A Metal–Ligand-mediated Intersubunit Allosteric Switch in Related SmtB/ArsR Zinc Sensor Proteins

A Metal–Ligand-mediated Intersubunit AllostericSwitch in Related SmtB/ArsR Zinc Sensor Proteins

Christoph Eicken1,2, Mario A. Pennella3, Xiaohua Chen3

Karl M. Koshlap3, Michael L. VanZile3, James C. Sacchettini1 andDavid P. Giedroc3*

1Department of Biochemistryand BiophysicsCenter for Structural BiologyTexas A&M UniversityCollege StationTX 77843-2128, USA

2Institute of Biosciencesand Technology, Texas A&MUniversity, HoustonTX 77030, USA

3Department of Biochemistryand BiophysicsCenter for AdvancedBiomolecular ResearchTexas A&M UniversityCollege StationTX 77843-2128, USA

The origin of metal ion selectivity by members of the SmtB/ArsR familyof bacterial metal-sensing transcriptional repressors and the mechanismof negative allosteric regulation of DNA binding is poorly understood.Here, we report that two homologous zinc sensors, Staphylococcus aureusCzrA and cyanobacterial SmtB, are “winged” helix homodimeric DNA-binding proteins that bind Zn(II) to a pair of tetrahedral, interhelical bind-ing sites, with two ligands derived from the a5 helix of one subunit,Asp84 Od1 (Asp104 in SmtB), His86 Nd1 (His106), and two derived fromthe a5 helix of the other, His970 Nd1 (His1170) and His1000 N12 (Glu1200).Formation of the metal chelate drives a quaternary structural switchmediated by an intersubunit hydrogen-binding network that originateswith the non-liganding N12 face of His97 in CzrA (His117 in SmtB) thatstabilizes a low-affinity, DNA-binding conformation. The structure of theZn1 SmtB homodimer shows that both metal-binding sites of the dimermust be occupied for the quaternary structural switch to occur. Thus, acritical zinc-ligating histidine residue obligatorily couples formation ofthe metal-sensing coordination chelate to changes in the conformationand dynamics of the putative DNA-binding helices.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: metal sensing; zinc; transcriptional regulation; allostery; x-raystructure; histidine*Corresponding author

Introduction

Zinc and other transition metal ions play essen-tial roles in the diverse array of cellular processes,either as structural components in proteins or asobligate cofactors in metalloenzymes.1,2 All metalions, however, become toxic at high concentrations.This has led to the evolution of homeostatic mecha-nisms that maintain the intracellular concen-trations of biologically required metal ions, e.g.Zn, Fe, Cu, Mn, Co, and Ni, in a range that iscompatible for cell viability through the involve-ment of specific metallochaperones that traffic

metals inside cells and/or the coordinate regu-lation of specific uptake and efflux systems.3,4

Heavy metal pollutants, e.g. Cd, Pb, Bi, and Hg,are removed efficiently from cells using the samegeneral mechanisms. Although the specific detailsdiffer, gene regulatory systems required for theefficient detoxification and resistance of high con-centrations of transition metal ions are ubiquitousin nature.

In prokaryotes, the SmtB/ArsR5 and MerR6

family of metal sensor proteins represent two gen-eral classes of metalloregulatory proteins. MerRproteins are transcriptional repressors in theabsence of inducing metal ions and become potentactivators when bound to their metal-ion coacti-vator;7 metal binding drives an underwinding ofthe promoter DNA, which repositions the 210and 235 sequences to allow for a favorable inter-action with RNA polymerase.8 Other non-metalregulated MerR family members include Escherichiacoli SoxR,9 which senses oxidative stress via a2Fe-2S redox center, and Bacillus subtilis BmrR,10

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

C.E. and M.A.P. contributed equally to this work.

E-mail address of the corresponding author:[email protected]

Abbreviations used: HSQC, heteronuclear singlequantum coherence; TROSY, transverse relaxationoptimized spectroscopy; NOESY, nuclear Overhausereffect spectroscopy; HMBC, heteronuclear multiple bondcorrelation; HTH, helix-turn-helix.

doi:10.1016/j.jmb.2003.09.007 J. Mol. Biol. (2003) 333, 683–695

which binds toxic drugs and regulates theexpression of a multidrug efflux transporter.Although metal-sensing MerR proteins have beencharacterized that respond to a wide range ofdivalent transition metals,6 how individual MerRregulators discriminate among distinct metal ionsand couple metal binding to DNA conformationalchanges is unknown.

