, although not in the predicted direction, may still favour thermostability in TLN. Other preferred exchanges in the TLN system mentioned by Sidler et al. (19863) are Asn to -4s~ and Ile to Val. Only one Asn to Asp exchange occurs between NP and TLN at Asp94 in TLX, which extends into the solvent where the charged side-chain may indeed be favoured. Two Ile to Val exchanges (at residues 140 and 192) and one Val to Ile exchange (at residue 1) occur without resulting in obvious structural perturbations.

5. Conclusion The general “cold-hot,” exchanges suggested by Argos et al. (1979) have limited relevance to NP and TLX. Obviously, not only the type of exchange but also its location in the three-dimensional structure of the protein must be considered when examining its effect on thermostability. There is no evidence that TLN is stabilized by a greater number of salt bridges, but some indications that TLN may have a greater number of intramolecular hydrogen bonding interactions than NP were found. Furthermore, the Gly to Ala exchanges seem to be important, as shown by the experiments of Imanaka et al. (1986). Finally, it seems that there are differences in the environment of the double calcium site which may have an impact on the overall stability of the enzyme. A recent collection of 19 A resolution data for NP at the DESY synchrotron source in Hamburg should ena.ble a more precise (by R.A.P.) comparison between NP and TLN in the future. We thank

Professor H. Zuber and Dr W. Sidler for

suggestmingthis project to us and for generous gifts of enzyme. J.N.J. is grateful to Professor B. W. Matthews, University of Oregon, for his hospitality and support, and for communicating unpublished material to us. We are grateful to Dr D. Tronrud, University of Oregon, for computing advice and permission to use the TNT program in Base]. Thanks are due to several cblleagues, especially Dr E. L. Mehler and Dr J. P. Priestle, for use of their computer programs, and to the University of Base1 Computing Centre (URZ) for computing facilities. R.A.P. is grateful for an EMBO short term fellowship which has allowed him to continue this project by collecting high resolution data at the DESY synchrotron source in Hamburg. This work was supported in part by grants 3.224-0.82 and 3.098-0.85 from the Swiss National Science Foundation to J.N.J.

References Argos, P., Rossmann, M. G., Grau, U. M., Zuber. H.. Frank, G. & Tratschin, J. D. (1979). Biochemi&~. 18, 5698-6703. Artymiuk; P. J. & Blake, C. C. F. (1981). J. ~!Iol. :3io;. 152, 737-762. Bernstein, F. C., Koetzle, T. F.. Williams, 6. J. B.. Meyer, E. F., Jr, Briee, M. D.. Rodgers, J. R.: Kennard, O., Shimanouchi, T. 8z.Tasumi, M. (1977). J. Mol. Riol. 112, 535-542. Bruschi, M., Cambillau, G.; Bovier-Lapierre, G., Bonicel, J. & Forget, P. (1986). Riochim. Biophys. Acta. 873. 31-37. Bryan P. N., Rollence, M. L., Pantoliano, M. W., Wood. J.; Finzel, B. C., Gilliland, G. L., Howard, A. J. & Poulos, T. L. (1986). Proteins: Struct. Fun& Genet. 1, 326-334. Colman, P. M., Jansonius, J. N. & Matthews; 5. W. (1972). J. bb.11.RioE. 70. 701-724. Creighton, T. E. (1983). In Proteins. Structures and Molecular Principles, p. 299, W. H. Freeman, New York. Crowther, R. A. (1972). In The Molecular Replacement Method (Rossmann, M. G.; ed.), pp. 173-178, Gordon and Breach, New York. Crowther, R. A. & Blow, D. M. (1967). Acta Crystailogr. 23, 544-548. Davies, D. R. & Segal, D. M. (1971). In Methods in Enzymology (Jacoby, W. B., ed.), vol. 22, pp. 2666 269, Academic Press, New York. Endo, S. (1962). Nakl%o Kogaku Zasshi, 40, 346-353. Feder, J., Keay, L.; Garret’& L. R., Cirulis, N., Mosely. M. H. & Wildi, B. S. (1971). Biochim. Biophys. Acta. 251> 74-78. Frank, G.; Haberstich, H. U., Schar, H. P., Tratschin, J. D. & Zuber, H. (1976). In Enzymes and Proteins from Thermophilic Microorganisms (Zuber, H.: ed.), Verlag, Base1 and Birkhauser pp. 375-389, Stuttgart. Grandi, C., Vita. C., Dalzoppo, D. & Fontana, A. (1980). Int. J. Peptide Protein Res. 16. 327-338. &titter, M. G., Hawkes, R. B. & Matthews, B. W. (1979). Nature (Lo&on), 277, 667-669. Hocking, J. D. & Harris, J. I. (1976). In Enzymes and Proteins ~frfromTherm,ophilic iMicsoorganisms (Zuber, H., ea.), pp. 121-133, Birkha,user Verlag. Base1 and St,uttgart. Holmes, M. A. & Matthews; B. W. (1982). J. Mol. Biol. 160, 623-639. Imanaka, T., Shibazaki, M. & Takagi. M. (1986). Xatwre [ LondorL) , 324, 695-697. Jones, T. A. (1978). ./ Appt. Crystallogr. 11: 268--272. Levy, P. L., Pangburn, M. K., Burstein, Y., Ericsson L. H.; Neurath, H. & Walsh. K. A. (1975). Proc. Xat. Acad. Sci., U.S.A. 72, 4341-4345. itlatsumura, M., Yasumura, S. & Aiba, S. (1986). Nature (London), 323, 356-358. Matthews, B. W. (1968). J. Mol. Biol. 33, 491497. Matthews, B. W., Jansonius, J. N., Colman, P. W., Schoenborn, B. N. & Dupourque, D. (1972a). aaiure LVewBid. 238, 3741. Matthews, B. W., Colman; P. M., Jansonius, J. S.; Titani; K.; W’alsh, K. A. & Neurath, H. (1972bj. ,Vature New Biol. 238, 4143. Matt,hews, B. W., Nicholson, H. 83 Becktel, W. J (1987). Proc. Nat. Acad. Sci., U.S.A. 84, 6663-6667. Nyborg, J. & Wonacott. A. J. (1977). In The Rotntion Method in Crystallography (Arndt, U. W. &

