Biochimica ef Biophysics Amt. 1089 (1991) 287-M Q 1991 Elsevier Science Publishers B.V. 0167-4781/9l/$fl3.50 ADONLS Lll67478lYIoOI66K

287

Review

BBAEXP 92274

Escherichia coli seryl-tRNA synthetase: the structure of a class 2

aminoacyl-tRNA synthetase Reuben Leberman, Michael Hgrtlein and Stephen Cusack European Molecular Biology Luboratoty, Grembk Otmtatim, Grenoble (France) (Received 7 May 1991)

Key words: Aminoacyl-tRNA

synthetase: Protein structure: Structural motif: Nucleotide binding: Evolution

Contents

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I.

Introduction

il.

.s.z % iz!y.!jtJ ..............................

Ill.

Cloning and gene sequence of SRSEC

IV.

Regulation of the gene for E. cd seryl-IRNAsynthetase

V.

Isolation of SRSEC

VI.

Crystallization of SRSEC

VII.

X-ray diffraction analysis of SRSEC

VIII.

Description of the structure

IX.

Classes of

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synthetases ...........................

Acknowledgements References

287

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I. Introduction

The aminoacyl-tRNA synthetases are a group of enzymes indispensible for ensuring the fidelity of the translation of the genetic code into protein gene products. The common property of these enzymes is the catalysis of the overall reaction AA,+ATP+tRNA”oAA,-tRNA”+AMP+PP,

Abbreviations: SRSEC, seryl-tRNA synthetase from Escherichia cd; MIR, multiple isomorphous replacement. Correspondence: R. Leberman, European Molecular Biology Laboratory, Grenoble Outstation, 156X. 38042 Grenoble Cedex, France.

292 294

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297

..

297

The product of aminoacyl-tRNA is the .means whereby a specific amino acid may be located with respect to its codon via a specific tRNA adaptor [l] in the process of translation of mRNA during protein biosynthesis. There are generally, but not universally, one enzyme for each of the 20 amino acids in any particular cell (or organelle) type. In their functional mode these enzymes must recognize, besides the common ATP, specifically a small substrate and a macromolecular substrate; the cognate amino acid and tRNA(s), respectively. Since the cognitive properties of the&e enzymes are central to their role in protein biosynthesis they are desirable candidates for studies by which their discriminatory powers might be defined in femw of tertiary

288 structure. Since their discovery by Hoagland [2], and until fairly recently, structural analysis of these enzymes has been mainly hampered by lack of material. The two enzymes whose three-dimensional structures have been extensively studied by X-ray diffraction analysis are tyrosyl-tRNA synthetase from Bacillus stearothermophilus and methionyl-tRNA synthetase from Escherichia coli; the former was isolated from large scale bacterial preparations [3] and the latter from an overexpressing episornal strain of E. coli [4]. The advent of modern recombinant DNA methodology has led, in the last decade, to a surge of activity in the structural analysis of aminoacyl-tRNA synthetases; the best example of this might be the primary and tertiary structure analysis of E. co~ glutaminyl-tRNA synthetase [5]. At the present time the primary structures of more than 50 aminoacyl-tRNA synthetases have been determined, and the recent sequencing of the gene for the E. coli cysteinyl-tRNA synthetase [6] has provided the means for completing the primary strucures of all the E. coli enzymes. Table I illustrates the variety in size and quarternary structure of these E. coli enzymes and we are just beginning to understand the relationships between this apparently diverse group of proteins. The determination of primary structure has, understandably, outstripped the determination of tertiary structures. However, the determination of the latter is essential for understanding the mechanisms of recognition and function at the molecular level, and the determination of this relationship between structure and

