J. Mol. Biol. (1990) 213, 167-186

Refined Crystal Structure of Type III Chloramphenicol Acetyltransferase at 1.75 A Resolution A. G. W. Leslie~ Blackett Laboratory Imperial College, London S W 7 2 B Z , U . K . (Received 27 October 1989; accepted 12 J a n u a r y 1990) High level bacterial resistance to chloramphenicoi is generally due to O-acetylation of the antibiotic in a reaction catalysed by chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) in which acetyl-coenzyme A is the acyl donor. The crystal structure of the type III enzyme from Escherichia coli with chloramphenicol bound has been determined and refined at 1"75 A resolution, using a restrained parameter reciprocal space least squares procedure. The refined model, which includes chloramphenicol, 204 solvent molecules and two cobalt ions has a crystallographic R-factor of 18"3% for 27,300 reflections between 6 and 1"75 A resolution. The root-mean-square deviation in bond lengths from ideal values is 0.02 A. The cobalt ions play a crucial role in stabilizing the packing of the molecule in the crystal lattice. CAT is a trimer of identical subunits (monomer M r 25,000) and the trimeric structure is stabilized by a number of hydrogen bonds, some of which result in the extension of a fl-sheet across the subunit interface. Chloramphenicol binds in a deep pocket located at the boundary between adjacent subunits of the trimer, such that the majority of residues forming the binding pocket belong to one subunit while the catalytically essential histidine belongs to the adjacent subunit. Hisl95 is appropriately positioned to act as a general base catalyst in the reaction, and the required tautomeric stabilization is provided by an unusual interaction with a main-chain carbonyl oxygen. somal loci have also been reported. In enteric bacteria in particular, it is often a component of plasmids conferring multiple drug resistance (Shaw, 1983). The type I CAT gene, as typified in the transposon Tn9, is widely used as a tool for studying gene expression in eukaryotic systems (Gorman et al., 1982). Characterization of the enzyme has focused on the type III variant as encoded by the plasmid R387 and purified from Escherichia coll. This enzyme has been studied using kinetic and chemical techniques (Kleanthous & Shaw, 1984; Kleanthous et al., 1985) and more recently by sitedirected mutagenesis (Murray et al., 1988; Lewendon et al., 1988). This work has led to the identification of an essential histidine residue (His195) and a proposed mechanism wherein His195 acts as a general base in catalysis (Kleanthous et al., 1985). A number of mutants of the type I enzyme have also been studied (Burns & Crowl, 1987). The principle reaction catalysed by CAT is the 3-O-acetylation of chloramphenicol using acetylCoA as the acyl donor. However, 3-acetyl chloramphenicol undergoes a non-enzymic and pH dependent re-arrangement to 1-acetyl ehloramphenicol, which can then be acetylated a second time to yield the 1,3 diacetyl derivative. The second acetylation step is significantly slower than the first, and is not required to inactivate the antibiotic as the mono-

1. Introduction

Chloramphenicol was one of the first broad spectrum antibiotics to be used clinically, although its initial widespread use has now been severely restricted due to serious toxic side-effects. It belongs to the class of antibiotics that act by inhibiting bacterial ribosome function. In the case of chloramphenicol this is achieved by binding to the peptidyl transferase centre, "effectively blocking bacterial protein synthesis (Gale et al., 1981). High levels of resistance to the drug have been shown to be due to the . enzyme chloramphenicol acetyltransferase (EC 2.3.1.28), which catalyses the transfer of an acetyl group from acetyl-CoA to the primary (C-3) hydroxyl of chloramphenicol (Shaw, 1967, Suzuki & Okamoto, 1967). The modified drug no longer binds to the bacterial ribosome, and loses its antibacterial activity (Shaw & Unowsky, 1968). In many cases the CATS gene is plasmid borne, although chromo~"Present address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K. :~Abbreviations used: CAT, chloramphenicol acetyttransferase; Cm, chloramphenicol; MPD, 2-methyl2,4-pentandiol; m.i.r., multiple isomorphous replacement; PHMB, para-hydroxymercuribenzoate; PICM, para-iodo chloramphenicol; r.m.s., root-mean-square. 0022-2836/90/090167-20 $03.00/0

167

© 1990 AcademicPress Limited

A. G. W. Leslie

168

acetyl d e r i v a t i v e is devoid o f antimicrobial activity. A t least 15 n a t u r a l l y occurring v a r i a n t s of C A T from both G r a m positive a n d G r a m negative bacteria have been described (for a review, see Shaw, 1983). T h e a m i n o acid sequences of ten of these have been d e t e r m i n e d (Alton & Vapnek, 1979; S h a w et al., 1979, 1985; H o r i n o u c h i & Weisblum, 1982; Charles et al., 1985; M u r r a y et al., 1988; I. A. M u r r a y & W. V. Shaw, personal c o m m u n i c a tion) and these show a high degree o f h o m o l o g y , r a n g i n g f r o m 30~/o to 77O/o identity. All v a r i a n t s have similar s u b u n i t molecular weights (M r 25,000), and crystallographic (Leslie et al., 1986) a n d h y d r o d y n a m i c studies ( H a r d i n g et al., 1987) h a v e shown t h a t the molecule is a trimer, an unusual oligomeric s y m m e t r y for a soluble enzyme. A p r e l i m i n a r y discussion of the s t r u c t u r e of C A T as d e t e r m i n e d by X - r a y diffraction has a l r e a d y been published (Leslie el al., 1988). In this p a p e r the details of the s t r u c t u r e d e t e r m i n a t i o n a n d refinem e n t of the b i n a r y complex of C A T + chlorampheuicol (Cm) at 1"75 A (1 A = 0"l nm) resolution is reported. The details o f the complex with CoA will be given in a s u b s e q u e n t paper.

2. Experimental

(a) Crystallization and derivative search Crystals were grown by microdialysis against 2 to 4 % (v/v) MPD, l0 mM-MES (pH 6.3), 0"5 mMchloramphenicol, 0"5 mM-cobaltic hexamine chloride at 4°C (Leslie el al., 1986). Crystals were harvested into the same solution as the dialysate but containing 8% MPD. The space group of the crystals was determined by precession photography as R32 (cell dimensions a = 74"5 A, = 92-4°), with one subunit (M~ 25,000) in the crystallographic asymmetric unit. This gives a specific volume (Matthews, 1968) Vm = 2-74 _~3 dalton-i. All computation was performed using the equivalent hexagonal cell with a = 107"6 A, c = 123"6 A. Potential isomorphous heavyatom derivatives were screened using either precession photography or a single oscillation photograph. In all cases derivative crystals were prepared by soaking native crystals in a solution of the appropriate heavy-atom compound. (b) Data collection and processing All X-ray data were collected photographically using an Arndt-Wonacott oscillation camera. During data collection the crystals were cooled to +5°C in a stream of cold air to minimize the effects of radiation damage. The 1"75 A resolution native dataset was collected on the Wiggler line at the S.E.R.C. synchrotron radiation source at Daresbury, U.K., using radiation of wavelength 0"90 A. Three film packs were used, with interleaving 150 ~m thick A1 foils to extend the dynamic range. All other data were collected using graphite monochromatized, CuKa radiation from a rotating anode source. The films were digitized on a Joyce-Loebl Scandig 3 microdensitometer, with a 50 #m raster and 0"0 to 2-0 units optical density range. Films were processed with the new extended version of the MOSFLM program suite developed at Imperial College, which includes options for auto-indexing "still" photographs to determine crystal orientation

(using an algorithm due to Howard, 1986) and incorporates a profile fitting algorithm (Leslie, 1987) to improve the accuracy of weakly recorded diffraction spots. As each film pack was processed the postrefinement program POSTCHK was used to monitor the crystal orientation so that any crystal slippage could be detected and the orientation corrected appropriately. Subsequent data reduction and scaling was performed with the CCP4 programs ROTAVATA and AGROVATA. At the AGROVATA step the reflection intensities were merged to give: I = ¢olprof + ( l - oJ)I z,

with ~o = l / ( l + ( I ~ / ( I ) ) 3 ) ,

where I, lp~of, I~ are the merged, profile fitted and summation integration intensities, respectively; ( I ) is the mean intensity for the entire data set. This procedure gives higher weight to the summation integration intensities for strong reflections where systematic errors in the profile fitting can give rise to large errors, but allows the profile fitted intensities to dominate for weak reflections, where the reduction in random error afforded by profile fitting is greatest. (c) m.i.r, refinement and phasing The Patterson function for the iodinated chloramphenicol (PICM) derivative was readily interpretable in terms of 2 iodine sites. All other derivatives were solved using difference Fourier methods. The correct hand of the structure was established using the anomalous data from the PICM derivative, and calculating a difference Fourier for the platinum derivative (Blundell & Johnson, 1976). The heavy-atom parameters were refined using a pha,~e refinement program. The parameters of each derivative were refined in turn, using phases based on the remaining derivatives (Blow & Matthews, 1973). Pha~ses based on this refinement were used to calculate a "best" native Fourier map (Blow & Crick, 1959). (d) Map interpretation and model building The path of the polypeptide backbone was traced initially from a "minimap" (scale 4 A -- l cm) plotted on acetate sheet. The interpretation was readily confirmed using a version of the interactive graphics program FRODO (Jones, 1978) which incorporates the chain tracing algorithm of J. Greet (1985), in which the skeletonized backbone is represented by a series of bonded "bones" atoms. A molecular model was built using the "fragment fitting" feature of the FRODO program (Jones & Thirup, 1986). In this procedure the user identifes a number of bones atoms that represent the positions of alpha carbon atoms for a short contiguous stretch of the polypeptide chain. The program then searches a database of well-determined protein structures (a total of 36 in the version used) to find a fragment of polypeptide chain that provides the best fit to the chosen bones atoms. To ensure a good fit, it was usually neccessary to adjust the positions of the "alpha carbon" bones atoms so that their separation was within 0"15 A to the correct value of 3"84 A. (Bones atoms are initially placed at grid points in the Fourier map, and can therefore be up to one-half grid interval away from the "best" alpha carbon position as judged by the appearance of the electron density.) Typically, segments of between 4 and 7 residues in length were fitted, and the r.m.s, deviation between the positions of the bones atoms and the alpha carbons of the best frag-

Structure of Chloramphenicol Acetyltransferase ment ranged from 0"3 to 0"6 A. This approach was most valuable in building the model in those regions where the electron density was rather poorly defined, usually in extended loops or turns in the polypeptide backbone. The stereochemistry of chloramphenicol was taken from the single crystal structure (Chatterjee el al., 1979).