Crystal

Structure

of B. cereus Neutral

Wonacott, A. J., eds), pp. 139-152, North Holland Publishing Company, New York. Pangburn, M. K., Levy, P. L., Walsh, K. A. & Neurath, H. (1976). In Enzymes and Proteins from Thermo(Zuber, H., ed.), pp. 19-30, phi& Microorganisms Birkhauser Verlag, Base1 and Stuttgart. Per&z, M. F. (1978). Science, 201, 1187-1191. Perutz, M. F. & Raidt, H. (1975). Nature (London), 255,

256-259. Pflugrath,

J. W., Saper, M. A. & Quiocho,

F. A. (1984). In

Methods and Applications in Crystallographic Computing (Hall, S. & Ashida, T., eds), p. 404, Oxford University Press, London and New York. F. S. & Brewer, J.-M. (1986). Mol. Cell. Biochem. 71, 121-127. Richardson, J. S. (1981). Advan. Protein Chem. 34, 167339. Rossmann, M. G. (1979). J. AppZ. Crystallogr. 12, 22% 238. Sidler, W., Kumpf, B., Peterhans, B. & Zuber, H. (1986a). AppZ. Microbial. Biotechnol. 25, 18-24. Sidler, W., Niederer, E., Suter, F. & Zuber, H. (19866). BioZ. Chem. Hoppe-Seyler, 367, 6433657. Singleton, R., Jr & Amelunxen, R. E. (1973). Bacterial. Qaw;

Rev. 37, 320-342.

Protease

537

Smith, J. L., Hendrickson, W. A., Honzatko, R. B. & Sheriff, S. (1986). Biochemistry, 25, 50185027. Sussman, J. L., Holbrook, S. R.; Church, G. M. & Kim, S. (1977). Acta Crystallogr. sect. A, 33, 800-804. Takagi, M., Imanaka, T. & Aiba; S. (1985). J. Bacterial. 163, 824-831. Titani, K., Hermodson, M. A., Ericsson L. H., Walsh, K. A. & Neurath, H. (1972). Nature New BioZ. 238, 35-37. Tronrud, D. E.: Ten Eyck, L. F. & Matthews, 13. W. (1987). Acta Crystallogr. sect. A, 43, 489-501. Weaver, L. H., Kester, W. R., Ten Eyck, L. F. & Matthews, B. W. (1976). In Enzymes and Proteins from Thermophilic Microorganisms (Zuber, H., ed.), pp. 31-39, Birkhauser Verlag, Base1 and Stuttgart. Wilson, K. S.; Stura, E. A., Wild, D. L., Todd, :R. J., Stuart, D. I., Babu, Y. S., Jenkins, J. A., Standing, T. S.; Johnson, L. N., Fourme, R., Kahn, R., Gadet, A., Bartels, K. S. & Bartunik, H. D. (1983). J. AppZ. Crystallogr. 16, 2841. Yang, M. Y., Ferrari, E. & Henner, D. J. (1984). J. Bacterial. 160, 1521. Zuber, H. (1976). Editor of Enzymes and Proteins from Thermophilic Microorganisms, Birkhguser Verlag, Base1 and Stuttgart.