The SmtB/ArsR family of homodimeric helix-turn-helix (HTH) proteins represses the expressionof operons associated with metal ion sequestrationor efflux in both Gram-positive and Gram-negativebacteria, allowing these organisms to survive whenchallenged with toxic concentrations of heavymetal ions.11 Human pathogenic bacteria, includingvirulent strains of Staphylococcus aureus and Myco-bacterium tuberculosis12 sometimes encode manysuch metal-resistance systems, potentially provid-ing a selective advantage for survival in the humanhost. Comparative biochemical and spectroscopicstudies of six SmtB/ArsR proteins reveal thatthese proteins harbor one or both of two structu-rally distinct metal coordination sites, denoteda3N or a5,5,13 named for the location of the metalsites in the known or predicted secondary struc-

ture of individual family members. The a3N siteis cysteine thiolate-rich, forming S3 or S4 complexeswith large, thiophilic metals including Cd, Pb andBi, as found in the cadmium sensor S. aureusCadC.13 – 15 Site-directed mutagenesis and aminoacid sequence comparisons suggested that the a5site is composed of a combination of carboxylateand imidazole ligands, interacting preferentiallywith harder transition metal ions including Zn, Coand Ni.12,16,17 Spectroscopic studies of a zinc sensorencoded by S. aureus, CzrA, and a Ni-sensor fromM. tuberculosis, NmtR, suggest that a change in thecoordination number from four in CzrA to six inNmtR in the a5 metal sites is sufficient to switchthe metal selectivity from Zn/Co in CzrA to Ni/Co in NmtR.17 Metal binding to both sites isknown to regulate operator-promoter bindingstrongly, negatively and allosterically, reducing theaffinity of the repressor for DNA by ^ 300-fold,depending on the system.15,17,18

Synechococcus SmtB regulates the expression ofthe divergently transcribed smt operon nearlyexclusively in response to Zn(II).12,19 In additionto the smtB gene, this operon encodes SmtA, aprokaryotic metallothionein that sequesters toxic

Figure 1. Structure of cyanobacterial SmtB. (a) Sequence alignment of Synechococcus SmtB and S. aureus CzrA, point-ing out the location of the proposed ligands to the a3N (red boxes) and a5 (blue boxes) metal-binding sites.16 (b) Rib-bon representation of the structure of homodimeric apo-SmtB solved to 1.7 A resolution, with the side-chains of theproposed ligands of one of the a5 metal sites indicated (see (a)). Secondary structural units (N-a1-a2-a3-aR(a4)-b1-b2-a5-C) are indicated for the gold-shaded protomer. In the gold-shaded subunit, C-terminal residues 119–121, includ-ing zinc ligand Glu120, are not visible in the electron density maps, indicative of significant disorder in the absence ofzinc. (c) Superposition of apo-SmtB (green and gold-colored protomers) with Zn2 a5-SmtB (blue and cyan-colored sub-units), the latter solved to 2.3 A resolution. The polypeptide chain could not be traced from residues 92–97 in the blue-colored Zn2 a5-SmtB subunit. The Figure was generated by performing a superposition of the Ca atoms of the a5helices of the green- and blue-shaded subunits of apo- and Zn2 a5-SmtB, respectively. This illustrates the large move-ment of the HTH and b-wings of one subunit relative to the other, represented by a movement of the Ser74 Ca atomspositioned toward the N terminus of the aR helix by 4.8 A. (d) Superposition of the metal-binding residues in theapo- and Zn2 forms of SmtB derived from the superposition shown in Figure 1(c). Figures were created usingSPOCK: http://mackerel.tamu.edu/spock

684 Allosteric Switch in Zinc Sensors SmtB and CzrA

http://quorum.tamu.edu/spock

metals.20 S. aureus CzrA regulates the expression onthe czr operon, the expression of which is inducedby Zn and Co.21,22 The czr operon encodes CzrB, apredicted cation diffusion facilitator (CDF) antipor-ter efflux pump23 that appears to be structurallyhomologous to E. coli ZitB.24 SmtB and CzrA share35% identity in overlapping regions, including pro-posed ligands to the a5 metal ion; however, CzrAlacks an N-terminal extension as well as otheramino acid ligands thought to comprise the a3Nmetal-binding site in SmtB (Cys14, His18, Cys610

and Asp640) (see Figure 1(a)).16 The 2.2 A structureof apo-SmtB reported previously25 reveals thathomodimeric SmtB is a highly elongated wingedHTH protein, with each subunit adopting anaaaabba structure. This structure provided littledirect insight into the regulatory mechanism, sincethe structure of the zinc-bound protein was notreported.

