Proc. Natl. Acad. Sci. USAVol. 90, pp. 2261-2265, March 1993Biochemistry

Metal-binding site in a class I tRNA synthetase localized to acysteine cluster inserted into nucleotide-binding fold

(aminoacyl-tRNA synthetase/zinc finger/metal binding domain)

JAMES A. LANDRO AND PAUL SCHIMMEL*Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Paul Schimmel, December 9, 1992

ABSTRACT The 10 class I aminoacyl-tRNA synthetasesshare a common N-terminal nucleotide-binding fold. Idiosyn-cratic polypeptide insertions into this fold introduce residuesimportant for activity, including those that interact with thetRNA acceptor helix. The class I Escherichia coli methionyl-tRNA synthetase (L-methionine:tRNAMet ligase, EC 6.1.1.10),a 676-amino acid homodimer, was shown previously by othersto contain zinc and to have an activity dependent on itspresence. We show here by atomic absorption spectroscopy andzinc titrations the presence of 1 mol of zinc per polypeptide.Replacement of zinc with cobalt yields an active enzyme witha visible absorption spectrum characteristic of tetrahedralcoordination to sulfur ligands and an intense metal-to-sulfurcharge-transfer band at 340 nm. Mapping of the metal-bindingsite by zinc blotting of recombinant and proteolytic fragmentslocalized the site to a polypeptide insertion between two strandsof a (3-sheet in the N-terminal nucleotide-binding fold thatcontains the catalytic site. Beginning at Cys-145, this insertioncontains a Cys-Xaa2-Cys-Xaa9-Cys-Xaa2-Cys motif. Site-directed substitution of these cysteines with serines yieldedproteins that were stable but generally devoid of activity. Withthis result there is now at least one example of a class I and ofa class I E. coli tRNA synthetase with a metal-binding domainimportant for activity inserted into the catalytic domain.

Escherichia coli methionyl-tRNA synthetase [L-methionine:tRNAMet ligase (AMP-forming), EC 6.1.1.10; MetRS] is amember of the class I tRNA synthetase family (1-3). Theseproteins interpret the genetic code by catalyzing the activa-tion of amino acids with ATP to form aminoacyl-adenylates,with subsequent transfer to the 3' end of the cognate tRNAs(4). The native protein is a dimer with an apparent subunit Mrof 76,000 (5) and contains one zinc atom per subunit that hasbeen implicated to be essential for activity (6, 7). Mildproteolysis with trypsin removes 130 amino acids from the Cterminus of the native protein to yield a fully active, zinc-containing, monomeric protein with an apparent Mr of64,000(8, 9).The N-terminal domain of the monomer consists of 360

amino acids and is organized into a nucleotide-binding (Ross-mann) fold (10). The fold has six strands of parallel ,3sheetwith two a-helices on either side of the ,B-structure (11). Thefirst 97 residues constitute the first half of the fold, which isfollowed by an insertion before the second half begins nearLeu-226. The intervening residues constitute connectivepolypeptide 1 (CP-1; ref. 12), which connects the third andfourth }3-sheets. Previous manipulations of CP-1 with se-quence insertions showed that some portions are importantfor function (13).The amino acid substrate and ATP are bound in the

nucleotide-binding fold (14), which is the site of amino acid

activation. The crystal structure of the trypsin-modifiedMetRS-ATP complex located the enzyme-bound zinc nearthe aminoacyl-adenylate site, with His-28, Thr-82, Asp-83,His-95, and possibly a water molecule serving as zinc ligands(11). However, in the absence of ATP, the conformation ofthe zinc-binding loop prohibited identification of the zincligands (14).Based on various combinations of four or more cysteine