mechanics is our goal. The enzyme with which we chose to start was seryl-tRNA synthetase from E. coil (SRSEC). An isolation procedure for this enzyme was first described by Katze and Konigsberg [7] and an alternative protocol was produced later by Boeker et al. [8]. Both groups established that the enzyme was a dimer of apparently identical subunits of molecular weight 50000. The amino acid composition determined by Katze and Konigsberg agrees well with that found by us from the derived amino acid sequence [9]. The value for the extinction coefficient; A2sa---l~7.7 determined by Boeker et al. [8] by ultracentrifugation is consistent with the value we find (A2a i~0 --8) from small angle neutron scattering studies (Leberman et al., unpublished data). The studies performed by both laboratories on the properties and interaction of the enzyme with the various substrates in general agree [7,8,10-12]. One exception to this is the pH optimum for the aminoacylation reaction where Boeker et al. [8] find 7.4 and Katze and Konigsberg [7] find 8.5, the latter is in agreement with observations made in our laboratory (Piantier and Leberman, unpublished results). However, the major anomaly that came out of these studies was that the dimeric enzyme only bound one equivalent of cognate tRNA [11], whereas it clearly bound two equivalents of the other substrates at apparently equivalent sites [10]. This was resolved in a later publication [12] where a stoichiometry of approaching 2:1 was found for the tRNA: enzyme complex. Using small ang!e neutron scattering to follow complex formation

TABLE i

Structures and sizes of the Escherich~a coil aminoacyl.tRNA synthetases Amino acid

Quarternary structure

Subunit size (amino acid residues)

Reference

Alanine Arginine Aspartic acid A~paragine C-~teine Glutamic acid Glutamine Glycine Histidine lsoleucine Leucine Lysine (lysS) L ~ i n e (lysU) Methionine Phenylalanine Proline

a4 a a2 a2 a a a

875 577 590 466 461 471 551

0202

303, 689

a2 a a a2 a2 a a2/~ ~ or2 a2 a2 a2 a2 a

572 939 860 505 505 677 795, 327 572 430 642 334 424 951

Pumey et al. [33i Eriani et al. 134] Eriani et al. [35] Anselme a n d H~irtlein [36] Eriani et al. [6] Breton et al. [37] Hoben et al. [38] Webster et al. [39) Freedman et aL [40] Webster ct al. [41] Hirtlein and Madern [42] Kawakami et al. [43] l.~v~que et ai. [44] Dardel et al. [45] M~chalum et al. [46] Eriani et al. [25] Hfirtlein et aL [9] Mayaux et al. [47] Hall et aL [48] Barker et aL [49] Hiirtlein et al. [50]

SeHne Threonine Tryptophane Tyrosine Valine

289 we also find a stoichiometry of 2 on titration of the enzyme with cognate tRNA (Leberman et al., unpublished data). !1. tRNA s ~ identity

Biochemical and genetic studies have provided much information about tRNA acceptor identity (for review see Normanly and Abeison [13]). In many cases the anticodon has been identified as a major identity determinant. However, the anticodon of tRNAS% is apparently not involved in recognition by seryl-tRNA synthetase, since there is no conservation of anticodon bases among its isoacceptors associated with the presence of two different types of codons, UCN and A G C / U [14]. Procaryotic tRNA s~r, like tRNA ~u and tRNAT~, classified as class II tRNAs possess a long variable arm showing variation in both length and sequence within the isoacceptors. The discriminator base, the fourth base from the 3' end of the tRNA is phylogenetically well conserved in tRNAs~r (G), implying an important role or this base in aminoacylation. Normanly et al. [15] have demonstrated that a leucine specific suppressor tRNA acquired serine specificity in vivo by a change of 12 bases, comprising the discriminator base, five bases in the acceptor stem one base pair in the D-stem. However, the resulting molecule was a weak suppressor indicating that the base changes did not generate all tRNAs~r identity elements. Himeno et al. [16] studied the long variable arm as identity element for tRNAs~r using a variety of in vitro transcripts. They showed that the insertion of only two nucleotides into the variable stem of tRNATyr generated serine charging activity. The acceptor activity of some of such mutant tRNAs was enhanced by changes in nucleotides which seem to influence the orientation of the long variable arm indicating that this structure might be involved in the recognition by seryl-tRNA synthetase. These observations are in agreement with findings of Leinfeider et al. [17] that the tRNA responsible for incorporation of seleno-cysteine in proteins has a long variable arm and is initially charged with serine which is then converted into seleno-cysteine. The corresponding codon for this tRNA is normally chain terminating (opal), but in the appropriate mRNA context seleno-cysteine can be incorpated into a polypeptide. HI, Cloning and gene sequence of SRSEC With the exzeption of the iysyl-tRNA synthetase, the E. coli chromosome contains only one gene for each aminoacyl-tRNA synthetase. Temperature semitive mutants for SRSEC have been described and the structural gene has been localized at the 20 rain position on the E. coli linkage map near serC, the gene for