Results

A metal-induced quaternary structuralconformational change inSynechococcus SmtB

A ribbon representation of the structure of apowild-type SmtB solved to higher resolution thanpreviously (1.7 A; see Table 1 for structure statis-tics) is shown in Figure 1(b). As expected,25 thestructure conforms to the winged helix class ofhomodimeric HTH (a3-turn-aR) DNA-bindingproteins.26 As previously found,25 the first 20 or soN-terminal amino acids of both subunits, denotedthe N-terminal arm, are missing from the electrondensity maps, presumably because they are disor-dered. However, it is clear from this structure thatthe N-terminal arm of one subunit is close to thea3 helix of the other, suggesting that the previouslycharacterized a3N Zn(II) in SmtB (Figure 1(a))16

site employs ligands drawn from both subunits.Previous biochemical studies showed that apo-

SmtB contained both a3N and a5 metal-bindingsites and that the occupancy and precise coordi-nation environment of each depended on thenature of the metal ion.16 Therefore, a triple-substi-tution mutant of SmtB was prepared in which allthree cysteine residues (Cys14 in the N-terminalarm, Cys61 in the a3 helix, and Cys121 near the Cterminus; Figure 1(a)) were converted to non-liganding serine residues, to create a5-SmtB. Thismutant, like S-methylated SmtB,16,18 retains thehigh binding affinity of the a5 metal ion, andstrong negative regulation of smtO/P binding invitro, but blocks metal binding to the a3N site.16

Figure 1(c) shows the global structural differencesbetween apo-SmtB and fully zinc-ligandeda5-SmtB, determined to 2.3 A resolution, asdepicted by a superposition of the a5 helices ofone of the two protomers (the subunits on theright) within each dimer. Strikingly, the overallstructural change induced by metal binding is

exclusively quaternary structural in nature, welldescribed as a rigid-body rotation/translation ofone subunit relative to the other. For example, inthis superposition (Figure 1(c)), the Ser74 Ca atomin the aR helix of the subunit shown on the leftmoves by 4.8 A, or an average of <2.4 A in eachsubunit within the dimer; this reduces the point-to-point distance between Ser74 Ca atoms onopposite subunits by 3.6 A, from 51.7 A to 48.1 A.Thus, the homodimer becomes more globally com-pact on Zn(II) binding, in qualitative agreementwith the results of previous sedimentation equi-librium experiments;27 this results in significantmovements of both the HTH motif and b-hairpinwings in opposite subunits relative to one another.

The two zinc ions are bound to the homodimeracross the dimer interface in a symmetry-relatedpair of interhelical a5 metal-binding sites thatstraddle the a5 helices, fully compatible with pre-dictions made from the primary structure (Figure1(d))16 as well as a preliminary analysis of aheavy-atom derivative of apo-SmtB obtained frommercuric acetate soaks.25 Both zinc chelates in Zn2

a5-SmtB adopt distorted tetrahedral coordinationgeometries (ligand-Zn-ligand bond angles rangingfrom 89–1248 with 109.58 perfect tetrahedral sym-metry; averaged Zn-ligand bond lengths of 2.05–2.14 A), with ligands donated by Asp104 Od1 andthe His106 Nd1 atoms of one protomer, and His1170

Nd1 and Glu1200 O11 of the other. The metal chelateis largely pre-organized, with the exception ofGlu120, which is in an “open” conformation in theabsence of metal (in one protomer, electron densitycould not be traced beyond L118) and swings in to“close the gate” on the metal complex (Figure1(d)). Mutagenesis of His106 to a non-ligandingGln abolishes negative regulation of DNA-bindingin vitro18 and, when replaced by Arg, abrogateszinc-sensing in vivo,19 revealing that this is in factthe functional zinc-sensing site in SmtB.

Crystallographic studies of S. aureus CzrA

A ribbon representation of the dimer of dimersof the related zinc sensor apo-CzrA found in thecrystallographic asymmetric unit, solved to 2.0 Aresolution, is shown in Figure 2(a). The first five toeight N-terminal residues and C-terminal three tofive residues are not visible in the electron densitymaps. Chemical shift indexing5 and the magnitudeof the heteronuclear 15N– 1H NOE (M.A.P. andD.P.G., unpublished results) reveal that theseN-terminal and C-terminal regions are disorderedin solution as well. In contrast to the extensiveinterface formed by the N-terminal a1 andC-terminal a5 helices on opposite subunits withinthe dimer, the interdimer interface is filled withH2O molecules with contacts largely limited tothe turn connecting the a3 and a4 helices and theN-terminal end of the a4 helix (primarily residuesAsn50, Ser54 and Gln55) in the HTH motif.This is likely a crystal packing interface, sincesedimentation equilibrium experiments show no

Allosteric Switch in Zinc Sensors SmtB and CzrA 685

evidence of self-association beyond the dimer.17

As expected from previous NMR studies, thesecondary aaaabba and tertiary structures ofapo-CzrA are identical with that of apo-SmtB(Figure 1(b)).5 The major difference between thecrystallographic dimeric structures of apo-SmtBand apo-CzrA is that apo-CzrA is more com-pact than either apo- or Zn2 a5-SmtB. Forexample, the point-to-point distance betweenSer54 Ca atoms in the aR helices on oppositesubunits in apo-CzrA (Ser54 is analogous toSer74 in SmtB; see Figure 1(a)) is 43.8 A, nearly8 A smaller than in apo-SmtB.

