.J. Mol. Rid. (1992) 223. 377-380

Crystallization and Preliminary Crystallographic Escherichia coli TEM 1 P-Lactamase C. Jelsch’, F. Lenfant2,

J. M. Masson

Data on

and J. P. Samama’t

1Laboratoire de Cristallographie Biologique IBMC, 15 rue RenC Descartes 67084 Strasbourg Cedex. France 21NSA, Centre de Transfert en Biotvchnologie et Microbiologic Laboratoire d ‘inge’nierie des Protdines. 7rA544 CNRS Avenue de Rclngueil, 31400 Toulouse C’edex, France (Received 22 ,July 1991; accepted 27 September 1991) Two crystal forms of Grambacteria TEM /?-lactamase have been obtained. The tetragonal form has a very large unit cell and diffracts to 3.0 A resolution. Orthorhombic crystals, grown using ammonium sulfate and a small amount of acetone as precipitating agents, belong to space group P2,2,2, with cell parameters a = 43.1 A, b = 644 A, c = 91.2 ii and has been designed that ensures diffract to I.7 A resolution. A seeding procedure reproducibility of the crystal properties. Molecular replacement, using a model reconst,ruct,ed from the C” co-ordinates from Staphylococcus auwus PC1 fl-lactamase. gives a solution that> satisfies crystal packing constraints. h’eywordx:

Class

crystallization:

fi-lactamase; Escherichin X-ray structure

p-lactamases (EC 3.5.2.6) from are plasmid-mediated enzymes. TEM enzymes. which are the most frequently encountered in Gram resistant clinical isolates, are highly active against penicillins and confer bacterial resistance t)owards fl-lactam ant,ibiotics by hydroIvzing the cyclic amide bond in the four-membered i-&g of penicillin molecules. Two TEM enzymes were initially described: they differ by a single amino acid substitjutSion Q39K (TEMl /TEM2) (Barthelemy et al.. 19%) and thus by their isoelectric points (Matt’hew $ Hedges. 1976). The emergence of novel resistance towards third generat,ion cephalosporins result’s from specific amino acid mutations in TEMl or TEM2 /?-lactamases. These enzymes are known as TEM3 to TEM9 B-lactamases (Collatz rt al., 1990). Class A /?-lactamases from E. coli are easily spread fin the transmissible R factor. It is then of clear clinical importance to describe, at the molecular level, the binding specificities of substrates to the enzyme. Threr classes of /?-lactamases (A, B and C) have been proposed on the basis of sequence alignments Eschrri&a

t Author addressed.

A

co/i

to whom all correspondence

should he

coli

TEMI

: antibiotics:

1980). Enzymes hydrolyzing oxacillin (Ambler, more rapidly than benzylpenicillin (OXA /?-lactamases: Hedges et al.: 1974; Dale et al., 1985) have been grouped as class D /?-lactamases (Joris et al., 1988). The class A enzymes probably evolved from a, common ancestral gene, while class B B-la&amases. of smaller size, carry a metal cofactor. It was init’ially proposed that only class C enzymes have the same evolutionary origin as DD-carboxypeptidases (Waxmann Cy Strominger, 1983). However. although sequence similarities between class A and C are weak, the X-ray structure of the class C B-lactamases from Citrobncter freundii (Oefner et al.. 1990) is related both to the class A /?-lactamase struct,ures (Herzberg & Moult. 1987; Dideberg rf al.. 1987) and to DD-carboxypeptidase (Kelly et al.. 1989). This finding supports the hypothesis of a common gene ancestor for all fi-lact’am target’ enzymes (Kelly et al., 1986; Samraoui et al., 1986). Class C P-lactamases bear a functionally important tyrosine residue at position 150 that corresponds t,o Serl30 in class A enzymes. a position highly conserved in this class and mutationally aensitive (Jacob et al.. 1990). Tt should also be mentioned. from substrate specificit?. considerations. that a point mutation at posltlon 166 in TKMl enzyme result,ed in a change in substrate