phosphohydroxy-pyruvate transaminase, an enzyme for the biosynthesis of serine [18]. A putative rho-independent transcriptional terminator separates the serS gen¢ from the anaerobic dimetbyisulphoxide reductase operon [19]. We used the serS temperature sensitive mutant K28 to clone the wild-type gene by complementation. K28 has a mutation in the structural gene for SRSEC which affects formation of its gene product at high temperature [20]. We transformed this mutant with an E. coli gene bank DNA containing chromosomal fragments of approx. 5 kb long, generated by partial digestion with Sau3A and Hpall cloned into pBR322 C/al and BamHl sites. Clones were selected on ampiciUin plates at 44 ° C. SRSEC activity in the crude extract of one of the isolated clones (pSerS1) was measured by the aminoacylation assay and compared with that of the host strain. The pSerS1 containing cell extract exhibited a greater than 50-fold enzyme activity compared to the control. To check if our construct contains an undeleted form of the enzyme and to estimate the level of expression we compared total cell extracts and 35S-labelled mini-cell extracts by SDS-PAGE. The overexpressed protein and a radioactive band in the mini-cell extract had the expected molecular weight (M r ~ 47000). The overexpressed protein reacted also positively with anti-SRSEC antiserum in a Western blot experiment. DNA sequencing was started by the subcloning of a 2 kb EcoRI-Hindlll fragment of pSerS1 bearing the total serS gene into pBR322. This construct was used to generate M13 clones enabling us to read the total serS gene in both directions. In this way 336 bp of the Y untranslated region, 1290 bp of the 430 amino acid coding region and 148 bp of the 3' untranslated region were sequenced. The 430 amino acid sequence derived from the sequence of the gene is shown in Fig. 1 and from this we can calculate a subunit molecular weight

of 48383. The identity of the derived protein sequence was confirmed by its corresponden,'e at the N-terminus of the first 25 residues determined by Edman degradation !

MLDPNLLRNEPDRVREKLRARGFKLqUD~L GRLEERRKVLQUKTENLQAE

51 RNSRSKSIGQRKRRGEDIEP LRLEUhgL~:~ ELDAAKR£LDRLQREIRDIR 101 LTIPNLPROEUPVGKOENDNUEU$RUGTPREFOFEURDHV]LGEMHS6LD 151 FAflRUKLTGSAFUUMKGQIRRMHRRLSQrMLPLHTEQHGYSE~YUPVION 2OI QDTLYGfGQLPI(FRGDLFHT RPLEEERDI$ NYRLiPTREVPLTNLURGEI 251

IDEDOLPIKMTRHTPCFRSE RGSYGRDTRGLIRm4QF01(UEMUQIURPED

301 SilRRLEEnTGHREgULOLLG LPYRI(IILCTGOI1GFGRCKTYOLEUWIPAQ 351 HTYAEISSCS(~UWOFQRRRfl QRRCRSK$OKrTRLUHTLNG $GLRQGRTLU 401

RUIIENYQQROGRIEUPEULRPVnNOLEVIO

Fig. 1. A m i n o acid sequence o f seryl-tRlqA synthetase from E. co//.