A global superposition of the structures of apoand Zn2 CzrA, the latter solved to 2.3 A resolution,shown as a protomer Ca superposition (Figure2(b)), reveals that in contrast to SmtB (Figure 1(c)),the global quaternary structural change uponmetal binding in CzrA is small, with the inter-subunit Ser54 Ca distance decreasing by only0.5 A. This may be due, in part, to crystal packingforces, since both Zn2 versus apo-CzrA crystal

lattices (vide supra) are stabilized by a similar setof interdimer interactions mediated by thea3-turn-a4 HTH motif. In apo-CzrA, these inter-dimer contacts may significantly alter the relativeorientation of one subunit relative to the otherin this dissociable homodimer ðKdimer ¼ 1:7£105 M21Þ:17 In the case of Zn2 CzrA, a zinc ion isbound between Ser52 (3.7–3.8 A) and Asn55(3.3 A) bridging two molecules related by crystallo-graphic symmetry (data not shown). In addition, asecond metal ion appears to bridge the bona fidea5-metal binding sites, mediating a second inter-dimer contact via a distorted square pyramidalcoordination sphere, the base of which is formedby His86 N12, His96 N12, His9600 N12 (from anotherdimer), and a water molecule, with the apicalposition of the pyramid donated by His8600 N12.Since there is no evidence for additional metal ionbinding beyond two per dimeric CzrA molecule insolution,17 these additional metal ions bound bythe a4 and a5 helices of Zn2 CzrA are likely crystal-lographic artifacts that play no role in vivo and may

Table 1. Data collection and refinement statistics

Data collectiona

Data setapo-SmtB

Zn2 a5-SmtBremote

(Zn MAD)Peak Inflection

Zn2 a5-SmtB(refinement)

Zn1

SmtBapo-CzrA Zn2 CzrA

Beam line APS14C

APS 14D APS 14D APS 14D APS 14D APS14C

Inhouse

APS 14C

Unit cell parametersa (A) 37.24 64.48 64.47 64.50 64.45 37.00 50.12 54.44b (A) 69.72 125.72 125.00 125.47 124.68 70.37 50.12 75.37c (A) 43.22 26.00 26.00 26.00 25.91 42.68 153.55 50.63b (deg.) 95.84 95.50

Space group P2(1) P2(1)2(1)2 P2(1)2(1)2 P2(1)2(1)2 P2(1)2(1)2 P2(1) P31 P2(1)2(1)2Monomers/ASU 2 2 2 2 2 2 4 2Wavelength (A) 1.00 1.2398 1.2828 1.2833 1. 2828 1.00 1.54 1.00Resolution (A) 60–1.7 62–2.8 62–2.8 62–2.8 50–2.3 60–2.0 18.9–2.0 60–2.3Redundancy 4.3 5.8 5.8 6.0 6.0 3.3 3.0 5.7Completeness (%) 92.5

(58.3)98.5 (94.5) 96.3 (87.2) 98.1 (92.5) 94.4 (85.8) 96.8

(85.7)96.7

(74.1)97.4 (91.4)

I/s(I) 19.9 18.8 22.6 20.8 12.6 27.4 13.6 15.4Rsym (%)b 2.9

(18.5)5.8 (12.8) 4.5 (10.1) 4.8 (11.5) 6.9 (27.1) 3.0

(7.4)3.5

(13.7)4.9 (12.2)

Refinement statistics

Data setapo-SmtB Zn2 a5-SmtB Zn1 SmtB

Apo-CzrA Zn2 CzrA

Resolution (A) 60–1.7 60–2.3 60–2.0 18.9–2.0 60–2.3No. reflections used 21,874 8841 14,328 20,625 9215Test set 2163 442 1458 2355 949Rcryst (%)c 21.6 22.9 20.4 20.2 19.5Rfree (%)c 24.9 29.0 23.6 25.6 24.9Average B-factor

(A2)66.3 35.5 53.7 33.8 37.6

No. of non-hydro-gen atoms

1640 1569 1712 3216 1638

rmsd. bond length(A)

0.004 0.028 0.005 0.022 0.005

rmsd. bond angle(deg.)

1.038 2.175 1.056 1.818 1.017

a Values in parentheses are for the highest-resolution shell.b Rsym ¼ ShSilIðhÞI 2 kIðhÞll=ShSikIðhÞil; where I is the observed intensity, and kIl is the average intensity of multiple observations of

symmetry-related reflections.c R ¼ SllFol2 lFcll=SlFol: Rcryst and Rfree were calculated using the working and the test reflection sets, respectively.


have been facilitated by the high pH of these par-ticular crystals (see Materials and Methods).

