The crystal structure of the β-lactamase of Streptomyces albus G at 0.3 nm resolution

Biochem. J. (1987) 245, 911-913 (Printedin GreatBritain)

The

crystal

structure

of the /-lactamase

of

Streptomyces

albus

G

at

0.3

nm

resolution

OttoDIDEBERG,* PauletteCHARLIER,* Jean-PierreWERY,* Philippe DEHOTTAY,t Jean DUSART,t ThomasERPICUM,t Jean-Marie FREREt and Jean-MarieGHUYSENt

*LaboratoiredeCristalographie, InstitutdePhysique, B5,andtServicedeMicrobiologie,Institut deChimie, B6, UniversityofLiege, B-4000SartTilman(Liege 1), Belgium

Thecrystal structure of the

fl-lactamase

of Streptomycesalbus Ghas been solved at 0.3 nm resolution by X-ray-diffraction methods. Theenzymeisatypicaltwo-domainprotein.One domain consists of fivea-helices, andthe other isfive-stranded f-sheet witha-helicesonboth sidesof the sheet. The active-siteserineresidue (Ser-48) is within acleft located between thetwo domains.

INTRODUCTION

Three classes, A, B and C, of/1-lactamases

(fl-lactam

hydrolases,EC3.5.2.6)have beenidentified onthe basis of their substrate specificity, amino acid sequence and

mechanistic properties. The

fl-lactamases

of class B possess azinc cofactor; those of class A and C operate

by an acyl-enzyme mechanism involving an active-site

serine residue. Several

fl-lactamases

of class A and C have been studied by X-ray-crystallographic methods (Knox et al., 1976; Aschaffenburg et al., 1978; Charlier et al., 1983; Dideberg et al., 1985; Moult et al., 1985; Kellyetal., 1986;Samraouietal., 1986). Asreportedby Kelly et al. (1986) and Samraoui etal. (1986), the class A

fl-lactamases

of Bacillus

licheniformis

and B. cereus on the one hand and theDD-peptidaseofStreptomycesR61 onthe otheraresimilar intheextentand distribution of secondary structures (though lacking relatedness in primary structure). In no case, however, have these studies disclosed thecompletetracingofthepolypeptide

chain.Recently,the geneencodingtheclass

A,f-lactamase

of StreptomycesalbusG hasbeencloned and sequenced,

thusgivingaccesstotheprimarystructure of the enzyme (Dehottay et al., 1987), and the active-site serine residue has beenidentified at position48 (i.e. position 89 inthe precursor) after

,l-iodopenicillanate

derivatization of the

protein(De Meester et al., 1987).Inturn, we here present

thethree-dimensionalstructureof thisStreptomyces albus

G

,J-lactamase,

solved at0.3nm resolution.

MATERIALS ANDMETHODS

The enzyme (purified to protein homogeneity as

describedbyDuez etal., 1981)wascrystallizedusingthe

vapour-diffusion technique (McPherson, 1976). The

protein[21 mg ml-'in 25mM-sodiumphosphate buffer, pH 7.0, containing 8% (v/v) glycerol and 8% ethylene

glycol] was dialysed against 50mM-Tris/HCl, pH 7.0,

containing 10mM-NaN3. After centrifugation, 10 d

drops ofthe protein solutionwere

(i)

mixed with3

,1

of

a

10%

solution of10000-Mr

poly(ethylene

glycol)

made

in the above buffer and (ii)

suspended

over 1 ml wells containing thesame

poly(ethylene

glycol)

solution. After

1 week ofequilibration at 12°C, well-formed crystals

were obtained whichgrew up to 1.2mm in

length

and

0.5mminwidth.When

exposed

tonitrocefin

(0.1

mM in the mother liquor), the

crystal

and the

surrounding

mixture became red after 3 min at 18 °C, as aresult of

thecatalysedhydrolysisof the

fl-lactam

amidebond.The space group was

p212121

and the unit cell paramdters were a=4.067 nm, b= 4.353 nm and c= 13.843 nm.

Assuming one 28500-Mr protein molecule per asym-metricunit,a

specific

valueof 0.00216 nm3(2.16

A3)/Da

Table1. Data-collectionandphasingparameters Abbreviation:pCMB,p-chloromercuribenzoate.

Soaking conditions Phasing

No. of power Resolution

Enzymederivative Concn. (mM) Period (days) RisO*(%) sites (FH/E) (nm)

Native K2Pt(C204)2 K3UO2F5 pCMB SmCl3 U02(NO3)2 K2PtCl4 XIFPH-FPI *Ri = hkl

klFpl

hkl 5 5 1 0.5 1 5 5 2 2 2 1 7 12 26 18 12 15 9 1 2 3 2.4 2.0 1.7 0.23 0.4 0.4 0.3 0.4 0.3 0.4 Vol. 245 911

0. Dideberg and others wascalculated.This value was within the range found for

proteincrystals (Matthews, 1968).Three native datasets werecollectedto aresolution of 0.23 nm.