and/or histidine residues, South and Summers (15) catego-rized short amino acid sequences containing these ligandsthat occur in a variety of proteins believed to bind zinc andto participate in protein-nucleic acid interactions. The N-ter-minal domain of MetRS harbors a cysteine-containing motif(Cys box; Table 1) first identified by Berg (18) as a potentialmetal-binding domain. The Cys-Xaa2-Cys-Xaa9-Cys-Xaa2-Cys sequence of MetRS differs from the "classical" zinc-finger sequence Cys-Xaa2-Cys-Xaal2.14-His-Xaa2-His (15) insome DNA-binding proteins as well as from "nonclassical"Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys (15) fingers present in ret-roviral nucleic acid-binding proteins and the Cys-Xaa2-Cys-Xaal3-Cys-Xaa2-Cys fingers of steroid-receptor-binding pro-teins (15).The class II alanyl-tRNA synthetase metal-binding motif

Cys-Xaa2-Cys-Xaa6-His-Xaa2-Cys (Table 1; ref. 16) occurs inthe N-terminal region of the protein. Although a Rossmann-like nucleotide-binding fold is not present in this synthetaseor other class II tRNA synthetases (19, 20), the metal-bindingsequence is within the ATP-binding domain (21). Zn(II)bound to Ala-tRNA synthetase can be substituted with Co(II)to generate a fully active enzyme with a spectroscopic probe.In the presence of tRNAAIa or a small RNA microhelix thatrecreates the 7-bp tRNAAla acceptor helix, the intensity ofthevisible absorption spectrum of the Co(II) enzyme increasesby >100%. There is no detectable change in the presence oftRNAMet or a noncognate RNA microhelix based on thesequence of tRNAHiS. These data suggest that interaction ofthe metal-binding domain with the acceptor stem is respon-sible for the effect (22).The Cys box sequence of MetRS resides within CP-1.

Although a metal ion is not present in the family memberglutaminyl-tRNA synthetase, the CP-1 insertion ensuresproper positioning of tRNAG0n within the active site byinteractions with the acceptor helix (23).These considerations suggest that the Cys box sequence is

properly positioned to play a role in recognition of substratetRNA. In addition, the importance of the Cys box region inMetRS for catalytic activity was suggested by site-directedmutagenesis (13), where a Cys-158 -* Ala mutation wasexpressed as a stable protein devoid of activity. For thesereasons we were prompted to determine whether the metal-binding site was located at the Cys box in CP-1 and whether

Abbreviations: MetRS, methionyl-tRNA synthetase; PAR, 4-(2-pyridylazo)resorcinol; PMPS, p-hydroxymercuriphenylsulfonate;CP-1, connective polypeptide 1; Cys box, cysteine-containing motif.*To whom reprint requests should be addressed.

2261

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2262 Biochemistry: Landro and Schimmel

Table 1. Representative sequences of metal-binding domainsType Sequence

Classical zinc finger C-X2-C-X1214-H-X2-HRetroviral NC binding protein C-X2-C-X4-H-X4-CSteroid hormone receptor C-X2-C-X13-C-X2-CAlaRS (E. coli) C-X2-C-X6-H-X2-CMetRS (E. coli) C-X2-C-X9-C-X2-CMetRS (Thermus thermophilus) C-X2-C-X13-C-X3-HRepresentative sequences of some known and putative metal-

binding domains. For more complete listing and discussion ofnomenclature, see South and Summers (15). A more complete list ofsequences occurring within the aminoacyl-tRNA synthetases can befound in Miller et al. (16) and Nureki et al. (17). NC, nucleocapsid;AlaRS, alanyl-tRNA synthetase.

it was important for structural stability and, additionally oralternatively, for enzyme activity.