Despite these additional interdimer contacts, thecoordination structure of the a5 chelate in CzrA isvirtually superimposable on that of SmtB (Figure2(c)), with the N12 atom of His100 donating thefourth ligand in CzrA, rather than the carboxylate

oxygen atom of Glu120 in SmtB, as anticipatedfrom the sequence alignment (Figure 1(a)). In Zn2

CzrA, Zn(II)-ligand coordination bond lengthsrange from 1.8–2.1 A, typical for tetrahedral Zncoordination to N/O atoms. The coordinationgeometry is less distorted than in Zn2 a5-SmtB,since ligand-Zn-ligand bond angles cluster more

Figure 2. Structure of S. aureus CzrA. (a) Ribbon representation of the structure of apo-CzrA solved to 2.0 A resolu-tion with all four subunits of the asymmetric unit shown. The secondary structural units for the gold subunit areshown. (b) Global Ca trace subunit-based superposition of apo-CzrA with Zn2 CzrA, the latter solved to 2.3 A resolu-tion. (c) Representative structure of the zinc coordination chelate of Zn2 a5-SmtB superimposed on that derived fromZn2 CzrA.

Figure 3. An intersubunithydrogen-bonding network in Zn2

a5-SmtB involved in allostericcoupling of Zn and DNA bindingsites. (a) Schematic of the inter-subunit hydrogen-bonding networkthat links the allosteric a5 zinc-binding sites to the DNA-bindingaR helices (shaded yellow) in Zn2

a5-SmtB. Only one of the coordi-nation chelates is shown (the zincion is indicated as a black sphere).The critical intersubunit His117N12–H12· · ·OvC Arg870 hydrogenbond is encircled. (b) Another viewof structural changes that occurupon Zn(II) binding to the a5metal sites of SmtB. Left panel,apo-SmtB; right panel, Zn2 a5-SmtB.Only one of the two metal chelatesis shown.


Figure 4 (legend opposite)

closely around 109.58 (the range is 102–1128), withthe exception of the H86 Nd1-Zn(II)-H97 Nd1, anglewhich is 1288.

A metal–ligand-mediated cross-subunithydrogen bonding network links the metal andDNA-binding sites

A detailed analysis of the crystallographic struc-tures of apo and Zn2 SmtB suggests that His117plays a critical role in establishing an intersubunithydrogen bonding network that may be importantfor allostery (Figure 3). This network originateswith the non-liganding N12 atom of this His, andextends, via just two hydrogen bonds, the full dis-tance to the DNA-binding aR helix via the aR-b1loop (Figure 3(a)). In Zn2 SmtB, but not in theapoprotein, this His forms a short, linearN12–H12· · ·OvC Arg870 side-chain to main-chainhydrogen bond (dN– O ¼ 2:67 �A versus 3.6 A in theapo-SmtB structure) across the subunit interface ofboth protomers. The immediately adjacent amide

group of Leu880 then forms a short backboneN-H· · ·OvC Leu630 hydrogen bond ðdN– O ¼ 2:95 �AÞto the carbonyl oxygen atom of Leu830, anunsatisfied a-helical hydrogen bond acceptornear the C terminus of the DNA-binding aR helix.Furthermore, on at least one side of the homo-dimer, the side-chain of Arg870 interacts intimatelywith the metal chelate, with the guanidino groupmoving into hydrogen-bonding distance fromboth the backbone carbonyl oxygen atom ofHis117 and the non-liganding O12 atom of Glu120upon formation of the zinc complex (Figure 3(b)).A comprehensive sequence alignment reveals thatonly a5 zinc sensors like SmtB and CzrA uni-versally conserve a basic Arg/Lys/His residue inthe position corresponding to Arg87 in the aR-b1loop of SmtB.5 We hypothesize that these reciprocalmain-chain and side-chain hydrogen bondinginteractions functionally link the a5 and aR helicesof opposite protomers and play a significant role inallosteric coupling of the metal and DNA-bindingsites in SmtB.

Figure 4. NMR evidence in support of a metal–ligand-mediated hydrogen-bonding network in Zn2 CzrA in solu-tion. (a) Structural representation of the NMR chemical shift perturbation map of CzrA, which reflects changesin 1H and 15N chemical shift induced upon zinc binding to the a5 metal sites in solution, whereDppm ¼

pðDd2

H þ ðDdN=7Þ2Þ:48 In each case, the ribbon is color-coded and ramped from white, through yellow togreen according to the magnitude of Dppm, with yellow set at the mean perturbation value for all amide groups(Dppm ¼ 0.127), and green reflecting large changes (Dppm ^ 0.35) in chemical shift of amide 1H and 15N resonances(see Materials and Methods). The worm diameter thickness also reflects these changes, going from thin to thick, indica-tive of small to large values in Dppm. (b) Complete side-chain assignment for His97 in Zn2 CzrA (signified by the blue bro-ken lines), revealing an unambiguous assignment of the N12–H12 correlation of His97 observed in 1H–15N HSQC spectrum(far left), obtained by linking the 2D [13C,1H]-TROSY-H(CDCG)CB44 13Cb–1Hd2 correlation (top) to the remainder of theHis97 side-chain via an 1H–15N HMBC (middle) and 3D 15N-separated NOESY spectra (15N ¼ 168.4 ppm) (bottom). A por-tion of the full 1H–15N HSQC spectrum is also shown (inset, top panel). (c) Chemical structure of His97, showing the scalarcorrelations indicated in (b). The red broken line links the 13Cb and 1Hd2 nuclei in the 2D [13C,1H]-TROSY-H(CDCG)CB spec-trum, the green broken lines link the non-exchangeable 1H and 15N nuclei identified in the 1H–15N HMBC spectrum, theorange broken line identifies the nuclei correlated in the 1H–15N HSQC spectrum, while the black broken linescorrespond to the cross-peaks observed in the 3D 15N-separated NOESY spectrum. (d) The same His97 N12–H12 correlationis not observed in 1H–15N HSQC spectrum of apo CzrA acquired under the same solution conditions (top panel) or apo-SmtB (bottom panel), but is present in Zn2 a5-SmtB (middle panel).