29O 1

of the isolated enzyme. The codon usage of the sets gene resembles in general that of highly expressed genes, with exception of the phenylalanine codons, where UUU is prefered in setS. For serine a rather equal distribution of all six possible codons is found.

2

3

4

IV. Regulation of the gene for E. cali seryl-tRNA syn-

thetase It is usually assumed that temperature-sensitive mutants produce proteins which are thermolabile at nonpermissive temperature. This turned out not to be so in the case of the serS ts-mutant K28. Although the mutant protein is more thermolabile than the wild-type enzyme at 57°(2 the striking discrepancy between the temperature-sensitivity for growth of this mutant and the unexpected thermostability of the altered seryltRNA synthetase at 45°C was examined by Hill and Konigsberg [20]. They found that the rate of formation of seryl-tRNA synthetase activity fell from 100% to near zero within ~ 4°(2 temperature range from 40 to 44°C. The temperature dependent rate of formation of SRSEC activity is reversible. Dropping the temperature from 44 to 37°(2 results, after a 2-3 rain delay, in a resumption of the initial rate of formation of enzyme activity. Addition of the E. coli RNA polymerase inhibitor rifampin just before the temperature shift down from 44 to 37°(2 inhibited the appearance of SRSEC activ!ty at lower temperature. The authors speculate that a base change in the structural gene might allow this portion of the DNA to be recognized as a transcriptional termination site ('attenuator region') at high temperatures, but not at 37 ° C. A different type of regulation of seryl-tRNA synthetase via a unlinked gene (serR located near leu) causing elevated levels of the enzyme has been described by Theall et al. [21]. The mode of action of the putative serR protein, its biochemical features and the reason why it effects only the expression of seryl-tRNA synthetase remain to be clarified.

SRSEC

EF-Tu

Q

Fig. 2. SDS-PAGEof samplesat variousstages in the purificationof seryl-tRNAsynthetase,stained with Coomassieblue. Lane !. bacterial cell extract after clarification;lane 2. pool of active fractions from DEAE-Sepharosecolumn; lane 3. pool of fractions from AcA 44 column:lane 4. pool of fractionsfrom Sepharose4B by hydrophobic interactionchromatography.

V. Isolation of SRSEC The isolation of SRSEC from the bacteria CSH26 pSerSl is facilitated by the high level of expression as judged by Coomassie blue staining of on SDS-PAGE gel (Fig. 2). This appears to be somewhat greater than the expression of polypeptide elongation factor Tu (EF-Tu) that usually constitutes 6 - 8 % of the soluble cell proteins of E. coii [22]. The isolation procedure for the enzyme used for the production of protein crystals consists of three steps which are carried out over a period of 3 days. The procedure is essentially that devised earlier for the isolation of EF-Tu [23] and is applicable to any similar overexpressed gene product in E. co//. The three steps are preparation of bacterial

cell extract, tractionation by anion-exchange chromatography and molecular sieving by gel-filtration. Frozen cell paste is suspended in 2-3 vol. (ml buffer per g paste) buffer (64 mM Tris, 50 mM HCl, 1 mM EDTA (pH 7.6) at room temperature) containing 1 m g / m l lysozyme. This suspension is gently stirred until the viscosity of the released DNA causes the apparent inversion of the meniscus of the solution. To complete lysis sodium deoxycholate is added, this is then followed by MgC! 2 (to 10 mM) and DNase I to digest the DNA and reduce the viscosity of the suspension. The cell extract is recovered by centrifuging the suspension at 15000 x g for 60 min and applied to a column of DEAE-Sepharose CL-6B preequilibrated with our