378

CT .J~lxch

profile. the mutant protein behaving much like a (Llass c‘ enzyme (Adachi et al.. 1991; Delaire it (~1.. 1991). The X-ray struct,ure analysis of several class A penicillinases from Gram+ bacteria have been solved. The P-lactamases from Bacillus licheniformis 749/C (Moews et al., 1990) and from Staphylococcus aureu* PC1 (Herzberg, 1991) were refined t,o 2 LA (1 ‘4 = 0.1 nm) resolution, the penicillinase from Streptomyces albus has been refined to 3 L% (Dideberg et al., 1987) and the enzyme structure from Bacillus cereus I compared to that of n-alanyl-n-alanine peptidase (Samraoui et al.. 1986). Crystallization and st,ructure determination of E. coli TEM j?-lactamase to 5.5 Lk resolution has been reported (Knox et al., 1973, 1976). In order to probe, through single and double mutations. the active site of TEM /&lnctamase and to understand the evolution of subst’rate specifici@ from penicillinase to cephalosporinase (Lenfant et al.. 1990, 1991), a program combining site-direct,ed mutagenesis and high resolution structure determination was undertaken. The gene of the TEMl /I-lactamase (263 amino acid residues) was expressed from the plasmid pGFTH T-Phe (Masson & Miller, 1986). The enzyme was extracted by osmotic shock from a six lit’er cult’ure, grown in a ferment’or at 37”(‘, on a saltenriched minimal medium supplemented with glucose. The protein extract was submitted to two successive ammonium sulfate precipitat,ions (at. 4506 and 75(+, saturation). After dialvsis, the protein was prepurified by fast-flow anion exchange chromatography on Q Sepharose (Pharmacia) and further dialyzed against water. At this stage, a 200 mg stock of partially purified fl-lactamase in 40 ml was obtained. The next step was fast liquid chromatography on a monoQ column (Pharmacia) in 20 mM-Tris (pH 7.5). with a 0 to 0.2 M linear gradient, followed by gel filtration on a Sephacryl HRlOO column (Pharmacia) in 50 mm-phosphate buffer (pH 7) and 0.1 M-NaCl. The enzyme fractions were pooled and concentrated using an Amicon YM-5 filter. /I-Lact,amase activity was measured spectrophotometrically at 37°C in 50 mm-sodium phosphate buffer with ampicillin as substrate (1 = 236 nm, As = 820 M-~ cm-‘). The purity of the protein can be estimated to be > 950/analysis, were obtained using the hanging drop method at, 6’C as follows. A solution of protein (20 mg/ml) in 60 mM-sodium phosphate buffer (pH 7%). containing 10:/b saturated ammonium sulfate solution was equilibrated against the same buffer containing 469; saturated ammonium sulfate solution and 4% acetone. Polyhedric crystals grew after a few weeks. In order to control nucleat,ion.and growth times a seeding procedure was designed. The droplet. prepared as above, was brought to supersaturation by vapor equilibrium against 0.1 Msodium phosphate buffer (pH 7.8) containing 41 ?(I saturated ammonium sulfate and 4O,;, acetone. A small crystal was then seeded. Two days lat,er. the ammonium sulfate concentration was raised to 43 y/o and three days later to 44(/,. The t’ypical crystal size was (h4 mm x 0.4 mm x 0.6 mm. Symmetry of t’he diffraction pattern and specific ext’inction conditions are (‘OIIsistent with the orthorhombic space group 1’2,2,2, with cell parameters a = 43.1 8. b = 64.4 4, c = 91.2 4. There is one molecule per asymmet>ric unit which gives a crystal volume per unit of molecular mass of 2.2 W3/Da. The crystals d#ract to 1.7 A resolution when exposed to rotating anode X-ray radiation. One heavy atom derivative was prepared by soaking native crystals in a 2 mM solution of PtCl, for two days. This derivative is isomorphous to t,he native data to 4.5 a resolution (Table 1). Structure determination of TEM j?-lactamase is currently being pursued using isomorphous replacement and molecular replacement methods. A model