Figure 5. Zinc binding induces adramatic dampening of the internaldynamics of CzrA. Ribbon repre-sentation of the raw H–2Hexchange rates ðkexÞ of all resolvableamide protons in apo (top) versusZn2 (bottom) CzrA (pH 6.0, 0.1 MNaCl, 40 8C). Those amide groupsfor which kex ^ 10 h21 or #0.005 h21

were assigned the lower and upperlimits of 10 h21 or 0.005 h21 for illus-tration purposes. The ribbon iscolor-coded from red to blue, withred indicative of fast exchange(no protection from exchange withsolvent) and blue indicative ofslow exchange (see the scale at theright). The width of the ribbonreflects the absolute exchange rate,with fast exchange thin and slowexchange thick.


Figure 6. Structure of Zn1 SmtB. (a) Monomer superposition of the structures of Zn1 SmtB with apo-SmtB, depicted as a Ca trace; the position of the single metal ion isshown as a blue sphere. (b) Superposition of the empty (left) and filled (right) zinc sites in Zn1 SmtB (shaded green) compared to that of the two filled sites in Zn2 a5-SmtB.Note that the coordination bond lengths to His106 and His117 are significantly longer in the Zn1 SmtB structure (see the text). (c) Binding of Zn(II) to apo-CzrA as monitoredby chelator competition assays with mag-fura-228 (top) and quin-216 (bottom). Top panel: The continuous line is a simultaneous, non-linear, least-squares fit of the changes inabsorbance measured at 365 nm (W) and 325 nm (X) to an independent site metal-binding model, and where K ¼ 4:2ð^0:9Þ £ 109 M21: The broken curve is a simulation with

Since the crystallographic structures of apo andZn2 CzrA are more similar than different (theaverage N–O distance for the analogous His97N12–H12· · ·OvC His670 interaction in CzrAdecreases by just 0.1 A on zinc binding, from 2.9 Ato 2.8 A) (Figure 2(b), insight into changes in con-formation and dynamics induced upon Zn(II)binding to apo-CzrA in solution was sought. Ahigh-resolution NMR perturbation map, in whichchanges in backbone 1HN and 15N amide frequen-cies that occur upon Zn(II) binding to apo-CzrAare color-coded on the ribbon representation ofthe structure, with the thickness of the worm alsoscaling with increased perturbation (Figure 4(a)).Large perturbations (Dppm ^ 0.35 ppm) aregenerally localized to the immediate vicinity ofthe bound metal ion in the N and C terminiof both a5 helices, with a notable exception beingthe amide group of Leu68 in the aR-b1 loop,analogous to Leu88 in SmtB (vide supra). Formationof a hydrogen bonding network in CzrA struc-turally analogous to that determined for SmtB(Figure 3) makes the prediction that in solution,the His97 H12 proton of CzrA would exchangeslowly with solvent and therefore should bevisible in an 1H– 15N heteronuclear single quantumcoherence (HSQC) spectrum. This is indeed thecase under our standard NMR solution conditions(40 8C, pH 6.0) (Figure 4(b) and (c)), but only inthe Zn2 conformation (1H12 d ¼ 12.4 ppm; 15N12

d ¼ 168.4 ppm) (Figure 4(d)). The same is true ofthe corresponding His117 H12 proton in Zn2

a5-SmtB (1H12 d ¼ 12.2 ppm; 15N12 d ¼ 167.9 ppm)(Figure 4(d)).