MATERIALS AND METHODSMaterials. [35S]Methionine and Rainbow molecular weight

markers were obtained from Amersham; 65ZnC12 was ob-tained from New England Nuclear; tRNAfmet was obtainedfrom Boehringer Mannheim; cobalt dichloride hydrate(99.999%) was obtained from Aldrich; p-hydroxymercuriphe-nylsulfonate (PMPS) and protease type XVII-B from Staph-ylococcus aureus strain V8 were obtained from Sigma; 4-(2-pyridylazo)resorcinol (PAR) was obtained from EastmanOrganic Chemicals (Eastman Kodak); Chelex 100 (100-200mesh) was obtained from Bio-Rad.Atomic Absorption Spectroscopy. These experiments were

done at the Massachusetts Institute of Technology CentralAnalytical Facility with a Perkin-Elmer 703 spectrometer.Samples submitted for analysis were treated with metal-freebuffer in Centricon 30 microconcentrators (Amicon; ref. 24)to remove adventitious metal.Enzyme Purification and Activity Assays. Expression and

purification of the full-length native and split (R367//M; ref.25) MetRSs were done as described by Burbaum and Schim-mel (25); expression and purification of the monomericMetRS and site-directed Cys box mutants containing theN-terminal 547 amino acids (fragment 547N) of the wild-typeenzyme were done according to Kim and Schimmel (26),except that protein was further purified on a Superose-12column (Pharmacia) equilibrated in Tris-HCl (50 mM, pH 7.5)containing 1 mM 2-mercaptoethanol and 250 mM NaCl.Aminoacylation and tRNAfMet charging assays were done asdescribed by Kim and Schimmel (26).

Reaction of MetRS with PMPS. To monitor Zn(II) release,MetRS (5 ,uM, dialyzed to remove 2-mercaptoethanol) in 0.33ml of 50 mM TrissHCl, pH 7.8/25 mM NaCl/0.1 mM PARwas titrated at ambient temperature over time with succes-sive additions of stock PMPS (0.5 mM by weight). Absor-bance at 500 nm was recorded 5 min after PMPS addition toallow for a consistent, stable reading. The spectrophotometerwas adjusted to zero absorbance units before the first PMPSaddition; therefore, subsequent readings representedAA500m. This absorbance change was converted into molesof zinc by using the reported extinction coefficient for theZn(II)PAR2 complex (A E = 6.6 x 104 M-1-cm-1; ref. 27). Tomonitor formation of a charge-transfer complex at 250 nm,MetRS (13.2 ,uM) in 0.33 ml of the above buffer was titratedat ambient temperature over time with successive additionsof stock PMPS (1.0 mM). The A250nm, indicative of sulfur --

Hg(II) charge-transfer absorption, was measured relative tothat of the start of the titration (AA25,m_).

In Vivo Cobalt(II) Substitution. Cells were grown in poly-propylene Erlenmeyer flasks in minimal medium containing(per liter) 2 g of (NH4)2SO4, 0.5 mg of FeSO4-7 H20, 1 ml of

1 M MgSO4, 2 ml of glycerol, 100 ml of 1 M phosphate buffer(pH 7.0), and 1 g of Casamino acids. The phosphate bufferwas passed over a Chelex-100 column to reduce the amountof zinc in the medium. Co2+ was introduced by CoCl2addition to a final concentration of 5 x 10-6 M. Co(II)-substituted fragment 547N was purified by the same methodused to purify the native protein, except that 1 mM EDTAwas included in all buffers.

Proteolytic Digestion. Cleavage of glutamyl bonds in frag-ment 547N was achieved with a 45-min treatment with S.aureus V8 protease at a ratio to substrate of 1:12 (mol/mol)in 100 mM ammonium bicarbonate (pH 8.25) at 37°C. At theend of this treatment, the sample was treated at 95°C for 3 minand then stored at -20°C until used.

Zinc Blotting and N-Terminal Sequence Determination. Thezinc-blotting procedure of Schiff and coworkers (28) wasfollowed with some minor modifications. Proteins and pep-tides were separated on a 15% SDS/PAGE gel and thentransferred to Immobilon-P poly(vinylidene difluoride) mem-branes (Millipore) with a Milliblot semi-dry electrode accord-ing to the directions of the manufacturer. Metal-bindingbuffer consisted of 100mM Pipes, pH 7.0/50mM NaCl. Zincblots were analyzed by using a Molecular Dynamics phos-phorimager interfaced with Image Quant software. TheN-terminal sequences ofbands on poly(vinylidene difluoride)membrane that bound zinc were determined by CharlesBurkins at the Whitehead Institute for Biomedical Researchon an Applied Biosystems model 470 sequenator by using theprocedure of Matsudaira (29).