379

Communications structure has been constructed from the 2.5 a C” co-ordinates of S. aureua found in t,he Brookhaven Protein Data Bank (entry: lblm.brk). There is 32:/o sequence identity between the proteins. Rotation (Crowther, 1972: Navaza, 1987) and translation (Crowther & Blow, 1967) functions give a solution consist’ent with one molecule per asymmetric unit and that satisfies crystal packing constraints. Details of thtase calculations will be reported elsewhere. A difference Fourier map of t’he platinum derivative using the phases derived from the molecular replacement solut)ion reveals peaks that satisfy the difference Patterson rect,or map. The major binding sit’e is found in the vicinity of Metld9. The comparison between high resolution crystallographic structures of TEM Gramand Gram+ p-lactamases might reveal the st’ructural a,nd/or funct,ional roles of residues conserved within each subgroup but of a quite different nature between subgroups. In this respect,. one could mention asparagine 245. aspartic acid 246. tryptophan or tyrosine 251 and proline 258 found in Gram+ enzymes, which become glycinch, isoleucine, glycine and arginine, respectively in (iram /I-lact,amases. We thank I). Moras for facilities placed at our disposal and for his interest in this project. The French Ministry of R,esearch and Technology is acknowledged for financial support, (cont,ract no. 89TO840). C.,I. is supported by a (‘?;RS-Biostru~trIrr SA grant.

References Adachi. H.. Ohta. T. Kr Matsuzawa. H. (1991). Site-directed mutants. at position 166. of RTEMl /?-lactamase that forms a stable acyl-enzyme intermediate with penicillin. J. Biol. Chem. 266. 3186+3191. Ambler. R. P. (1980). The structure of /I-lactamase. Phil. Trans. Roy. Sot. ser. B. 289, 321-331. Barthelemy. M.. Peduzzi. ,J. & Labia, R. (1985). Distinction entre les structures primaires des b-lactamases TEMl et TEMZ. Ann. Inst. Pasteur Microbial. 136A. 31 l--321. (‘ollatz, E.. Labia, R. & Gutmann, L. (1990). Molecular evolution of ubiquitous /?-lactamases towards extended-spectrum enzymes active against newer fl-lactam antibiotics. Mol. Microbial. 4, 1615-1620. Crowther. R. A. (1972). The fast rotation function. In Thr Molecular Replacement Method (Rossmann. M. G., ed.). pp. 173-178, Gordon & Breach, Pu’ew York. (‘rowther, R. ,\. & Blow, D. M. (1967). A method of positioning a known molecule in an unknown crystal structure. Acta Crystallogr. 23, 544-548. Dale, ,J. W., Godwin, D., Mossakowska, D.. Stephenson, I’. $ Wall, S. (1985). Sequence of the OX82 fl-lactamase: comparison with other penicillin-reactive enzymes. FEBS Letters, 191, 39-44. Delaire, M., Lenfant, F., Labia, R. & Masson, J. M. (1991). Site-directed mutagenesis on TEMl b-lactamase: role of Glu166 in catalysis and substrate binding. Protein Engin. 4, 805-810. Dideberg. 0.. Charlier, P., Wery, J. P.. Dehottay, P..

Dusart, J., Erpicum, T., Frere, J. M. & Guysen, ,J. M. (1987). The crystal structure of the /?-lactamase of Streptomyces albus G at @3 nm resolution. Biochwn. J. 245, 911-913. Hedges, R. W., Datta. N.. Kontomichalou. I”. & Smith, ,J. Y. (1974). Molecular specificities of R factor determined b-lactamases: correlation with plasmid compatibility. J. Bacterial. 117. 5ci-62. Herzberg. 0. (1991). Refined crystal structure of /?-lactamase from Staphylococcus aureus PC’1 at, 2.0 A4 resolution. J. Mol. Biol. 217, 701-719. Hrrzberg, 0. & Moult, J. (1987). Bacterial resistance t’o b-lact,am antibiotics: crystal struct,urr of a-lactamase from Staphylococcus aurws PC’1 at 2.5 1% resolution. Scirnxr. 236. 694-701.