In addition, if the amide group of Leu68 in theaR-b1 loop in CzrA donates a hydrogen bondpropagating this hydrogen-bonding network insolution, the main-chain amide hydrogen–deuter-ium (H– 2H) exchange rates of both Leu68 andLeu63 would be expected to be among the slowestin the CzrA, but again, only in the Zn2 form.Although the exchange rate of Leu63 in the Zn2

conformation could not be obtained due to spectraloverlap, the kex for Leu68 NH is reduced by 30-fold(0.77 h21 to 0.024 h21) on zinc binding. Strikingly,the H–2H exchange rates of Leu68 in the aR-b1loop, and nearly all of the buried hydrogen-bonding amide protons in the core of a-helices a1,a2, aR and a5, as well as those at the base of theb-hairpin wing, uniformly decrease substantially( ^ 20-fold) in the Zn2 versus apo forms of thehomodimer (Figure 5). A striking exception to this

is the b-hairpin wings, whose exchange ratesremain rapid in both conformations. These sametrends in internal dynamics in solution mirror thecrystallographic temperature factors for bothforms of CzrA, with mobilities within the core ofZn2 CzrA strongly dampened, again with theexception of the b-wings, which become evenmore highly mobile on Zn(II) binding (data notshown). Note that in the structure of Zn2 a5-SmtB(Figure 1(c)), the polypeptide chain of one of theb-hairpin wings within the homodimer could notbe traced, indicative of considerable disorder inthe crystal in this region.

Both a5 Zn(II) sites in SmtB must be filled todrive the quaternary structural switch

The crystallographic structure of Zn1 wild-typeSmtB, solved to 2.0 A resolution, where just one ofthe a5 zinc binding sites was occupied by a metalion, was obtained (Figure 6(a)). The empty site onthe homodimer is easily distinguished from thefilled site in this structure due to the distinct con-formations of the Glu120 “gating” residue (Figure6(b)). Strikingly, the quaternary structure of Zn1

SmtB is essentially superimposable on that of apo-SmtB, with the aforementioned hydrogen-bondingnetwork clearly not established on either side ofthe homodimer. Consistent with this, the inter-subunit distance between the aR Ser74 Ca atoms is51.3 A, very close to the 51.7 A found for apo-SmtB. Interestingly, the metal chelate appears onlypartially formed, with very long coordinationbonds to both His106 (2.55 A) and His1170 (2.57 A)in this complex (Figure 6(b)), relative to Zn2 SmtB(dZn-H106d1 ¼ 2.08 A; dZn-H1170Nd1 ¼ 2.05 A). Theseresults suggest that both a5 metal-binding sitesmust be loaded in order to drive a concerted qua-ternary structural conformational transition. Thisfinding makes the prediction that zinc binding tothe a5 metal sites of the CzrA or SmtB homodimermight be negatively cooperative (stepwise metalassociation equilibrium constants, Ki; whereK1 . K2), since some of the free energy of bindingto the second site ðDG2Þ must be used to drive thequaternary structural change in conformationand/or dynamics. This is what is found for apo-CzrA (Figure 6(c)). A quantitative analysis ofmetal-binding affinities using two fluorescent-chelator competition assays16,28 reveals that Zn(II)binding to apo-CzrA is extremely tight at equi-librium (25 8C, 0.1 M NaCl, pH 7.0), and strongly

K ¼ 5:1 £ 1012 M21 (see below), shown for comparison. [Mag-fura-2] ¼ 12.3 mM; [apo-CzrA] ¼ 28 mM monomer.Bottom panel: The continuous line represents a simultaneous, non-linear, least-squares fit of the changes in absorbancemeasured at 366 nm (W) and 265 nm (X) to a two-step, metal-binding, non-dissociable dimer model, where K1 and K2

are the step-wise zinc-binding affinities and K1 @ K2: Here, K1 ¼ 5:1ð^2:1Þ £ 1012 M21; and K2 # 109 M21: Other simu-lated curves are shown for comparison: continuous curve to right, K1 ¼ K2 ¼ 5:1ð^2:1Þ £ 1012 M21; red broken curve,K1 ¼ 5:1ð^2:1Þ £ 1012 M21; K2 ¼ 4:2 £ 109 M21 (K derived from the mag-fura-2 titration, top panel); black dot-dashcurve, K1 ¼ K2 ¼ 4:2 £ 109 M21: [Quin-2] ¼ 28 mM; [apo-CzrA] ¼ 17 mM dimer (34 mM monomer). The data revealthat the apo-CzrA homodimer binds two Zn(II) ions with strong negative cooperativity, since K1 .. K2 (see the textfor details).


negatively cooperative, with K1 ¼ 5:1 £ 1012 M21;with upper and lower limits obtainable only forK2 (109 M21 # K2 # 1010 M21 (Figure 6(c)).

Discussion

The X-ray crystallographic and NMR studiesreported here provide new molecular level insightinto the mechanism of negative allosteric regu-lation of operator/promoter binding by evolu-tionarily related zinc-sensing metalloregulatorytranscriptional repressors. Utilization of both Nd1

and N12 faces of a critical liganding histidineresidue (His97 in CzrA; His117 in SmtB) drives aquaternary structural switch in the global confor-mation and/or internal dynamics of the repressorthereby stabilizing a low DNA binding affinityconformation. In the case of SmtB, Zn(II) inducesa quaternary structural compaction of the homo-dimer that spatially alters the positions of the puta-tive DNA recognition (aR) helices relative to oneanother (Figure 1(c)). In the case of CzrA, Zn(II)binding simply functions to globally “freeze out”the internal dynamics of the dimer (Figure 5),such that the high-affinity operator/promoter-binding conformation is not energetically acces-sible to the repressor. In contrast, both apoproteinsmaintain a substantial degree of global quaternarystructural flexibility that may help maximize theaffinity of the repressor for various specific DNAbinding sites.