Site-Directed Mutagenesis. Site-directed mutants of cys-teine residues within the Cys box sequence of MetRS wereconstructed by using the Amersham mutagenesis system onthe phagemid pJB104 described by Kim and Schimmel (26).Mutants were screened by ampicillin resistance and se-quenced by using Sequenase (United States Biochemical).The mutant phagemids were transformed into E. coli strainMJR (K-12 1EA metG(Km)recA-), a methionine auxotrophdue to the Pro-14 -3 Ser mutation (30) that elevates Km formethionine (5), and were tested for in vivo complementationwithout methionine.Immunoblot Analysis. The synthesis of mutant MetRS

proteins in E. coli JM109 [recAl, supE44, endAl, hsdR17,gyrA96, relAl, thi A(lac-proAB)] was induced by 1 mMisopropyl j3-D-thiogalactopyranoside. Induced cells from 1 mlof LB medium were harvested by centrifugation, resus-pended in 100 ,ul of gel loading buffer, treated at 95°C for 3min, and centrifuged to remove cell debris. Proteins wereseparated on a 10% SDS/PAGE gel. Subsequent to transferof proteins to poly(vinylidene difluoride) membrane, as de-scribed above, the membranes were treated with anti-MetRSpolyclonal antibodies, as described by Kim and Schimmel(26).

RESULTSZn(II) Content of MetRS. The zinc content of full-length

native MetRS and the monomeric N-terminal fragment 547Nwere determined by atomic absorption spectroscopy at var-ious concentrations ofprotein (2-7 ,uM; data not shown). Ourdata corroborate the results of earlier investigators (6, 7),which indicated, on average, 1 mol of zinc per mol ofpolypeptide chain.

1, 10-Phenanthroline has been used to remove zinc from thetruncated monomer (7). This reaction is slow and generallyirreversible. As an alternative to remove endogenous Zn(II)from MetRS, the strongly dissociating sulfhydryl reagentPMPS was used. Fig. 1 illustrates the release of zinc fromfragment 547N promoted by PMPS. Metal released during thePMPS reaction was monitored with the zinc-binding dyePAR, which forms a Zn(II)PAR2 complex with an absorption

Proc. Natl. Acad. Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 2263

the same with respect to adenylate synthesis and aminoacy-lation (data not shown).

Fig. 2 gives the UV and visible absorption spectrum ofCo(II)-substituted fragment 547N and clearly suggests asulfur indentity for the ligand donor atoms. Three discreted-d electronic transitions positioned at 733 (e = 777), 702 (e= 660), and 651 nm (e = 310), together with an intense

_ _0.3 absorption band at 340 nm (e = 3450; E in units of M-1-cm-1)are present. The positions and magnitudes of the d-d tran-

-0.25 ~~~~~sitions of Co(II)-fragment 547N are consistent with this/ 0.2 Co(II) occupying a tetrahedral or distorted tetrahedral envi-

iQ _ /ronment (31). The position and magnitude of the absorptionL .o15- / 1 j band at 340 nm indicate S -- Co(II) charge transfer, consis-

c o.1 / tent with cysteine ligands to the intrinsic Co(II). The spec-(A o /trum in Fig. 2 can be virtually abolished by adding 6 equiv-g < 0.05 / alents of PMPS (data not shown).

_o_/_,_,_,_,_Localization of the Metal-Binding Domain. To localize the0 2 4 6 8 10 metal-binding site within MetRS, we dissected the protein byEquivalents gene manipulations and proteolysis and probed these prep-

/ _ _ | l arations by the 65Zn-blotting technique of Schiff and cowork-ers (28). By this technique, the full-length polypeptide (676-

0 2 4 6 8 1 0 mer) clearly binds radiolabeled zinc, as does the truncatedEquivalents PMPS fragment 547N (Fig. 3).