(e) Model refinemenl The model was refined using a modified version of the restrained least-squares structure factor refinement program PROLSQ (Hendrickson & Konnert, 1980) kindly supplied by E. J. Dodson. In this version the structure factor amplitudes and their derivatives" are calculated using the fast Fourier transforms (Agarwal, 1978; Isaacs, 1982). The refinement required 25 min/cycle at 2"5 A resolution {1680 atoms, 9500 reflections) or 44 min/cycle at 1-75 _~ resolution (1932 atoms, 27,300 reflections) on a VAX 11/750. In the initial stages of refinement X-ray data to 2"7 A resolution were included. Typically, 10 cycles of refinement were followed by manual rebuilding using an Evans and Sutherland PS300 interactive graphics display. Rebuilding was performed using Fourier maps with coefficients ( 3 [ F J - 2 [ F J ) and calculated model phases. Segments of polypeptide missing from the initial model were built using the bones option when the electron density had improved sufficiently to allow an unambiguous interpretation. Initially only an overall temperature factor was refined, but atomic temperature factors were refined when the R-factor had dropped to 32"9% for data between 10 A and 2'5 A resolution. For the first 2 rounds of refinement and rebuilding, the entire potypeptide chain was examined to detect errors in the initial model. In all subsequent rebuilding, attention was restricted to those parts of the model that gave rise to large features in the difference electron density map. A peak search routine located the largest peaks in the difference map, and a subsequent program due to T. Skarzynski associated these peaks with the nearest protein atoms. The list of peaks were examined in order of

169

decreasing magnitude until a level was reached at which the peaks were no longer interpretable, typically between 3 and 4 times the r.m.s, level of the electron density map. This approach facilitated rapid correction of the most significant errors in the model. During the final rounds of refinement, residues with poor stereochemistry were also examined during the rebuilding step. Solvent molecules were introduced when the R-factor had dropped to 27"8% (10 to 2"5 A resolution). Initially only water molecules in the 1st hydration shell were included (those with at least 1 hydrogen bond to a protein atom) although in the later stages a few waters in the 2nd hydration shell were introduced. Solvent atoms were only introduced if they made at least l plausible hydrogen bond with either a protein atom or another solvent atom. Following the 1 lth round of refinement (R -- 18"7°/o for data between 6 and 1"75 A) the solvent structure was completely re-examined. All solvent atoms were deleted from the model, and a difference map with coefficients [Fo[--[F~[, a¢ was calculated. Solvent molecules were introduced at the positions of positive peaks greater than 3 times the r.m.s, level of the electron density, providing that (1) they were capable of forming at least l hydrogen bond and (2) the site could not be interpreted as a partially ordered side-chain position. Additional solvent atoms were added in subsequent rounds of refinement, again subject to the criteria given above. (f) Solvent-accessible area Solvent-accessible areas were calculated using the algorithm of Lee & Richards (1971), with a probe radius of 1.4 A. (g) Residue numbering A standard numbering scheme for CAT has been adopted on the basis of an alignment of 7 CAT sequences.

Table 1

Diffraction data statistics Resolution (A)

Unit a

Native Native Native

2'5 1-75 1-75+2-5

107-6 107-6 107-6

123'6 123-6 123-6

40,533 94,171 36,931

9661 27,270 28,038

KAu{CNh 5 mM for 6 days KAu(CNh 2 mM for 6 days PICM 1"75mM for 3 days PHMB 66% saturated for 5 days K2PtCt4 90/~M for 16 h Sm(N03) 3 1-25mM for 16 h

2"7 2'7 2'7 2"7

107"6 107'6 107"8 107-4

123'6 123"6 123"6 123-5

26,192 26,936 17,839 14,663

2"7 2"7

107"8 107"7

122"8 123-5

13,278 17,498

Data set

Cell (A)t Number of a measurements

Unique data

R~,rg~$

Rderiv§

Percentage complete

7417 7411 7386 6797

5"9{14'0) 5"8{14.7) 4"5(5"2 at 2"5 A) 4"4(8"7) 4"4(8'1) 4"5(14"5) 4"6(9'7)

99"5 96-7 99-5 17"0 13'2 10'1 12"1

95-8 95"7 95"1 87'8

6321 6978

7"9(29"4) 4"2(14"4)

16"0 9"1

81'6 90"1

3?The cell parameters given are those for the hexagonal unit cell equivalent to the true R32 cell.

ZZIl(h)j-l R~,,,. =

ZZI-(h b

'

where I(h) is the measured diffraetion intensity and the summation includes all observations. The Rr*~,8,in the outermost resolution bin is given in parenthesis. ZF.,,

'

where F,,, and Fn,,l, are the native and derivative structure factor amplitudes.

A. G. W. Leslie

170

For the type III enzyme described here, residues 1 to 73 and 74 to 213 of the linear sequence (Murray et al., 1988) are numbered 6 to 78 and 80 to 219, respectively. 3. Determination of the Structure

(a) Diffraction data Crystals of CAT diffract to at least 1"7 A resolution ~nd show little radiation damage after prolonged exposure to X-rays. As a result each of the derivative data sets was collected from a single crystal, and only two crystals were required for each of the two native data sets. With the exception of the K2PtCI4 derivative, which diffracted rather weakly, the quality of the X-ray data was excellent (Table 1). This certainly contributed to the quality of the final m.i.r, phases, particularly as anomalous differences were used for three of the derivatives and the anomalous signal is typically of about the same magnitude as the errors in the observed structure factors. The effect of merging the intensity estimates derived from profile fitting and summation integration as described in Experimental, section (b), can be illustrated for the 1"75 A native data set. The merging R factor was 6"1°/o (20"7% at 1"8 A) for summation integration intensities, 6"4~o (14"7~/o at 1"8 A) for profile fitted intensities, and 5"8~o (14"7~/o at 1"8 A) for the merged intensities. (b) Heavy-atom derivatives and multiple

isomorphous replacement Over 40 compounds were screened and full data sets were collected from five of these (Table 1). The refined heavy-atom parameters and the residues

close to the heavy-atom positions in the final refined structure are shown in Table 2. The occupancies are those obtained with the native data on an absolute scale (derived from a Wilson plot) and should therefore correspond to the true occupancy of the site. (This assumes that the heavy-atom site is unoccupied in the native crystals, which is not true for PICM where iodine replaces a nitro-group on chloramphenicol, nor where the heavy-atom reagents displace ordered solvent. In these cases the refined occupancies will underestimate the true occupancy of the site.) With the exception of site 1 for the 5 mM-gold derivative the occupancies are generally rather low. This reflects the considerable difficulty experienced in dealing with the K2PtCi4, PHMB and Sm(N03)3 derivatives, for which the reagent concentrations and the soak time were critical. Longer soak times or higher reagent concentrations invariably led to the crystals shattering. These difficulties did not arise with the iodinated chloramphenicol derivative, and it is not clear why the site occupancy is so low in this case, particularly since PICM is a good substrate for CAT (W. V. Shaw, personal communication). It is interesting to note that iodinated chloramphenicol binds not only in the active site, but also in a hydrophobic pocket near Ala176, which accommodates the adenine moiety of CoA in the structure of the CAT-CoA complex (Leslie et al., 1988). There is no evidence for chloramphenicol binding at this site in the refined structure of the CAT-Cm binary complex, and the iodine position is in fact occupied by a well-ordered water molecule (War234). This suggests that the derivative binding is due to the presence of the iodine atom and not because this pocket represents an alternative chloramphenicol binding site.

Table 2

Heavy-atom parameters used in the 2"7.7t m.i.r, phasing Derivative PICM KzPtCI4 2 mM-KAu(CN)2

Site

X

Y

Z

Occ.

B (A2)

Binding sitet

1 2 1 2 1

0"0932 0-0848 0'1380 0'1459 0"0503

0-1224 0-1754 0'3116 0.3111 0"1149

0'0939 0"2505 0"1943 0"2128 0"1033

0"45 0"53 0"35 0"33 0'57

14"3 32-4 20"05 20"0:~ 15"3

Tyr168, Ile172, Cm (N20, 0-8 A) Phe55, Ala176 Va195, Met125 (SD, 2'6 _k) Met125 (SD, 3"4 A) Cys31 (SG, 2-4 A), Leul60, Vail62, Cm

2

0.0309

0.1104

0.1168

0'35

8"7

3 4

0"0944 0.0296 0'0508 0.0302 0"0950

0'2228 0"1818 0'1149 0'1104 0-2219

0'2227 0-1992 0'1024 0.1166 0"2225

0"31 0"15 0'87 0'54 0"37

10"4 32"0 20.4 9'0 18"8

Cys31 (SG, 2.4 A), Leul60, His195, Cm (C9, 3"2 A) Tyr56, Aia149, Prol51 Ala149, Prol51, Trp152, Phe201 As above As above As above

0-0471 0"0511 @3283 @3333

0"1035 0'1193 0"1587 @2131

0"1015 0'1011 0-1480 0-1667

0"38 0"44 0"41 -0"18

22"9 32"3 77"1 27-2

Cys31 (SG, 2-6 A), Val162, Cm (C10, 2"4 A) Cy831 (SG, 2"3 A), Leu29, Cm (C10, 1'9 A) Asp71 (OD1, 2"9 A; OD2, 2"8 A). Asn68 (2nd cobalt site)

(Ol0, 1-8 A)

5 mM-KAu(CN)2

I

2 3 PHMB

I

SIn(NO3) 3

l 2

X, Y, Z, are the fractional co-ordinates in the hexagonal unit cell. 0co. is the site occupancy with the native data placed on an absolute scale using a Wilson plot, and B is the heavy-atom temperature factor. t Residues within 3"5 A of the heavy-atom position in the native structure. :~Not refined.