Howard. A. J.. (Niland. 0. L., Finzel. H. (‘. & Poulos, T. L. (1987). The use of an imaging proportional counter in macromolecular crystallography. .I. Appl. Crystallogr. 20. 383-387. ,Jacob. F.. Joris. B.. Lepage, S.. Dusart,. J. B F&e. *J. M. (1990). Role of the conserved amino atsids of the SDN’ loop (Ser13’. Azs~‘~‘. hsn’32) in a class A fl-lactamasr studied by site-directed mutagenesis. Riochrm. J. 271. 399-406. Joris. B.. Ghuysrn, ,J. M.. Dive. G., Renard. ,I., Dideberg. 0.. Charher. P.. Frkre. J. M., Kelly. ,J. -1.. Boyington.

.J. (‘.. Morws. P. C. & Knox, ?J. R. (1988). The activesite-srrinr penicillin-recognizing enzymes as members of the Streptomycrs R61 DD-peptidasr family. Biochem J. 250. 313-324. Kelly. .J. A.. Dideberg. 0.. (‘harlier. I’.. \Vtbry. J. I’.. Lib&. M.. Moews. 1’. C’.. Knox. .J. K.. Duez, C‘.. Fraipont. (‘I.. Joris. K.. Dusart’. .J.. Fri~re. ,J. M. & Ghuysrn. !I. &I. (1986). On the origin of bacterial resistance to penicillin: comparison of a /5lactamase and a penic*illin target,. Sciencr. 231, 14% 1431. Kelly. A. J.. Knox. J. R., Zhao. H.. Frtre. .J. M. & (Ghuysm. J. M. (1989). C’rystallographic mapping of p-lactams bound to a n-alanyl-u-alanine peptidase target rnzvme. .I. ,Mol. Hiol. 209. 281~ 295. Knox. ,J. R.. ‘Zorsky. P. E. & Murthy. ?u‘. S. (1973). Preliminary crystallographic data for Escherichia coli b-lactamasr. .J. Mol. Bid. 79, 597 598. Knox, tJ. R.. Kelly. ,J. A.. Morws. P. (‘. &Z Nurthg, S. I%. (1976). 5.5 .% crystallographic structure of penicillin /?-lactamasr and radius of g,vration in solution. J. Mol. Hid. 104, 865%%5. Lenfant. F.. Labia, R. & Masson. ,J. $1. (1990). Probing the active site of p-lactamase R-TEMl by informational suppression. Hiochimir, 72. 195AO3. Lenfant. F.. T,abia. R,. 8 Masson. .J. M. (1991). Replacement of lysine 234 affects transition state stabilization in the active site of /I-lactamase TEM 1. J. Bid. (‘hvm. 266. 17187~17194. Mason. ,J. M. &I Miller. ,I. H. (1986). Expresston of synthetic suppressor tRNA genes under the c*ontrol of a synthetic promoter. Cena. 47. I79 183. Matthew. Il. & Hedges, R. 11;. (1976). Analytical isoelectric focusing of R factor-determined fl-lactamases: correlation with plasmid rompat~ibility. .I. Ba,cterioZ.

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P. C’.. Knox, J. R.. Didebrrg, 0.. C’harlier, 1’. 8: FrPre. ,J. 11. (1990). /I-lactamase of Bacillus lichenqormis 749/C. Proteins, 7. lTii%lil. Piavaza, ,J. (1987). On t’he fast rotation timc+ion. .-l&a Crystallogr. sect. A 43. 645-653. Oefner. 0.. I)‘Arcg. A., Daly, ,J. ?J.. (iubrrnator. K.. Charnas. R. L., Hienzr, T.. Hubrrrhwerlen. C. & FVinkler, F. K. (1990). Refined (arystal structurr of