The metal-binding domain was localized to the N-terminalQuantitation of Zn(II) release from E. coli MetRS with 366 amino acids by using the "split" protein of Burbaum and

[etRS (5 ,uM) in 0.33 ml of 50mM Tris HCl, pH 7.8/25 mM Schimmel (25). This protein was constructed by replacementmM PAR was titrated at ambient temperature with suc- of the codon for Arg-367 of metG with the 15-nt sequencedditions from a stock solution of 0.5 mM PMPS. The corresponding to the intersubunit region of E. coli glyS,absorbance change at 500 nm corresponded to 1.05 mol of which consists of a stop codon, a ribosome-binding site, and

r mol of MetRS. (Inset) Formation of a charge-transfer a methionine start codon. The split protein is expressed as ain MetRS with PMPS. MetRS (13.2 ,uM) in 0.33 ml of the separate N-terminal (a) fragment and a C-terminal ((3) frag-ffer was titrated at ambient temperature with successive ment consisting of 366 and 310 amino acids, respectively. Asfrom a stock PMPS solution (1.0 mM) to give the indicated shown in Fig. 3, the a fragment, which contns the entireif equivalents of PMPS relative to MetRS. The A25onm, shown indig fold, contains the entof S -* Hg(II) charge-transfer absorption, was measured nucleotide-binding fold, contains the metal-binding domain.that of the start of titration (AA25Onm). The arrow indicates In an attempt to localize the metal-binding domain withinabsorbance after addition of 2 mM 2-mercaptoethanol. the nucleotide-binding fold, we probed proteolytic fragments

of fragment 547N for their abilities to bind metal. A numbermaximum at 500 nm. PMPS titration of fragment 547N in thepresence of PAR exhibits a linear release of Zn(II), where-upon color development plateaus. This plateau correspondsto 1.05 mol of Zn(II) released per mol of protein. In addition,the formation of an S -+ Hg charge-transfer complex can bemonitored at 250 nm (Fig. 1 Inset). The data show the linearincorporation of 5-6 equivalents of PMPS per mol of proteinand suggest a sulfhydryl identity for the metal-binding li-gands. [There are eight cysteines in fragment 547N (5).] Theabsorbances at 500 nm and 250 nm can be abolished by theaddition of 2 mM EDTA (data not shown) and 2 mM2-mercaptoethanol, respectively. Abolition of absorbance at250 nm by the addition of thiol indicates the reversibility ofthe covalent modification.

Visible Absorption Spectroscopy of Co(ll)-MetRS. Substi-tution of Co(II) for Zn(II) provides a spectroscopic probe inthe visible region that is sensitive to metal ion coordinationand geometry. The direct in vitro substitution of Co(II) forZn(II) in fragment 547N proved difficult, as attempts toexchange the endogenous metal with Co(II) by dialysisprecipitated protein. Similar precipitation occurred whenprotein was treated with excess PMPS, treated with 2-mer-captoethanol to reduce modified sulfhydryls, and, subse-quently, dialyzed against Co(II)-containing buffer.

Alternatively, Co(II) substitution was achieved by in vivoincorporation, as described in Materials and Methods. TheCo(II)-substituted fragment 547N was purified by the sameprocedure used to purify the zinc metalloenzyme. Atomicabsorption analysis of the Co(II) content of this enzymepreparation indicated 0.85 mol of Co(II)/mol of protein. Thecatalytic activities of the Co and Zn enzymes are essentially

E0

i

.04-0

0

0

0

wavelength (nm)

FIG. 2. UV/visible absorption spectrum of Co(II) fragment

547N. The absorption spectrum of a solution of Co(II) fragment 547N

(10.7 mg/ml) in 50 mM Tris.HCl/25 mM NaCl was recorded and

corrected by subtracting out the absorption spectrum of the native

Zn(II) protein with a Beckman DU-64 spectrophotometer. The d-d

electronic transitions and presumed Cys -. Co(II) charge-transfer

transition are clearly present.