Structure of Chloramphenieol Acetyltransferase

171

Table 3

The 2"7 A resolution heavy-atom phasing statistics Resolution Figure of merit Derivative: 5 mM-KAu(CN)2 A fx E E~.o Phasing power Cullis R 2 mM-KAu(CN)2 A

f. E Ea. o

Phasing power Cullis R

7"5 0"90

6"0 0"90

5"0 0-85

4"2 0"77

3"7 0"76

3"3 0"72

3"0 0"66

2"7 0"62

Overall 0"72 552 696

748 t216 171 1I0 6"6

711 1000 261 93 3"7

666 911 232 93 3"7

609 771 290 103 2"5

538 687 240 87 2"7

491 633 230 90 2"6

451 577 222 92 2"4

389 540 153 89 3"0

534 841 179 91 4'4

524 722 208 95 3"3

512 670 209

473 575 261

421 515 230

388 477 227

111

120

118

116

3-0

2-1

2-1

2"0

353 437 198 102 2"1

299 414 146 96 2"5

422 518 206 107 2"3 58"1

223

92 2"9 51"3

PICM

A fa E Ea,o Phasing power Cullis R

331 420 173 110 2"2

357 393 158 98 2"3

348 349 182 97 1"8

387 323 238 102 1"3

336 288 215 96 1"2

298 248 179 97 1"2

276 218 163 94 l'l

236 193 134 ! 16 l'l

319 269 178 102 1"3 61"2

PHMB

A fu E Phasing power Cullis R

609 734 293 2"4

516 621 237 2'5

461 552 284 1"9

447 460 289 1"5

399 387 293 1"3

364 316 246 1-2

333 254 215 1'0

274 207 182 0"9

413 369 242 1"4 65"6

K2PtC14

A fn E Phasing power Cullis R

619 532 337 1"5

481 470 312 t-4

495 396 399 1-0

546 340 496 0"7

499 291 419 0-7

Sm(NO3) 3

A fn E Phasing power Cutlis R

327 325 253 1"2

311 255 241 l'0

296 193 240 0"8

313 166 276 0'6

286 149 252 0"6

524 387 423 0"9 82"6 261 124 224 0"5

238 96 213 0"4

263 75 241 0"3

281 142 238 0'6 91"l

A, is the r.m.s, isomorphous difference; fn, is the r.m.s, heavy-atom structure factor amplitude; E, is the r.m.s, lack of isomorphism; Ea,o, is the r.m.s, anomalous lack of isomorphism. Phasing power =lillE. Cullis R factor = 100 x Z[FPHcalc-FPH°bsl

ZIFP Ho~,- FP]

for centric reflections only, where FPH and FP are the derivative and native structure factor amplitudes, respectively.

The protein environment of the major binding sites for the other derivatives are similar to those observed in other protein structures (Blundell & Johnson, 1976). The platinum sites are both very close to Met125 (in agreement with the results of Petsko et al., 1978), while the mercury and two mQor gold sites all probably involve an interaction with Cys31. The proximity of the latter to the chloramphenicol binding site (Table 2) suggests that the heavy-atom reagents bind competitively with respect to chloramphenicol. The third gold site lies in a deep cleft, which forms part of the acetyI-CoA binding site (Leslie et al., 1988), while the fourth site lies at the surface of this cleft, and will necessitate a movement of the side-chain of Trpl52. If the occupancies of the sites for the two gold derivative concentrations are compared (Table 2) the results are incompatible with a simple binding model. For sites 1 and 2 the occupancies for the higher reagent

Table 4

The contribution of individual derivatives to m.i.r, phasing r.m.s, phase errort Including all derivatives Omitting derivatives 5 mM-KAu(CN) 2 2 mM-KAu(CN)2 5 mM and 2 mM-KAu(CN)2 PICM PHMB Sm(N03) 3 K2PtCI 4 Including only 5 mM-KAu(CN)2 and PICM All derivatives, but excluding anomalous data

57"3 60'4 57'0 66"3 64"6 59"0 58"0 57"6 59"7 61-7

t The r.m.s, phase error is defined as the r.m.s, difference between the m.i.r, phases, and phases calculated from the final model (R = 15"6°/o for data between 6 and 2.7 A). The resolution limits are 10 A and 2"7 A.

A. G. W. Leslie

172

concentration are greater by about 50%, while for site 3 the increase is about 20~o and the fourth site decreases in occupancy so that it was not detected. This suggests that chemical modification of either the protein or the heavy-atom reagent occurs during the six day soaking period. The samarium derivative bound near AspT1 (Table 2), but this resulted in the displacement of one of the cobalt ions that is present in the native crystals (see section (I), below) and which lies on a crystallographic 2-fold axis 6 A from the samarium site. This cobalt lies between adjacent molecules in the crystal lattice, and the crystal cracking observed on soaking in high concentrations of samarium nitrate is probably due to the displacement of this cobalt ion. For the purposes of heavy-atom parameter refinement the displaced cobalt was modelled as a samarium atom with negative occupancy. The statistics of the m.i.r, phasing (Table 3) show that 5 mM-gold cyanide is a particularly good derivative, with an overall phasing power of almost 3. In spite of the relatively low scattering power of iodine, the PICM derivative was valuable because it gave two unique sites and was also relatively isomorphous. By contrast the K2PtC1 , derivative was highly non-isomorphous, and only provided useful 180

+

I

4

phasing to 4 A resolution. The samarium derivative was quite isomorphous but only weakly substituted. Following refnement of the structure at 1-75 A resolution, the model phases between l0 A and 2'7 A were used to evaluate the quality of the m.i.r. phases, and the contribution that each derivative made to the overall phasing. The results, summarized in Table 4, verify that the 5 mM-KAu(CN)2 and the PICM derivatives dominated the phasing, and that the inclusion of the 2 mM-KAu(CN)2 derivative actually degrades the phases. This may be the result of overweighting effectively the gold derivative in the phasing, since the two gold derivatives will give rather similar phasing information. These tests also demonstrated the significant improvement in phases due to the inclusion of the anomalous scattering for three of tbe derivatives (5 mm and 2 mM-KAu(CN)2, PICM). (c) Map interpretation and model building The 2-7 A resolution m.i.r, electron density map was of sufficiently high quality to allow the entire polypeptide backbone to be traced from the minimap with only one point of ambiguity in the connectivity. This ambiguity was resolved by fitting

-~-.\

+÷

+

~-r-. + ~

+"tl:.%.,~;"

*+" ~4 ; * ++,++x

+~

+-*-* x t

=E °°s| °* S

x*

90

+

÷

°°°°o°°.~.o~°°°°°

+

+

+/"

X

÷" +

÷ :

\,+ O.

o

"-,+

a. + ,~.L+

")' +

. . . ,' r. ............ j 1

-I-

+ . _=1.-

1-.

+

+ N:..I- """.

2 ÷.,~. "11-

+

X

-,,, ,

!

+

-90 re

m

+ +

,,............... ~ .......... ~.......... .,, --180

-90

0 Phi

(a)

Fig. 1.

,,I 90

180

Structure of Chloramphenicol Acetyltransferase the known amino acid sequence to the electron density map using the fragment fitting option in FRODO. With the exception of a short loop (residues 78 to 85), where the density was particularly poor, it was possible to build a complete molecular model. One of the advantages of using the fragment fitting option is a p p a r e n t in the distribution of main-chain torsion angles (Fig. l(a)), which shows very few residues lying outside the fully allowed regions of the R a m a c h a n d r a n plot.

173

atoms. This behaviour persisted until the resolution of the data was extended to 2 A. A total of 15 rounds of alternate model refinement and rebuilding were carried out. The final R-factor was 18.3% for all d a t a between 6 A and 1-75 A resolutiont. The r.m.s, shift during the course of the refinement was 0"87 A (0"69 A and 1"03 A for main-chain and sidechain atoms, respectively). The final model contains 1932 non-hydrogen atoms including 204 water molecules, the 20 atoms in chloramphenicol and two cobalt ions. The stereochemistry of the final model and the weights used in the refinement are summarized in Table 5. The mean isotropic t e m p e r a t u r e factor is 19-5 A 2 for all atoms, 16"8 A z for protein atoms only and 42-3 A 2 for solvent atoms only. The largest peaks in the final difference map (0.3 to 0"5 e/A 3) were all associated with disordered residues (see section (j), below). In some cases two alternative conformations were clearly visible, but

(d) Refinement of the model The initial model gave an R-factor of 44"3% for d a t a between l0 and 2"7 A resolution. Individual atomic t e m p e r a t u r e factors were refined when the R-factor had dropped to 32"9% (10 to 2"5 A resolution). The temperature factor refinement was poorly behaved, with m a n y atoms refining to the minimum allowed B value of 2 A 2, and almost half of the residues had a lower average t e m p e r a t u r e factor for side-chain atoms than the corresponding main-chain

t See note added in proof.

1 8 0 ,,

+

i'

~,+#~

+

i

+x

90

+

i i

+%

+

x..

.

'~

+i

++ +

,: ,4

{

*s ***

............

il

i

0

|

1• L

i i

-",.,;+ ~#'+-IX"-..

i' l

'

E '

-90

m []

...... -4---~ ..... "4: -tso

, -90

',. 0

90

~80

Phi

(b)

Figure 1. Ramachandran plots of the main-chain torsion angles ¢, go, for (a) the starting model built using the fragment fitting option in FRODO and (b)the final model. The symbols used are (0)glycine, (X)asparagine, ( + ) others. The continuous lines enclose areas that are fully allowed conformational regions for z(Ca) of 110 degrees and the broken lines show the areas of acceptable van der Waals' contacts for v(Ca) of 115 degrees (Ramakrishnan & Ramachandran, 1965).