291 standard buffer (64 mM Tris, 50 mM HCI, 10 mM MgC! 2, 1 mM D T r , 0.1 mM PMSF (pH 7.6)L The column is developed at 4 ° C with a linear salt gradient of 0-0.3 M NaCI in equilibration buffer. l h e fractions collected from the column are assayed for enzyme activity by measuring the charging of tRNA with J4Clabelled serine according to reaction 1. Since the concentration of enzyme in the fractions is high the samples used for assay have to be considerably diluted (generally 2000-fold) before a meaningful decision can be made on which fractions should be taken for the activity pool. This pool is then brought to 70% saturation in ammonium sulphate and after 30-60 rain at room temperature the precipitated protein is recovered by centrifugation. The precipitate is redisoived in a minimum volume of standard buffer and applied to a column of AcA 44 preequilibrated and developed in the same buffer. Active fractions are collected and pooled and maybe concentrated by ammonium sulphate precipitation. The protein at this stage although not completely pure (see Fig. 2) may be used for the production of crystals. Further purification maybe carried by hydrophobic interaction chromatography from ammonium sulphate solutions on Sepharose 4B. VI. Crystallization of SRSEC After many unsuccessful crystallization trials of the the enzyme alone and with substrates it was decided to examine the effect of detergents. A sample of what was believed to be practical quality octyl glucoside was available in the laboratory and some of this was added to crystallization trials with ammonium sulphate. Crystals (Fig. 3) appeared within 2 days and preliminary measurements showed that these crystals diffracted to at least 2.7 ,g, and were suitable for X-ray diffraction

FiB. 3. C~/'~tals of Set~I,tRNA slmthetase from 52% saturated ammonium sulphate, 1% practical ~ (Pfanstiehl). Bar represents I ram.

i:il

ili,

,ilJ Fig. 4, Gas chromatograph of practical octyl-glucoside used for crystallization of seryl-tRNA synthetase superimposed on chromatogram of pure /3-octyl glucoside (large peak indicated by arrow). Courtes)' of M. Zulauf.

analysis. By defining the crystallization conditions more closely, the time between the start of an enzyme preparation ant! the production of crystals is generally 1 week. The octyl glucoside sample we have used turned out to contain very little/3-octyl glucoside as judged by gas chromatography (Fig. 4) and could not be replaced with pure/3-octyl glucoside or other samples of practical quality material provided by the manufacturers (Pfanstiehl). The manufactures did, however, provide free of charge a sufficient amount of the particular batch (Lot No. 14711) to ensure the continued production of crystals for our studies. So far the composition of the major component of this detergent has defied analysis. Under the conditons of crystallization nucleation occurs very readily so that unless the majority of the nucleii are removed many small crystals will be rapidly formed. These nuclei are removed by preparing a solution containing protein, detergent and ammonium sulphate at a lower concentration required for crystallization and centrifuging this in an Airfuge (Beckman) for 5 min. Crystal growth is carried out by equilibrating by vapour diffusion samples, as sitting drops, of the clarified solution against 52% saturated ammonium sulphate solutions. Crystals 0.2 to 1 mm in the maximum dimension can be grown routinely. Crystals can also be grown from sodium citrate solutions in the presence of the detergent but these offer no advantage over those obtained from ammonium sulphate solutions and have not been used in the structure analysis.