1.2

1Ec00"- 0.8

0)

*b 0.6

cro 0.4R

0.2

0

FIG. 1.PMPS. MNaCl/0.1cessive almaximumZn(II) peicomplex iabove buladditionsnumber aindicativerelative toobserved

Biochemistry: Landro and Schimmel

2264 Biochemistry: Landro and Schimmel

B 547

026367

H2i

D CIC02H H

367E s132H2N I

l< tC02H

FIG. 3. Localization of the zinc-binding site to the Cys box in MetRS. (Center) Amido black-stained gel. (Left) Lanes: A, molecular sizemarkers (46, 30, 21.5, 14.3, 6.5, and 3.4 kDa); B, full-length monomer (676-mer); C, truncated monomer (fragment 547N); D, split protein; E,S. aureus V8 protease digestion of fragment 547N. (Right) Phosphorimage (16 hr) of a 65Zn blot of the same gel before staining. Arrows in Centerleft gel mark locations of protein that binds zinc in Center right gel. The molecular size marker at 30 kDa corresponds to carbonic anhydrase,a zinc metalloenzyme. Arrows and numbers associated with various permutations of the structure in the diagrams B-E correspond to cleavagesites introduced by proteolysis or genetic engineering. The filled circle associated with the C terminus of the fragment 547N digest product ispresent to indicate the general location of the fragment end on the basis of SDS/PAGE gel mobility.

of proteases were tested for their abilities to release a

relatively small peptide, which retained metal-binding capac-ity. The S. aureus V8 protease, when used under conditionsthat cleaved glutamyl bonds (32), yielded such a fragment.Treatment of fragment 547N with this protease released a

metal-binding peptide with a molecular weight of< 10,000. Asdetermined by direct sequencing from poly(vinylidene fluo-ride) membrane (see Materials and Methods), the N-terminalsequence of the first five residues of this fragment is Lys132-Gly-Met-Phe-Leu136. This sequence occurs within CP-1 ofthe nucleotide-binding fold and is close to Cys-145. Thisresult, together with the spectroscopic data of Fig. 2, suggeststhat Cysl45-Pro-Lys-Cys-Lys-Ser-Pro-Asp-Gln-Tyr-Gly-Asp-Asn-Cys-Glu-Val- ys6 is the metal-binding domain inMetRS. Tfiis assignment contrasts with the location of themetal-binding domain based on a crystallographic analysis(His-28, Thr-82, Asp-83, His-95). None of these ligands are

expected to give rise to the spectrum of Fig. 1 and, inparticular, the segment His-28-His-95 is missing from thezinc-binding 10-kDa peptide shown in Fig. 3.

Substitutio

Stability

Compleme

143

Site-Directed Mutagenesis in the Cys Box. The functionalimportance of the cysteine residues in the putative metal-binding domain was investigated by site-directed mutagene-sis (Fig. 4). Individual Cys -* Ser substitutions were intro-

duced via oligonucleotide-directed mutagenesis (Fig. 4). Sub-sequent to construction of mutants in phagemid pJB104,phenotypes were tested with E. coli strain MJR, whichharbors a Km mutant for dimeric MetRS (5, 28). Althoughthese four mutants are expressed as stable proteins (byimmunoblot analysis, data not shown), only the Cys-148 --

Ser mutant exhibited complementation. This protein waspurified to homogeneity from E. coli JM109 by the sameprocedure used to purify the wild-type fragment 547N. It has20% of the adenylate synthesis and aminoacylation activitiesof the native enzyme and contains 0.19 mol of Zn(II)/mol ofprotein.