A . G . W . Les~e

174

Table 5

Summary of the restrained least-squares refinement of CAT Target at Final value

a2

C

N

(a)

R-factor Resolution range (A) Number of reflections (% of total) r.m.s, co-ordinate shift on last cycle (A) r.m.s. B shift on last cycle (A2) Number of atoms protein chloramphenicol solvent cobalt Number of parameters Distances (h) bond angle intraplanar Planar group Chiral centre Torsion angle (°) Staggered (Xl aliphatic) Transverse (X2 aromatic) Non-bonded contact (A) Single torsion Multiple torsion Thermal factor (A2) Main-chain bond Side-chain bond Side-chain angle

18"3% 6-1"75 A 27,303 (99"5%) 0-019 0"76 1706 20 204 2 7729 0.02 0"03 0"05 0"02 0"15 15"0 20"0

0.02 0-04 0"05 0-02 0"17 14'5 26.8

0"20 0"20

0' 1fi 0-22

2"0 3"0 4"0

4-2 7"0 9-2

~'The target a values determine the relative weights of each restraint in the refinement.

C

D

A

(b)

F i g u r e 2. A schematic representation of the structure of (a) the monomer and (b) the trimer of CAT. fl-Sheets are represented by arrows, ~-helices by helical ribbons. The Figures were produced by the program R I B B O N (Priestle, 1988).

no a t t e m p t w a s m a d e to m o d e l m o r e t h a n one c o n f o r m a t i o n for a s i d e - c h a i n . A Ramaehandran p l o t o f t h e refined m o d e l (Fig. l ( b ) ) s h o w s t h a t o n l y f o u r n o n - g l y c i n e r e s i d u e s h a v e p o s i t i v e (I) v a l u e s (Asp80, A s p 8 1 , G l u l 0 1 , Asp167). All o f t h e s e r e s i d u e s a r e l o c a t e d in t u r n s in the polypeptide chain. The average deviation from p l a n a r i t y o f t h e p e p t i d e b o n d s is v e r y s m a l l (r.m.s.

7 F

H F i g u r e 3. A topology/packing diagram of the CAT monomer. Each fl-strand is represented by a triangle whose apex points up or down depending on whether the strand is viewed from the N or C terminus, e-Helices are represented by circles. The fl-strand labelled H' belongs to an adjacent subunit of the trimer.

Structure of Chloramphenicol Acetyltransferase

Helix number 1 2 3

4 5

Table 6

Table 8

Parameters of helices

Residues involved in fl-sheet structures in C A T

Residues

(¢)t

(~)t

19-26

--69(11)

--39(14)

42-49 55-67

--60(4) -- 58(3)

--45(5) -- 49(6)

--61(3) --62(5) - 61 (7) -62 --71(14) --71(17) --71(15} -71 -87(24)

--45(7) --43(5) - 45(8) -41 --24(12) --20(19) --22(16) - 18 -38(17)

114-127 200-214 Overall

Standard helix:~ 31o helices Standard helix n helices

175

67-69 71-73 Overall 24-28

Residues

Strand

A. 7-stra~uted sheet 31-40 90-96 103-107 144-149 157-162 172-181 184-194

fib fie flF flo fin fl~ flj

B. 3-stramled sheet 8-10 75-78 82-86

fl^ tic tip

t The r.m.s, deviation from the mean is given in parentheses. :~Values from Barlow & Thornton (1988).

d e v i a t i o n , 3-7 °) w i t h a m a x i m u m (His144).

d e v i a t i o n o f 12 °

t h r e e a n t i p a r a l l e l s t r a n d s (B, F , J ) . F i v e a - h e l i c e s p a c k a g a i n s t o n e face o f t h e f l - s h e e t t o g i v e a t w o l a y e r e d s t r u c t u r e t h a t h a s b e e n d e s c r i b e d as a n o p e n - f a c e d s a n d w i c h ( R i c h a r d s o n , 1981). A f u r t h e r s m a l l t h r e e - s t r a n d e d a n t i p a r a l l e l f l - s h e e t is f o r m e d by the N terminus and the extended loop linking helix a 3 t o s t r a n d fie (this s h e e t w a s n o t s h o w n e x p l i c i t l y b y L e s l i e et al., 1988). A l t h o u g h t h i s

(e) Overall description o f the structure T h e s t r u c t u r e o f C A T is s h o w n s c h e m a t i c a l l y i n F i g u r e 2. T h e s i x - s t r a n d e d f l - s h e e t is m a d e u p o f t h r e e c e n t r a l p a r a l l e l s t r a n d s (E, G, I) f l a n k e d b y

Table 7

Parameters for turns in C A T

Residues sequencer

¢2

¢3

A, Reverse turn (Ot "-*Nl +J hydrogen bond) 16-19 WVRR -67 78-82 K_DDE 55 128-131 YKSD -64 135-138 FPQG -55 150-153 LPWV --72 165-168 FT_DY -46 181-184 EGDR 68 194-197 HHAV -63

-27 50 -32 -39 --12 130 --119 -22

--95 54 -93 -80 -89 54 --90 -58

21 23 -7 -12 -8 25 --8 -40

3"2 2"7 3"0 3'0 3"6 3"2 3"1 3'3

140 155 155 152 177 159 155 143

I I' I I I II II III

B. Reverse turns azsociated with ct helices 48-51 LDDS -- 55 213-216 LCNS - 58

-- 44 - 39

-86 -67

28 -34

2"9 3"2

136 161

III

2"9 3"2

127 153

3"1 3"4

121 129

3"1 3"0 3"2 2"9 3"0

159 153 129 125 160

C: ~-Turn (0 i -.* Ni+ ,t hydrogen bond) 97-101 HQETE 194-198 HHAVC D. Inverse ?-turn (0 i ~ Ni +2 hydrogen bond) 11-13 FDV -76 142-144 ENH -93 E. Asx turns} Asp40 O D 2 . . . Thr42 NH Ser51 O G . . . T y r 5 3 NH S e r l l l OG . . . Aspll3 NH Aspl t 3 0 D 1 . . . Aspll5 NH Aspl31 O D I . , . Lysl33 NH

83 76

W3

N...0 N-H...0 distance (A) angle (°)

W2

Type:~

I

t Positions that show a preference for glycine are underlined. :[:The turn type is assigned on the basis of the ¢2 ~P2, Ca "Pa conformation angles (Crawford et al., 1973). § Hydrogen bonds involving side-chain atoms were assigned on the basis of an N . . . O distance less than 3-5 A and an N - H . . . O angle greater than 120° .

A. G. W. Leslie

176

Figure 4. Stereo view of the C~ backbone of the CAT trimer and bound chloramphenicol looking down the trimer axis. One subunit has been drawn boldface to clarify the position of the subunit interfaces. The chemical structure of chloramphenicol is shown in Fig. 12.

a r r a n g e m e n t of a-helices and fl-sheet is common in protein structures, the precise topology found in CAT (Fig. 3) has not been reported previously. Three subunits associate to form a very stable, discshaped trimer a p p r o x i m a t e l y 60 A in d i a m e t e r and 45 A thick. I n the trimer (Figs 2 and 4) the extended polypeptide strand flu forms an extension to the six-stranded sheet of the adjacent subunit, resulting in a seven-stranded fl-sheet t h a t spans the subunit boundary. This feature p r e s u m a b l y helps to stabilize the structure of the trimeric form of the enzyme. The extension of a fl-sheet between subunits of an oligomeric protein is rather common (Richardson, 1981) b u t CAT is a rare example when the intersubunit hydrogen bonding is not between equivalent strands related b y a local 2-fold axis. The substrate chloramphenicol binds in a deep pocket located at the interface between subunits (Fig. 4). H

B

J

I

G

E

F

A

D

C

)aT

i74 O--N

( ,-.o~

'

(~'W'N

¢ o--N

",,

N--O

"

(f) Secondary structure Secondary structure was assigned on the basis of main-chain hydrogen bonding using the algorithm of K a b s c h & Sander (1983). However, these authors adopted a cut-off energy of 0"5 kcal/mol (1 cal -- 4.184 J) in their definition of a hydrogen bond interaction, which can lead to d o n o r - a c c e p t o r distances of up to 5 A. This is s o m e w h a t g r e a t e r t h a n would n o r m a l l y be considered as a hydrogen bond, and indeed K a b s c h & Sander s t a t e t h a t the relatively low energy cut-off was a d o p t e d a t least in p a r t to allow for errors in a t o m i c co-ordinates. In the case of a refined high-resolution structure such as t h a t reported here, the co-ordinate errors will be significantly smaller t h a n those in m a n y of the structures examined by K a b s c h & Sander, and it seems a p p r o p r i a t e to increase the energy cutoff from 0"5 to l'0 kcal/mol, corresponding to a m a x i m u m d o n o r - a c c e p t o r distance of 4-1 A. In the CAT structure, the lower energy cutoff g a v e three hydrogen bonds with d o n o r - a c c e p t o r distances g r e a t e r t h a n 4 A, and l l instances where the N - H . . . O bond deviated from linearity b y more t h a n 70 °. T h e 1 kcal/mol cutoff g a v e a m a x i m u m bondlength of

,

)O-'N(i ~7o

N--O-

157( O-.N ~N"OI

(~N" ~ TO, T

O--N

~ O'.'~N(~

~N

4t N-"O(~

r

IS4!

~

('~N"O~ "~'O--N"

S7(li);"O( 102

--

Figure 5. Main-chain hydrogen bonding scheme of the 2 fl-sheet.s in CAT. The, amino acid residues are indicated using the l-letter code. The strand labelled H belongs to an adjacent subunit of the trimer.