292 VII. X-ray diffraction analysis of SRSEC The crystal structure of SRSEC was solved by the method of multiple isomorphous replacement (MIR). A large number of heavy metal compounds were screened before sufficient independent derivatives were found. In the final phase calculation, three derivatives were used: a single-site dysprosium derivative (from dysprosium chloride), a twin-sited mercury derivative (from etbyimercury chloride) and a uranyl-derivative (from uranyl acetate) with a cluster of nine sites. The rare-earth site, in which the metal ion is chelated by Glu-355 and Asp-342 in the enzyme active site, could also be occupied by erbium, but not samarium; dysprosium was used because of its high anomalous scattering signal es~cially close to the LII edge at a wavelength of 1.444 A near to which the derivative data were measured using synchrotron radiation. The two mercury sites, obtained by soaking crystals for only 4 h with ethyl mercury chloride at 0.01 mM to prevent crystal cracking, are close to Cys-338 and Cys-374, respectively. The former site could also be occupied by gold (soaking with ammonium tetrachloroaurate) and both sites also by bismuth (soaking with bismuth chloride). A third mercury site, at Cys-266 in the active site of the enzyme, is occupied upon more prolonged soaking in ethyl-mercury chloride but crystals often crack under these conditions. The uranium derivative was only obtained by reducing the pH of the mother liquor from the normal pH 7.5 to pH 6.8 to avoid precipitation of uranyl hydroxide. Unravelling the positions of the nine uranium atoms which form a compact cluster in the active site was only possible with the phase information from the other two derivatives. Subsequent phase calculatious were dominated by the strong phasing power of this derivative for which the anomalous signal was also included. The final MIR ma~ was calculated with 16054 phased reflections to 2.8 A with a mean figure of merit of 0.69. The map could be improved by phase extension to 2.5 ,A by solvent flattening procedures using the high solvent content of the crystals (about 65%). The quality of the final map was sufficient to trace the chain with few ambiguities. The structure has been refined using 2.5 ,~, data collected on the image-plate detection system developed at the EMBL Hamburg Outstation at DESY. Refinement was done principally with the powerful molecular dynamics method using the GROMOS package [51]. Subsequently several cycles of HendricksonKonnert refinement (PROLSQ) were used to obtain a fmal model with improved geometry (e.g., bond length deviations of 0.012 ,~). The R-factor is 19.1% for the 24212 reflections with l > 2 s in the resolution range 6-2.5 ~ and 20.9% for all measured data (20161 reflections) between 25-2.5 ,~, (Nassar and Cusack, unpublished data). The final model includes all of the

Fig. 5. Ribbondiagramof the seryl-tRNAsynthetasemonomer.

protein apart from three disordered loops between residues 223-229, 272-279 and 377-381, 96 ordered water molecules and one putative detergent molecule (see below). The atomic temperature factors of the molecule are generally rather high with an average of 33 .~2 for all protein atoms. VIII. Description of the structure The ribbon diagram of the monomer of SRSEC (Fig. 5) shows clearly that the molecule is divided into two domains, the N-terminal helical arm domain, and the C-terminal catalytic domain. The former comprises residues 1-100 and is folded into a remarkable structure containing a 60 ,~ long, solvent-exposed, antiparallel coiled-coil. The rest of the molecule forms a large, globular a-/] domain built around a sevenstranded antiparallel E-sheet and contains both the putative active site and the dimer interface. The helical arm of seryl-tRNA synthetase, due to its length and solvent accessibility, is a completely unique structure. The sequences of the two major helical seg-

293 ments (H3 and H4) show features typical of coiled-coil structures, for instance the regular occurrence of hydrophobic residues at positions 1 and 4 of successive heptads. These residues form a central inter-helical hydrophobic core which contains several exan, ples of leucine-(iso)-leucine bridges. Approx. 40% of the residues in the arm are charged, with an overall net charge of - 2; several of these residues are involved in inter- or intra-helical salt links and hydrogen bonds which presumably stabilise the structure; others are free in solution and disordered. In the crystal, helix H3 makes important crystal contacts with one other molecule in the unit cell and the end of the arm is close to the active site of a neighboring molecule. The question therefore arises whether the arm exists in solution in the ordered conformation observed in the crystal, or whether the crystal environment has led to its stabilisation. No definitive answer can be given at the moment to this question although two observations are relevant. Firstly, the radius of gyration ofothe dimer calculated from the crystal structure is 33.1 A, whereas that measured by small angle neutron scattering under low salt conditions is 31.2 A (Leberman et al., unpublished data). If this difference is not simply due to the different solvent conditons in solution and in the crystal, it could be accounted for by disordering some or all of the arm since the calculated radius of gyration with residues 48-77 removed (i.e., half the arm) is 29.7 ,A. Secondly, circular dichroism measurements on a synthetic peptide corresponding to the helical ann (re-