DISCUSSIONRecent investigations of MetRS from T. thermophilusshowed that this protein is an a2 dimer that contains one

Ala

t 163

Gly Thr|Cys Pro Lys Cys Lys Ser Pro Asp Gln Tyr Gly Asp Asn Cys Glu Val Cy Gly Ala

n Ser Ser Ser Ser

+ + + +

Mnt _ + _ _

FIG. 4. Site-directed mutagenesis of the Cys box sequence. Amino acid substitutions introduced in this study are indicated below thesequence, whereas the Cys-158 -* Ala substitution, introduced in a previous study (13), is indicated above sequence. Mutant proteins werejudged

for activity on their abilities to complement E. coli MJR, a methionine auxotroph due to an elevated Km for methionine (5, 30). The Cys-148-. Ser mutant was, in addition, purified from E. coli JM109.

C

'I

Proc. Natl. Acad. Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 2265

tightly bound zinc ion per subunit, that is essential foraminoacylation activity (33). The metal-binding site has beenlocalized to the N-terminal domain of the protein (34), aCys-His box residing in CP-1 has been identified (Table 1),and a Cys-127 -- Ser mutant (the N-terminal cysteine in Table1) is devoid of activity yet is structurally stable (17). In viewof our assignment of the Cys box in E. coli MetRS as themetal-binding site, the Cys-His box in the T. thermophilusenzyme is the probable metal coordination motif. Both metal-binding sites are inserted into the same region, and thecorresponding Cys -* Ser substitutions at the N-terminalcysteine (positions 127 and 145 in the T. thermophilus and E.coli proteins, respectively) result in the expression of stable,but inactive, proteins. The production of stable, yet inactive,protein for the Cys -- Ser mutants at positions 158 and 161of E. coli MetRS adds further evidence for the importance ofthis domain for catalytic activity. Additionally, the presenceof 0.2 mol of Zn(II) per mol in the Cys-148 -+ Ser mutant,which retains 20% of the catalytic activities of native enzyme,also suggests a direct relationship between Zn(II) content andcatalytic activity.Due to the disparity between our and the crystallographic

assignment of the metal-binding site, the structure of thisdomain remains unknown. However, the three-dimensionalstructure of the Cys box sequence Cys-Xaa2-Cys-Gly-Tyr-Xaa-Tyr-Asp-Xaa8_15-Phe-Xaa6-Trp-Xaa-Cys-Pro-Xaa-Cysconserved in the iron-containing rubredoxins present in anumber of anaerobes has been investigated from Pyrococcusfuriosus by using NMR with the zinc-substituted protein (35).The protein contains a three-stranded antiparallel (3-sheet,several tight turns, and a hydrophobic core. In addition, thebackbone conformation of the region of the N-terminalcysteine-pair of a retroviral-type zinc finger (36) is identicalto the folding of relevant residues in the iron domain ofrubredoxin, raising the possibility of structural similaritieswith the metal-binding domain in E. coli MetRS.Although the present investigation has not specifically

probed the functional significance of the metal-binding do-main in MetRS, some insight has been obtained. Our obser-vation of an electron-rich, tetrathiolate coordination complexis expected to restrict expansion of the coordination sphereby electron-donating ligands as well as solvent access to theZn(II) ion. This hypothesis is supported by the observationthat the absorption spectrum of Co(II)-substituted fragment547N is not perturbed by the addition of substrates (data notshown). Therefore, although the metal-binding region maynot play a direct role in catalysis by coordination to sub-strate(s), it may be required for the maintenance of a con-formation crucial for catalytic activity. The production ofstable Cys -3 Ser mutant proteins suggests that the presenceof metal is not a protein-folding requirement and that theeffect that metal has on conformation is highly localized. Amore extensive mutagenic analysis is currently underway toinvestigate the significance of the metal in this protein.

We thank Professor Michael Summers (University of Maryland-Baltimore County) and Eric Schmidt (Massachusetts Institute ofTechnology) for reading and commenting on the manuscript beforesubmission for publication. This work was supported by Grant GM15539 from the National Institutes of Health and by an AmericanCancer Society Postdoctoral Fellowship Grant PF-3755 to J.A.L.

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