Table 9

Asn or Gin side-chains involved in a pair of hydrogen bonds to main-chain atoms Side-chain atoms

Main-chain atoms

Asn68 OD1 ND2 Gln137 OE1 NE2 Gln192 OEl NE2 Gin211 OE1 NE2

Val89 N O Serl07 N 0 Leul60 N O Asp40 N 0

Distance Angle (N... O) (A) N-H... 0 (o) 2-9 3"1 2"8 2-8 3"0 3"0 2"8 2.8

168 177 143 167 170 163 154 173

Structure of Chloramphenicol Acetyltransferase L

177

It

Figure 6. Stereo view of the hydrogen bonding between the side-chain of Gln192 and the main-chain of Leul60. Carbon, nitrogen and oxygen atoms are represented by circles of increasing size.

both a-helices and 31o helices are very close to those reported by Barlow & T h o r n t o n (1988). With the exception of al, which has two i ~ i + 5 hydrogen bonds at its C-terminal end to give a short length of helix, the alpha helices are remarkably regular. There are two segments of polypeptide t h a t a d o p t a 31o helical conformation, residues 67 to 69 (at the C-terminal end of a3) and residues 71 to 73. Helices a 1 and a2 are made up of predominantly polar side-chains, whereas c% is quite buried in the structure and consists of almost exclusively hydrophobic residues characteristic of helices packed against a fl-sheet. The turn parameters of all reverse turns are listed in Table 7 with their classification according to Crawford et al. (1973). The preference for glycine at a particular position in the turn is not strongly expressed. There are two examples of an inverse ?-turn (Matthews, 1972), both of which show highly non-linear hydrogen bonds. The Asx turns (Baker & H u b b a r d , 1984) display conventional geometry.

3"65 A, and a maximum deviation from linearity of 59-3 ° . The higher energy cutoff resulted in 24 fewer hydrogen bond interactions, and shortened the lengths of seven elements of secondary structure (3 helices, 4 strands) by one residue and one element (%) by two residues. In all but one case (fir) the shortening was at the C-terminal end of the strand or helix, suggesting t h a t distortions of both types of secondary structure are more common at the C terminus than the N terminus. This is in agreement with observations made by Barlow & T h o r n t o n (1988) in their analysis of helical geometry in a n u m b e r of well-determined structures. The higher energy cutoff thus has the a d v a n t a g e of eliminating what would normally be considered as gross distortions from helical, sheet or turn geometry while maintaining the objective approach of the Kabsch & Sander algorithm. (i) Helices and turns There are five alpha-helices in the monomer of CAT involving a total of 58 residues or 27% of the polypeptide. The average conformation angles are summarized in Table 6. The helical parameters for

fl-Sheet structure

(ii)

The residues involved in the two fl-sheet structures are listed in Table 8. The hydrogen-bonding

4

÷

60-

+ +

+ 4

4

4

4

÷

4 4

4 4

o~

4

+~ L4 J

÷~

r#t

1

,

,

*/]4

4,

L .. 444

t

I

20

I

jl÷

•

+~

+

I

40

t

. .., I t

t

,, I

80

4

1

4.44

60

4

+4

i

+

I

IO0

+

+,',

E

.I, 4-

+ +

44-,/i

4 4-

÷ A I~

4 +

*

4.

,.**

,fl

4 +

~

4

: .Af. l i.: , : ff "

I

I

120

I

I

140

I

+4

1

]60

I

It I

180

÷ 4

+ ++

I

4 ~, 4

I

200

•

-

I

220

Residue number

Figure 7. Variation of the average main-chain (continuous line) and side-chain. There is a break at position 79 as this residue is a deletion in the type III sequence.

A . G . W . LesKe

178

Table 11 Analysis of hydrogen bonds involving water

Table I0 Interactions involving ion-pairs H-bonding distances (A)

Solvent accessible area (A2)~

Intrasubunit NZ Lysl0-OE1 Glu82 NH2 ArglS-OD2 Aspl99 NE Argl8-ODl Asp199 NH2 Arg26-OD1 Asp167 NE Arg26-OD2 Aspl67 NZ Lys38-OD1 Aspl56 NZ Lys45-ODl Asp49 NZ Lys45-OD2 Asp49 OD1 Asp71-NH2 Arg74 OEl Glu72-NH2 Arg205 NZ Lys78-OE1 Glul42 NZ Lys78-OE2 Glu142 NH1 Arg209-OEl Glu212

3-1 2-7 3"0 3"0 3"I 2"8 3"3 3"3 2'9 2"6 3.2 3-3 2-5

57 0 1 37 0 1l 57 52 28 l0 60 54 29

Intersubunit OE1 GIul01-NH1 Arg205

3.2

37

Residues

t The value given is the sum of the solvent accessible area of the 2 atoms directly involved in the interaction.

pattern within the major seven-stranded fl-sheet (which includes one strand from an adjacent subunit of the trimer) and the small three-stranded sheet are shown in Figure 5. Both fl-sheets show the characteristic right-handed twist when viewed along the strand direction. The mean main-chain conformation angles ((I), W) for all residues involved in fl-sheet structure are - 1 1 7 ° and 141 °, in good agreement with average values for other highly refined structures. There is a "wide" fl bulge (Richardson, 1981) involving residues Lys177, Tyrl78 and Leul87. (iii) Main-chain to side-chain hydrogen bonds There are numerous main-chain to side-chain hydrogen bonds in the CAT structure, but one type of interaction involving Asn to Gin side-chains appears to be unusual. In their analysis of hydrogen bonds in proteins, Baker & Hubbard (1984) found that these side-chains make a relatively large proportion of "long-range" interactions, providing important crosslinks in protein structures. However, they do not comment on the fact that these side-chains can form a pair of hydrogen bonds, with excellent geometry, involving the main-chain amide and carbonyl group of a residue in an extended conformation (see Fig. 6). There are four such interactions in CAT (Table 9), and in three of the four examples the side-chains involved are conserved in eight of the ten known sequences, suggesting that these interactions play an important role in stabilizing the folded structure.

(g) Temperature factors The variation of the average main-chain and sidechain atomic temperature factors along the poly-

Mean

Number distance (A) To To To To To

main-chain 0 main-chain N side-chain 0 side-chain N water

109 41 95 35 137

2-93 (0.26) 2"97 (0"17) 2"86 (0"25) 2.94 (0.27) 2'85 (0-30)

Mean

angle (°)

( B ) (A2)

127 160 124 148 --

39 32 38 41 34

The distance given is to the water oxygen, and the r.m.s. deviation is given in parentheses. The angle given is tbe C ~ O . . . O(HOH) angle for bonds involving protein oxygens and N - H . . . O(HOH) angle for protein nitrogens when the position of the proton is unambiguous.

peptide chain is shown in Figure 7. As expected, the temperature factors are greatest for loop regions connecting elements of secondary structure, reflecting the greater conformational freedom of these residues. An important exception to this is the loop (residues 195 to 199) connecting strand flj to a 5, which includes the active site histidine (Hisl95). The thermal factors for this loop and for the residues immediately proceeding it are amongst the lowest in the whole structure. For residues 6, 12 to 15, 81, 99, 129-130, 138-139, 164-165, 181 to 183 and 219 the main-chain temperature factor exceeds 25 A2, making a confident assignment of the mainchain conformation difficult. (h) Ion pairs There are a total of 46 charged polar residues (Arg, Lys, Asp, Glu) in the CAT III sequence, and Table 12 Residues with incomplete side-chain density in CAT Residue

Atoms omitted from model

Lysl4 Asnl5 Arg28 Lys46 Asp50 Gin69 Gin98 Glnll6 Glu126 Lys129 Lys133 Asn164 GlnlS0 GlulSl Aspl83 Argl84 Lys217 Lys219

CG, CD, CE, NZ OD1, ND2 CD, NE, CZ, NH1, NH2 CG, CD, CE, NZ ODI, OD2 CD, OEl, NE2 CD, OE1, NE2 OE1, NE2 CB, CG, CD, OEI, OE2 CE, NZ CE, NZ CG, OD1, ND2 CD, OE1, NE2 CB, CG, CD, OE1, OE2 CB, CG, ODl, OD2 CZ, NH1, NH2 CD, CE, NZ NZ

Solvent-accessible area (A2) 68 102 87 52 118 63 93 59 46 98 76 68 92 37 75 77 81 155

The solvent-accessible area calculations exclude the a t o m s listed as being omitted from the model, and will therefore be an underestimate of the solvent-accessibility of the complete residue.