Six heavy-atom derivatives

[K2Pt(C204)2,

K3UO2F5, SmCl3, U02(NO3)2, K2PtCl4 and

p-chloromercuri-benzoate] of the enzyme were studied by collecting the intensity data on a Stoe-Siemens diffractometer with

graphite monochromated CuKa radiation. Two

equi-valentreflections weremeasured with the nativeenzyme and, for the derivatives, the Bijvoet mate of each

reflectionwascollectedat -20. Decaywasmonitoredby using two standard reflectionsfor every 100 reflections. Lorentz,polarization and absorption (Northetal., 1968) corrections were applied. The main data-collection and

phasingparameters are shown in Table 1.

The difference Patterson functions with coefficients

(IFPHI-IFPI)2

or

(IFI-_IFi1)2

for the

p-chloromercuribenzo-atederivative wereeasily interpretable. The heavy-atom positions and occupancies were refined by using the lowest estimate of theheavy-atom structure

amplitude,

FHLE.Theprotein phases thus derivedwerethen usedto

calculate a cross-phaseddifference Fourier map and to locate the heavy-atom-binding sites of the other derivatives. Each solution was compared with the two Patterson functions. Only two derivatives [SmCl3 and

U02(NO3)2]

gave aconsistentsetof

heavy-atom

sites(so that three derivatives were used for the

phasing

procedure).

Positional, thermal and occupancy parameters were varied in thephase refinement.Minorsiteswerefoundby inspecting the difference Fourier map. The overall figure-of-meritwas0.79for4462reflections.Amini-map (scale 0.4nm/cm) was calculated on a

grid

of

32x32x100using the phase obtainedandtheamplitude weighted by theindividual

figure-of-merit.

Protein and solventregions appeared wellin themap. Approximate

C.,

positions weredirectly read off themapusinga grid chart, and the

C.

positions served as guide points to

construct the polypeptide backbone. The polyalanine modelwasfittedtothe 0.3 nm electron-density map with a PS300 Evans andSutherland colour graphics display system usingthe program FRODO (Jones, 1985).

RESULTS ANDDISCUSSION

The residuenumbering used below refers to the mature enzyme (whose N-terminus is Gly-40 of the precursor

protein). Except for the residues-77-91 amino-acid stretch, which lacked continuous density, thetracing of the chainas derivedfrom theelectron-density map was

unambiguous (Fig. 1). The two-domain protein has an

overall dimension of 5.5 nmx4.3 nm x 3.8nm. One of

thedomainshasacentral structural coreconsisting ofa

five-stranded

f-sheet

withthree a-heliceson oneface and

onea-helixonthe other. Thestrands S3,S4 and

S.

form

a

fl-meander,

averystablestructurethat is also foundin the active-site serine proteinases of the trypsin family (Schulz & Schirmer, 1979). The strands

S,

and S2 have

a hairpin connection, and the

fl-sheet

has the usual left-handedtwist witharather smallQangle. The second domain consists of five a-helices. Stabilizing salt bridges

occurthroughout thestructure.Inparticular, Glu-16of helixH1 interacts with Arg-39of strand

S2*

The active site (*, Fig. 1), identified as the cleft

possessing theserine residue at position 48 and known to be labelled by

fl-iodopenicillanate

derivatization of the

protein(seetheIntroduction),istopologically defined by the N-terminal portion of helixH3on one side, strand S3 onthe other side,helix H2 at the back, a loop connecting helicesH7andH8onthe top and aloop connecting helices

H5 and H6 atthe bottom (Fig. 2).The active-site Ser-48 is at the N-terminus of helix H2, a position that may

facilitatecatalysis (Hol, 1985). Other residueswhich, in concertwith Ser-48,mayplayimportantroles in catalysis

and/or substrate binding are Lys-51 (on H2), Glu-150

andAsn-1 54(onaloop between

H.

andH,),and Lys-218

N

Fig.1. Schematicrepresentation of the three-dimensionalstructureof theStreptomycesalbusG0-lactamase

,f-Strands

(Si)arerepresented byarrows,andhelices(Hi)bycylinders. Nis theN-terminus; Cis the C-terminus.

1987 912

Crystal structure of Streptomyces,6-lactamase 913

XA218 A218

CA5i CA5

C A4 A2 20 CA4 CA220

Fig. 2. Astereoview showing potentially important residues in the active site of theStreptomycesalbus GfJ-lactamase

Theorientation of the molecule is similar to that Fig. 1. The labelled residues are: Ser-48, Lys-51, Ser-l14,Glu-150,Asn-154,

Lys-218 andGly-220. 0,C<t(CA) atom.

and Gly-220 (on S3). Lys-51, Lys-218 and Gly-220 are conserved in the /-lactamases of class A and the DD-peptidases/penicillin-binding proteins of known primary structure.

The arrangement of secondary-structure elements in theStreptomyces albus G ,B-lactamase is comparable with those found in B. licheniformis and B. cereus Ienzymes (Kelly et al., 1986; Samraoui et al., 1986), except that one helix is missing in the two latter structures (H8 in B. licheniformisenzymeand H5in the B. cereusenzyme).

The work was supported bythe Fondsde Recherchede la Facultede Medecine, the Fonds de laRecherche Scientifique Medicale, Brussels(contractsno.3.4522.86 and3.4507.83),the Gouvernementbelge (actionconcerteeno. 86/91-90) and the Region wallonne (C2/C16/CONV.246/20428). J.D. is Cher-cheur qualifie of the Fonds National de la Recherche Scientifique(FNRS, Brussels).

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Received3 May 1987/15 May 1987;accepted27May1987