sidues 27-99) show that at pH 7 and 15°C the peptide is approx. 65% helical, the helix content decreasing with temperature the mid-point of the helix-coil transition being about 30°C (Biou et al., unpublished results). Although for the peptide, unlike the native protein, there is no constraint holding the C and N-termini in proximity which might promote helix formation, taken together these results are suggesting that under physiological conditions there may be some partial unfolding of the helical arm. The biological function of the helical arm is as yet unclear although it can b~, speculated that it plays a role in tRNA binding. Some supporting evidence for this comes from a mutant in which the majority of the arm has been truncated by deletion, however, leaving the N-terminal residues intact that pack against the globular domain. This mutant, so far characterised only as a fusion protein with /3-galactosidase, appears to have lost aminoacylation activity (Hiirtlein et al., unpublished results). The globular domain comprising residues 101-430 is built around a seven-stranded antiparallel/3-sheet, denoted sheet A in Fig. 5. A large insertion between strands hA1 and bA7 is responsible for many of the interactions holding the dimer together. These include helix H7 which has several polar interactions with its symmetry related counterpart across the- d~mer interface and two short inter-monomer antiparailel 0-sheets. One of these is connected via a short parallel/3-strand (AS) with the major/3-sheet which is thus in reality an inter-monomer ten-stranded sheet. The second ¢)ermits

Fig. 6. Carbon backbone representation of the seryl-tRNAsynthetase monomer showing the positionof a detergent moleculein the putative activesite.

294 help to test the following hypothesis: crystallization of SRSEC requires two conditions to be fulfilled, (1) a particular conformation in the active site which can be induced either by ATP (or ATP and serine) or a hexylor heptyl-glucoside, and (2) the presence of additional detergent possibly to assist in crystal packing. More importantly, this new crystal form may provide a means of revealing the mode of binding of ATP and serine to the enzyme. This has hitherto been impossible since we have not been able to bind the small substrates to the enzyme in the original monoclinic crystal form, either by soaking or co-crystallization. This maybe due to several factors, for instance the presence of the putative detergent molecule near the active site and the constraints on flexibility of the active site imposed by crystal contacts.

projection of the disordered loop LI (residues 222-229) towards the active site of the opposite monomer. The functional role of this loop which has a highly negatively charged end (224-E-E-E-A-D) remains to be elucidated. Although there is, as yet, no crystallographic evidence for the location of the binding sites of any of the enzyme suhstrates, a putative active site can easily be identified as a deep cave in the protein over the central section of the ,0-sheet A and rimmed by a number of loops. Of particular interest is the second disordered loop L2 (residues 271-279), invisible in the electron density map, but which appears that it might form a flexible flap over the entrance to the active site. The most significant feature in the final difference electron density map is strong positive density near the putative active site (see Fig. 6). Its shape and environment (like a tadpole with a narrow tail buried in a hydrophohic pocket and its head protruding into the active site) is inconsistent with ATP or seryl-adenylate but it could correspond to an amphiphilic detergent molecule arising from the impure mixture of detergents absolutely required for crystal growth. A five-membered sugar ring with a six carbon chain, i.e., a hexylribofuranoside, fits the density reasonably well and refines stably although a definitive identification cannot be made. The hydrocarbon chain of octyl-glucoside would be too long to fit into the same pocket. This observation led us to resume crystallization trials of the enzyme in the presence of various pure ,8-acyl-glucopyranosides leading to a new orthorhombic crystal form. Native protein crystals grow only with //-hexyl- and fl-heptyl-, but not jS-octyl-glucopyranosides; similar crystals grow in the presence of ATP or ATP and serine with //-hexyl-, /3-heptyl- and /3-octyl- glucopyranoside. Further X-ray analysis of these crystals will c(((