Structure of Chloramphenicol Acetyltransferase

179

Table 13 Residues with two distinct side-chain conformations

Residue

Solvent-accessible areas (A) '

Modelled side-chain conformation (Xl, •2, Z~)

Alternative side-chain conformation (Xl, ~, Z~)

Met6 Leu35 Ile84 Val85 Val 124 Leul45 Leul50 Ser157

27 0 l 8 7 O 8 3

(-61, --76, --72) (90, 62) (69, 160) (180) (180) (71, 143) (-76, 76) (81)

(60, -60, 180) (180, --60) (60, 180} (-60, 180) (60) (60) (180, 60) (180,60) (180)

Metl75 Ile207 Gln212

3 4 99

(-65, 179, -62) ( - 69, -- 179) (--62, -178, 12)

(60, 180, 60) ( -- 60, -- 60) (180, 180, 0)

At intermolecular contact At subunit interface

At subunit interface At subunit interface; cannot have ~1 = 180° in all 3 subunits simultaneously At subunit interface

exception of the two cobalt sites (see section (1), below), there was no evidence from the final electron density m a p t h a t a n y of the solvent sites were occupied by molecules other t h a n water, and in the following description it will be assumed t h a t all solvent sites represent bound w a t e r molecules. Over a q u a r t e r of the solvent molecules are arranged in hydrogen-bonded networks, which are qualitatively v e r y similar to those observed in other highly refined structures (see, for example, K a r p l u s & Schultz, 1987; Smith et al., 1988). In the CAT structure these networks involve up to a m a x i m u m of nine distinct sites. An analysis of the hydrogen bond interactions involving solvent (Table 11) shows the usual preference of hydrogen bonding to oxygens (main-chain or side-chain) rather t h a n nitrogens, in the ratio of a b o u t 3 : 1. The average hydrogen bond g e o m e t r y is v e r y similar to average values reported by B a k e r & H u b b a r d (1984). An analysis of the coordination of the 204 solvent a t o m s reveals t h a t 116 (57%) have three or four near neighbours. The g e o m e t r y is a p p r o x i m a t e l y t e t r a h e d r a l (mean bond

20 of these are involved in a total of 13 i n t r a s u b u n i t and one intersubunit ion-pair interactions (Table 10). On the basis of a s u r v e y by Barlow & T h o r n t o n (1983), only eight ion-pair interactions would be expected for a protein of M r 25,000. The larger n u m b e r found in CAT m a y have a bearing on the observed t h e r m o s t a b i l i t y of the enzyme; CAT can be incubated for long periods a t 70°C w i t h o u t significant loss of a c t i v i t y (Lewendon et al., 1988). E x a m i n a t i o n of the solvent accessibility of a t o m s directly involved in the ion-pairs shows t h a t only the A r g l 8 - A s p l 9 9 pair is fully buried. As discussed in a later section, this salt bridge is located in the active site region and plays an i m p o r t a n t role in stabilizing the conformation of the active site loop (residues 195 to 199). This is the only ion-pair t h a t is conserved in the nine known CAT sequences. (i) Solvent A total of 204 solvent molecules were located, of which 29 lie in the second hydration shell. With the

|

Comment

i

|l

Figure 8. An example of alternative side-chain conformations. The electron density of the 3Fo-2F¢, ac map is shown with the 2 alternative positions for atom CD1 (labelled CD1 and CDI') of Ile207. The contour level is 0.2 e/A a.

A. G. W. Leslie

180

L

11161A ~."'"'~

tllellB

R

Y ~3""

N18tN15OA A

l~l,e~,"

Figure 9. Stereo view looking down the trimer axis (indicated by a dot) of the intersubunit hydrogen bonds involving residues Asn159, Asnl61. Different subunits are indicated by using the suffix A, B or C.

angle is 110 °) but there is a considerable spread in bond angles for even the best ordered water molecules (temperature factors less than 20 A 2) with angles lying between 60 ° and 160 °. A total of 13 solvent molecules have zero solvent-accessible area and a further nine have less than 5 A 2 accessible area. However, none of these sites are located more than a few AngstrSm units from the surface of the protein, and these water molecules are therefore more correctly described as occupying crevices in the protein surface rather than being truly buried. With one exception, all of these water molecules

b

make at least three hydrogen bonds. The exception, War240, is a well-ordered water t h a t is located in a loop in the main-chain, forming hydrogen bonds to the carbonyl oxygens of residues 128 and 131. Although the hydroxyl of Serl04 and NE2 of His97 lie within hydrogen bonding distance of War240 the geometry precludes any significant interaction. The fact t h a t this site is well ordered indicates t h a t there is a strong energetic preference for this site to be occupied rather than vacant even when the bound water only satisfies half of its hydrogen-bonding potential.

g

Figure 10. Stereo view of the C~ backbone of 2 CAT trimers packed face to face in the crystal lattice, showing the location of the cobalt ions (open circles) sandwiched between the 2 trimers.

Structure of Chloramphenicol Acetyltransferase In other structures it has been observed that water molecules are frequently located at the end of secondary structures (Richardson, 1981). This observation is supported in this structure, where 24 water molecules are associated with the N or C termini of a-helices, although only two (Wat223 and Wat299) are involved in bridging the ends of fl-strands. While the majority of the solvent sites had well-defined, approximately spherical density in the final electron density maps, in a significant number of cases the density was weaker and markedly elongated, indicating the presence of more than one possible site. This type of discrete disorder is commonly found in protein structures (Smith et al., 1988). (j) Disordered residues There are many side-chains that exhibit some type of conformational disorder, and these fall broadly into two categories depending on the solvent accessibility of the residue. The first category, consisting exclusively of surface polar residues, show very weak (less than 0"2 e/A3) or uninterpretable electron density for atoms towards the end of the side-chain (Table 12). The second category involves predominantly buried, non-polar residues, for which there is clear evidence in the electron density for two distinct conformational states (Table 13 and Fig. 8). For side-chains that showed the first type of disorder, all the affected side-chain atoms were deleted from the model. No attempt was made to model more than one confor-

181

marion for side-chains in the second category, and the side-chain was positioned in the conformation corresponding to the stronger density in the electron density map. With one exception these residues behaved normally during structure refinement. However, Leu35, which is located at the trimer interface, refined to give side-chain torsion angles of 90 ° and 62 °, whereas the density is best modelled by equally populated conformations of 180°, - 6 0 ° and 60 °, 180°. This example illustrates the need to examine the electron density very carefully in the case of side-chains with an unusual conformation, to see if the density can be modelled equally well by two distinct conformations with the more usual staggered torsion angles. (k) Subunit interface The binding site for chloramphenicol is located at the interface between subunits (Fig. 4), as is the essential catalytic histidine (Hisl95). Consequently, the interface is important not only in maintaining the structural integrity of the enzyme, but also in substrate binding and catalysis. Solvent-accessible area calculations show that an area of 2200 A2 (20% of the surface area of the monomer) is buried on formation of the trimer. Hydrophobic residues (including 9 phenylalanines) account for 1080 A2, or 52% of the total. There are eight charged polar side-chains at the subunit interface, but only two of these (Glul01 and Arg205) are involved in an inter-

ss *~*%

Figure 11. Stereo view of the tetrahedral co-ordination of the cobalt ion by the imidazole nitrogen ND1 of His27 (2.1 A), the carboxylate oxygen OE2 of Glu23 (1-8 A) and their counterparts related by the crystallographic 2-fold axis.

A. G. W. Leslie

182

subunit ion-pair, while the remainder participate in intrasubunit interactions (Table 10). There are a total of 16 intersubunit hydrogen bonds. Eight of these are between main-chain atoms on the extended strands fin and fill, which result in the extension of the fl-sheet across the subunit interface (Fig. 4). There are an additional four hydrogen bonds between side-chain and main-chain atoms, and four involving only side-chains. The latter include a particularly intricate hydrogen-bonding scheme involving Asn159 and Asnl61, which results in a continuous network of hydrogen bonds around the 3-fold axis of the trimer (Fig. 9). Solvent molecules also play an important role in stabilizing the subunit interface. A total of l l solvent molecules, five of which are buried (accessible area < 5 A2) participate in a total of 13 bridging hydrogen bonds between protein atoms. (l) Crystal packing A cobalt ion, derived from cobaltic hexamine chloride, which was an essential component of the crystallization medium, plays a crucial role in the organization of the CAT trimers in the crystal lattice. The cobalt site lies on a crystallographic 2-fold axis between two trimers that are packed "face to face" with their 3-fold axes coincident with the crystallographic 3-fold axis (Fig. 10). There are, therefore, three cobalt ions sandwiched between the two trimers, and the resulting hexamer has 32 point group symmetry which is reflected in the crystallographic symmetry (space group R32). The cobalt ion is co-ordinated by the imidazole nitrogen ND1 of His27 (2"1 A) and the carboxylate oxygen OE2 of Glu23 (I-8 A), and the same atom from the second 2-fold related trimer (Fig. 1 l) resulting in a tetrahedral co-ordination geometry that is very similar to that observed in the structure of bis(imidazole)bis(acetato) cobalt(II) (Gadet, 1974). This cobalt ion has been stripped of its co-ordinating ammonia ligands and apparently changes its valence state during crystallization. (The trivalent ion shows a strong preference for octahedral co-ordination.) Excluding the interactions mediated by the cobalt ion, there are only two contacts less than 4 A between these two stacked trimers, involving CG2 of Vall39 with CB and CG of AspS0 both at a distance of 3"7 A. The cobalt is therefore effectively "gluing" the two CAT trimers together in a very specific interaction. The effect of the cobalt salt on crystallization and its role in the crystal packing is similar to the role played by a zinc ion in the crystal structure of the serine protease tonin (Fujinaga & James, 1987). There is a second cobalt site in the crystal lattice which, on the basis of its peak height in the electron density map compared with that of the first cobalt site, is only partially occupied or is subject to positional disorder. The nearest protein atoms to this site, which also lies on a crystallographic 2-fold axis, are the carbonyl oxygens of Asn68 and Asp87 at distances of 4'0 and 4.2 A, respectively. These

Table 14

Intermolecular interactions Residues involved

Hydrogen bond distances (A)

A. Protein-protein interactions N Met6-O Lys217 O Met6-NE2 Gin65 NE2 Gln65-OD1 Asp87

3"1 2-7 3'0

B. Hydrogen bonds via a M.ngle bridginff water OEl Gln65-Wat243-N Met6 ODl Asp87-Wat244-O Gin65 O Asn68-Wat282-O Asp87 OG Ser112-Wat286-O Asn7 OG Ser112-Wat286-O Ser88 OG Ser112-Wat286-N Ser88 N Met6-Wat295-OH Tyr61 N Met6-Wat295-O Leu218 N Ala52-Wat296-O AspS0 OD1 Asp87-Wat373-OH Tyrl I0 ND2 Asn143-Wat374-OG Ser112 O Ser51-Wat385-O SerS1 0 TyrS-Wat386-NH1 Arg209 O TyrS-Wat386-NH2 Arg209

2-7, 3-0 3"5, 2-9 3"5, 3"2 3"3, 2"6 2-9 2-7 3-0, 3-0 2"6 2"8, 3"5t 2'7, 2'7 3"2.2"6 3"3, 3"1~ 3"0, 3"1 2'7

van der Waals' interactions; atoms involved in contacts less than 4 A. Met6 CA, C, CG Ala52 CBt Ass7 CB Gin65 CG. CD Thr9 CG2 AspS0 CB, CG Arg28 CG Asp82 OD2 Asp49 O Ser112 Of Asp50 CA, C Vall39 CG2t Ser51 N. O Arg209CZ, NH2t. All interactions unless otherwise indicated are generated by the symmetry operation - X +], Y - X + ½ , - Z + ~ . tGenerated by symmetry operation X-Y+13, - Y + ] ,

-z+~.

distances are consistent with the site being occupied by a cobaltic hexamine ion, but the electron density gives no indication of the orientation of the octahedral complex, again suggesting the possibility of positional disorder. There are a number of other protein-protein contacts in this region (Table 14), which suggests that this second cobalt is less important than the first in determining the crystal packing. It is the occupancy of this second site that is significantly reduced in the samarium derivative (section (b), above). There are only two distinct areas of contact between adjacent molecules in the crystal lattice and both of these are extensively hydrated, resulting in relative few protein-protein contacts. There are only three protein-protein hydrogen bonds (Table 14), with another 14 involving a single bridging water. A total of 21 atoms are involved in van der Waals' contacts (Table 14). With the possible exception of the very specific interactions involving the first cobalt ion, it seems unlikely that the molecular conformation is significantly affected by lattice forces. (m) The chloramphenicol binding pocket During the course of the refinement, difference electron density maps consistently gave weak nega-

Structure of Chloramphenicol Acetyltransferase CL1\

/c,, C3-H

I

o,.\

/ N20

o,,/

C12

\

C13

..... Cll

.. C8--C7

\ clo

/

c9

I --

C5--

!,.

t

H

. C8-- H

!., 1 H

Figure 12. The chemical structure and atom numbering scheme of chloramphenicol (after Chatterjee et al., 1979). Ol5(H) is the primary (C-3) hydroxyl that acts as the acetyl acceptor in the forward reaction.

tive density over the chloramphenicol site, although the density for chloramphenicol was well defined in (31FoJ- 21Fd, a¢) electron density maps. In addition, the atomic temperature factors refined to values significantly greater than those expected for a tightly bound substrate. Adjusting the occupancies of the chloramphenicol atoms to 0"66 eliminated the features in the difference maps and also resulted in more reasonable atomic temperature factors, suggesting that the chloramphenicol site is indeed not fully occupied in the crystal. This result is unexpected in view of the fact that chloramphenicol was present at a concentration of 0-5 mM in the crystallization medium, which is considerably in excess of the binding constant (Kd) of 13 #~. The partial occupancy of the site is unlikely to give rise to any apparent disorder of the enzyme, as the apoenzyme is isostructural with the binary complex of CAT+Cm (P. C. E. Moody & A . G . W . Leslie, unpublished results). Chloramphenicol (chemical structure depicted in Fig. 12) binds in a deep pocket located at the inter-

183

face between adjacent subunits of the trimer (Fig. 4). For each binding site, residues involved in substrate binding are largely located on one subunit while the catalytic histidine resides on the adjacent subunit, so that the monomer alone could not be catalytically competent. Many of the residues lining the pocket are hydrophobic in nature (Fig. 13) and hydrophobic interactions make an important contribution to substrate binding. However, one side of the pocket is made up almost entirely of polar residues (Gln92, Thr94, Asn146, Ser148, Tyrl68 and Thr174) and these are involved in an extensive network of hydrogen bonds involving several ordered water molecules and chloramphenicol itself. The binding pocket thus reflects the amphiphillic nature of the substrate. A total of 284 A2 of protein surface is buried on binding chloramphenicol, and 49 °/o of this is due to buried non-polar residues. There are only two direct hydrogen bonds between Cm and CAT (NE2, His195-O15, Cm 2"8 A and OH, Tyr25-O14, Cm 2.9 A). with an additional bond via a bridging water molecule (OG1, Thr174-Wat252-O16, Cm 2.7/l, and 2-5 A). Nevertheless, all the polar groups of Cm, with the exception of one of the nitrate oxygens, are involved in one or more interactions with ordered solvent or with the enzyme. Two water molecules (Wat308, 313) lie within hydrogen bonding distance of the primary (C-3) hydroxyl of Cm, and presumably one or both of these are displaced during the acetylation reaction. Three further water molecules (War249, 252, 360) interact with the C-1 hydroxyl of Cm, and these will be displaced by the 1-acetyl group of chloramphenicol in the secondary reaction to yield the 1,3 di-acetyl product. Two residues, Leu29 and Phe135, partially cover the entrance to the Cm binding pocket (Fig. 13). Modelling studies sugest that one or both of these side-chains must be displaced, probably by rotation about the C~-C~ bond, to allow Cm to enter or leave the binding pocket. As both residues are located at

g4.&__ Figure 13. Stereo view of residues forming the chloramphenicol binding pocket. Ordered water molecules are shown as double circles and possible hydrogen bonds are indicated by broken lines. Residue names proceeded by # belong to an adjacent subunit of the trimer.

184

A . G. W. Leslie

the enzyme surface, the required side-chain rotation can be achieved without any steric hindrance. Only five of 17 residues shown in Figure 13 are strictly conserved among the ten known CAT sequences, and some residues that have extensive contacts (more than 20 A 2 buried surface on Cm binding) show considerable variation. Two examples are Leu29, which in other variants can be Ala, Ile, Val, T h r o r Gin, and Asn146, which can be Phe, Pro, Ser or Asp. In spite of the non-conservative nature of these substitutions the apparent Michaelis constants for Cm are remarkably similar for a large number of CAT variants with the great majority having Km values between 10 and 20 #~ {Shaw, 1983). The type I variant of CAT binds the steroid antibiotic fusidic acid (Bennett & Shaw, 1983). The binding is strong and competitive with respect to chloramphenieol (Ki, ll'7 #M) but is not shown by the type III enzyme whose structure is reported here. The fact that the Cm binding pocket of the type I enzyme can accommodate the much bulkier steriod is somewhat surprising. Nine of these 17 residues lining the pocket are identical in the type I and type III sequences, but substitutions Leu29 -~ Ala, Phe24 -* Ala will open out the pocket while other substitutions such as T y r 2 5 ~ P h e , Gin92-* Cys, Asnl46-* Phe, Tyrl68-~ Phe will make the binding site less polar, both of which would favour binding of the steriod. However, these differences also have to be reconciled with an unchanged K m value for Cm. It is hoped that model building studies will throw more light on this novel aspect of the Cm binding site. (n) Active site The use of non-specific inhibitors and thiolspecific reagents identified a cysteine residue (Cys31) and a histidine residue (His195) as potentially important in the acetylation reaction (for a review, see Shaw, 1983). In both cases modification led to loss of activity and protection was afforded by the presence of Cm, but not by CoA. However, Cys31 is not conserved in known CAT sequences, and moreover the susceptibility to inhibition varies considerably among variants of CAT, suggesting that this residue is unlikely to play a role in catalysis. 3-Bromoaeetyl chloramphenicol, an analogue of the product of the forward reaction, is an extremely potent inhibitor that uniquely modifies the NE2 position of His195 leading to complete loss of activity (Kleanthous et al., 1985). His195 is absolutely conserved and lies in a sequence of nine residues (192 to 200) that shows a very high degree of sequence conservation. These observations led to the proposal that Hisl95 plays a central role in catalysis. In view of the kinetic evidence for a ternary complex rather than a ping-pong mechanism (Kleanthous & Shaw, 1984), it has been proposed that the histidine acts as a general base catalyst, accepting a proton from the 3' hydroxyl of

Cm and promoting nucleophilic attack on the carbonyl of the thioester of acetyl-CoA to form a tetrahedral oxyanion intermediate (Kleanthous et al., 1985). This has been supported by site-directed mutagenesis of the type I enzyme; a mutant in which Hisl95 was replaced by tyrosine shows no detectable activity (Burns & Crowl, 1987). Hisl95 is located deep within the Cm binding pocket (Fig. 13) and the NE2 imidazole nitrogen is within hydrogen bonding distance (2.8 A) of the primary (C-3) hydroxyl of Cm, while the NDl imidazole nitrogen is suitably placed to form a hydrogen bond with the main-chain carbonyl oxygen of the same residue (N-O distance 2-85 A, N - H . . . 0 angle 138°). This later interaction presumably maintains the required tautomeric ibrm of the histidine observed in the modification experiments using 3-bromoacetyl Cm, rather than hydrogen bonding to a carboxylate group as originally proposed (Kleanthous et al., 1985) and as observed, for example, in serine proteases. The imidazole NDl-carbonyl oxygen interaction can only be achieved with energetically unfavourable side-chain torsion angles (:~1=--145 °, ~(2=--36°), which result in short contacts between ND1 and the a-carbon (3"08 A). A search of the Brookhaven database revealed no other examples of this type of hydrogen bond suggesting that this interaction alone is not sufficient to compensate for the steric hindrance that results from the unfavourable sidechain torsion angles. Presumably some other feature of the CAT active site is responsible for the observed conformation. In any case, the observed disposition of chloramphenicol and His195 are entirely consistent with the proposed role of the histidine in the reaction. The binding site for the second substrate in the transfer reaction, acetyl CoA, will be discussed in a subsequent paper. The cysteine residue that is susceptible to chemical modification (Cys31) is located to one side of the binding pocket (Fig. 13), with its sulphydryl group at a distance of 7 A from the primary hydroxyl of chloramphenicol. Its location is consistent with the protection from modification afforded by Cm binding, and also with the conclusion that this residue is not directly involved in catalysis. The hydroxyl group of a conserved serine residue (Serl48) lies at a distance of 4-3 A from the primary hydroxyl of chloramphenicol (Fig. 13). Although there is no clear indication from the present structure of a role for this serine in catalysis, its proximity to the reacting groups and its strict conservation strongly suggests that some involvement is likely. Site-directed mutants of Ser148 are being investigated in an attempt to clarify any possible role in catalysis (Lewendon & Shaw, personal communication). Another feature of the active site is a conserved and completely buried salt bridge between Argl8 and Asp199 (Table 10), with additional hydrogen bonds between the guanidinium group of Argl8 and the main-chain carbonyl oxygens of residues 195 and 196. In their analysis of ion-pairs in proteins,

185

Structure of Chloramphenicol Acetyltransferase

Barlow & Thornton (1983) found that buried ionpairs are likely to be "functionally important", particularly if they are conserved between a number of sequences; the Argl8-Aspl99 ion-pair falls into their definition of functionally important by virtue of being within 8 A of the catalytic His195. There is no structural evidence that either residue is directly involved in catalysis, although the possibility of an electrostatic interaction between Asp199 and His195 cannot be excluded. The mutants Asp199-~ Asn, Asp199-* Ala, Argl8-* Val have been constructed by site-directed mutagenesis (Lewendon et al., 1988). The Asp199-+Ala and Argl8-~ Val mutants are still active with a reduced kc~t value (13-fold and 9-fold reduction, respectively), but both mutants are significantly more thermolabile than wild-type (Lewendon el al., 1988). These results support the notion that the primary role of the Arg18-Asp199 ion-pair is structural rather than catalytic. The Aspl99-~ Asn mutant suffers a dramatic loss of activity (1500-fold reduction in kcat). The X-ray structure of this mutant shows a significant rearrangement of residues in the active site with movement of up to 1 A in the position of the polypeptide backbone, and in particular the orientation of the imidazole ring of the active site histidine is quite different (M. R. Gibbs, personal communication). The loss of activity can therefore be interpreted as a consequence of the structural changes rather than the loss of the ion-pair. 4. C o n c l u s i o n s

CAT is the first acetyltransferase for which detailed structural information is available, and it remains to be seen whether any structural features observed in CAT are shared by other members of this general class of enzymes. It is likely, however, that a distinction should be made between acyl transferases that employ a covalent acyl-enzyme intermediate and those like CAT that catalyse acyl transfer by a ternary complex (sequential) mechanism. The lack of.structural homology with known protein structures make it difficult to speculate on any evolutionary ancestor for CAT, although some sequence homology has been detected with the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex (Guest, 1987). The present structure provides detailed information on the role of ehloramphenicol binding, which helps to explain previous results on binding and acetylation of chloramphenicol analogues (Shaw, 1983), and has provided a firm structural basis for the proposed mechanism involving His195 acting as a general base, in which the tautomeric stabilization is provided by an unusual interaction with a carbonyl oxygen rather than a side-chain carboxylate group. The roles of individual amino acids in binding and catalysis are being explored further using site-directed mutagenesis techniques and the determination of the structures of selected mutants by X-ray diffraction.

The co-ordinates of the CAT-Cm binary complex have been deposited with the Brookhaven Databank. I would like to thank all my colleagues at Imperial College for their support and encouragement and particularly P. Brick and P. C. E. Moody for critical discussion of the manuscript. Computer programs were generously provided by E.J. Dodson, P. R. E. Evans and T. A. Jones. I am indebted to Professor W.V. Shaw for suggesting the project, for providing the CAT enzyme used in this work, and for many fruitful discussions of the CAT system. I am grateful to Jane Austin for help in preparation of the manuscript. This project was supported by the Medical Research Council (UK) by project grants and a Senior Fellowship. References

Agarwal, R. C. (1978). Acta Crystallogr. sect. A, 34, 791-809. Alton, N. K. & Vapnek, D. (1979). Nature (London), 282, 864-869. Baker, E. N. & Hubbard, 1%. E. (1984). Prog. Biophys. Mol. Biol. 44, 97-179. Barlow, D. J. & Thornton, J. M. (1983). J. Mol. Biol. 158, 867-885. Barlow, D. J. & Thornton, J. M. (1988). J. Mol. Biol. 201, 601-619. Bennett, A. D. & Shaw, W. V. (1983) Biochem. J. 215, 29-38. Blow, D. M. & Crick, F. H. C. (1959). Acta Crystallogr. sect. A, 12, 794-802. Blow, D. M. & Matthews, B. W. (1973). Acts Crystallogr. sect. A, 29, 56-62. Blundell, T. L. & Johnson, L. N. (1976). Protein CrystaUography, Academic Press, London. Burns, D. K. & Crowl, R. M. (1987). In Protein Structure, Folding and Design 2, UCLA Symposium of Molecular and Cellular Biology (Oxender, D. L., ed.), vol. 69, A. R. Liss & Co, New York. Charles, I. G., Keyte, J. W. & Shaw, W. V. (1985). J. Bacteriol. 164, 123-129. Chatterjee, C., Dattagupta, J. K., Saha, N. N., Saenger, W. & Muller, K. (1979). J. Cryst. Mol. Struct. 9, 295-300. Crawford, J. L, Lipscomb, W. N. & Schellman, C. G. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 538-542. Fujinaga, M. & James, M. N. G. (1987). J. Mol. Biol. 195, 373-396. Gadet, A. (1974). Acta C~Tstallogr. sect. B, 30, 349-353. Gale, E. F., Cundliffe, E., 1%eynolds, P. E., Richmond, M. H. & Waring, M. J. (1981). The Molecular Basis of Antibiotic Action, 2nd edit., pp. 460-468, Wiley, London. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982). Mol. Cell. Biol. 2, 104-105. Greet, J. (1985). Methods Enzymol. 115, 206-224. Guest, J. t%. (1987). F E M S Microbiol. Letters, 44, 417-422. Harding, S. E., Shaw, W. V. & Rowe, A. J. (1987}. Biochem. Soc. Trans. 15, 513. Hendrickson, W. A. & Konnert, J. H. (1980). In Computing in Crystallography •(Diamond, R., Ramasehan, S. & Venkatesan, K., eds), pp. 13.01-13.23, Natl. Acad. Sci. India, Bangalore. Horinouchi, S. & Weisbtum, B. (1982). J. Bacteriol. 150, 815-825. Howard, A. (1986). Proc. EEC Cooperative Workshop on Position-Sensitive Detector Software (Phases I and II), Lure, Paris, 26 May-7 June 1986, pp. 89-94.

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Isa~cs, N. (1982). In Computational Crystallography (Sayre, D. ed,), pp. 398-408, Clarendon Press, Oxford. Jones, T. A. (1978). J. Appl. Crystallogr. l l , 268-272. Jones, T. A. & Thirup, S. {1986). EMBO J. 5, 819-822. Kabsch, W. & Sander, S. (1983) Biopolymers, 22, 2577-2637. Karplus, P. A. & Schulz, G. E. (1987). J. Mol. Biol. 195, 701-729. Kleanthous, C. & Shaw, W. V. (1984). Biochem. J. 223, 211-220. Kleanthous, C., Cullis, P. M. & Shaw, W. V. (1985). Biochemistry, 24, 5307-5313. Lee, B. K. & Richards, F. M. (1971). J. Mol. Biol. 55, 379-400. Leslie, A. G. W. (1987). In Computational Aspects of Protein Crystal Analysis (Helliwell, J. R., Machin, P.A. & Papiz, M.Z., eds), pp. 39-50, S.E.R.C Daresbury Laboratory, Daresbury, U.K. Leslie, A. G. W., Liddell, J. M. & Shaw, W. V. (1986). J. Mol. Biol. 188, 283-285. Leslie, A. G. W., Moody, P. C. E. & Shaw, W. V. (1988). Proc. Nat. Acad. Sci., U.S.A. 85, 4133-4137. Lewendon, A., Murray, I. A., Kleanthous, C., Cullis, P. M. & Shaw, W. V. (1988). Biochemistry, 27, 7385-7390. Matthews, B. W. (1968). J. Mol. Biol. 33,491-497.

Matthews, B. W. (1972). Macromolecules, 5, 818-819. Murray, I. A., Hawkins, A. R., Keyte, J. W. & Shaw, W. V. (1988). Biochem. J. 252, 173-179. Petsko, G. A., Phillips, D. C., Williams, R. J. P. & Wilson, I. A. (1978). J. Mol. Biol. 120, 435-459. Priestle, J. P. (1988). J. Appl. Crystallogr. 21,572-576. Ramakrishnan, C. & Ramachandran, G. N. (1965) Biophys. J. 5, 909-933. Richardson, J. S. (1981). Advan. Protein Chem. 34, 167-339. Shaw, W. V. (1967). J. Biol. Chem. 242, 687-693. Shaw, W. V. (1983). CRC Crit. Rev. Biochem. 14, 1-46. Shaw, W. V. & Unowsky, J. (1968). J. Bacteriol. 95, 1976-1978. Shaw, W. V., Packman, L. C., Burleigh, B. D., Dell, A., Morris, H . R . & Hartley, B.S. (1979). Nature (Lo~lon), 282, 870-872. Shaw, W. V., Brenner, D. G., Le Grice, S. F. J., Skinner, S. E. & Hawkins, A. R. (1985). F E B S Letters, 179, 101-106. Smith, J. L., Corfield, P. W. R., Hendrickson, W. A. & Low, B.W. (1988). Acta Crystallogr. sect. A, 44, 357-368. Suzuki, Y. & Okamoto, S. (1967). J. Biol. Chem. 242, 4722-4730.

Edited by R. Huber

Note added in proof. Subsequent examination of the refinement results have revealed an error in the program used to refine the structure, which primarily affects the atomic temperature factors. The use of an alternative procedure has reduced the R-factor from 18"3% to 15"9~/o for all reflections between 6 and 1"75 A resolution. The resulting changes in atomic co-ordinates are very small (r.m.s. shift 0"12 A for all atoms, 0-08 A for main-chain atoms) and do not significantly affect the results quoted. The r.m.s, change in temperature factors is 3"6 A 2 (2-4 A 2 for main-chain atoms and 4-3 A 2 for side-chain atoms).