Utilization of both sides of a key imidazole metalligand ensures that formation of the a5 metal-sensing coordination chelate is coupled directly toformation of the hydrogen bonding network thatlinks the allosteric inducer site to the DNA-bindingaR helices some 15 A away. Characterizationof CzrA substitution mutants in which His97 isreplaced with either a non-liganding but hydrogenbonding glutamine residue (H97Q CzrA) versus apotentially metal liganding but poorly hydrogenbond donating thiolate group (H97C) will providea critical test of the proposed allosteric model.Consistent with our expectations, H97Q CzrAmaintains high-affinity, wild-type operator/promoter binding activity in vitro, but is com-pletely unresponsive to metal ions (M.A.P. andD.P.G., unpublished results). Efforts are underwayto determine how this coupling network has beenmodified to require six metal coordination bondsin the specific Ni-sensing site in M. tuberculosisNmtR,12 rather than four as in CzrA/SmtB, sincethe same set of four a5 metal ligands are proposedto be common to both CzrA and NmtR metalchelates.12,17 In addition, Cd and Pb binding toS. aureus CadC results in negative regulation ofDNA binding via the a3N site in that sensor,13

despite maintaining a high-affinity a5 Zn site.This requires either a key interruption in the flowof communication from the a5 metal-sensing sitesto the DNA-binding helices beyond the first shellof the metal ligands, and/or evolution of another

pathway(s) of allosteric coupling in these homo-logous SmtB/ArsR sensors.

Materials and Methods

Protein expression, purification and crystallization

Wild-type Synechococcus PCC7942 SmtB28 and S. aureusCzrA17 were overexpressed in E. coli BL21(DE3) andpurified to homogeneity as described. The expressionplasmid encoding the triple substitution mutant a5SmtB (C14S/C61S/C121S SmtB) was prepared usingPCR-based mutagenesis of the C14S/C61S overexpres-sion plasmid as template and C121S mutagenicprimers.16 The integrity of the nucleotide sequence ofboth strands of the plasmid was confirmed by DNAsequencing. a5 SmtB was purified to homogeneity usinga method essentially identical with that used for wild-type SmtB. All preparations of SmtB, a5 SmtB and CzrAwere devoid of bound zinc ((#0.05 mol equiv metal, asdetermined by atomic absorption spectroscopy) andthus were designated apoproteins; Zn2 complexes wereobtained upon the addition of 1.1 monomer-equivalentsof ZnSO4 to apoproteins.

Initial crystallization conditions were obtained usingthe hanging-drop method employing Crystal Screensfrom Hampton Research and Wizard Screens fromEmerald Biostructures. Crystallization conditions wereas follows: apo-SmtB, 1.6 M Na/K phosphate, 0.1 MHepes (pH 7.0); Zn2 a5 SmtB, 1.6 M Na/K phosphate,0.1 M Hepes (pH 7.5); apo-CzrA, 2.5 M NaCl, 0.1 M Na/K phosphate (pH 6.2); Zn2-CzrA, 10% PEG 8000, 0.1 MChes (pH 9.5), 0.2 M NaCl. Crystals of Zn1 SmtB wereobtained from stoichiometric mixtures of Zn(II) andapo-wild-type SmtB obtained under the same conditionsof apo-SmtB. Cell parameters, symmetry and monomersper asymmetric unit (ASU) of all crystals are listed inTable 1. All crystals were soaked in paraffin oil for afew seconds before flash-cooling to 100 K and subjectedto X-ray diffraction.

Data collection, structure determinationand refinement

Diffraction data were collected at beamline 14-BM-Cand D (MAD) at the Advanced Photon Source (APS),Argonne National Laboratory on a charge-coupled-device area detector (ADSC Q4). Apo-CzrA data werecollected with a rotating anode and a Rigaku IPDSIVþþ system. Data collection parameter and statisticsare listed in Table 1. Reflection data were processed andscaled using DENZO and SCALEPACK.29 Initial phaseswere determined by molecular replacement using thesearch models based on the previously published struc-ture of apo-SmtB.25 Molecular replacement solutionswere found using CNS30 or programs from the CCP4suite.31 Multi-wavelength anomalous dispersion (MAD)32

using the Zn-edge of the derivative was used to solvethe phase problem for Zn2 a5-SmtB (see Table 1). Theprogram SOLVE33 was used to locate the metal ion sitesin the asymmetric unit. Refinement was carried outagainst the peak data set. Initial phases of all formswere improved by solvent flattening as implemented inDM,34 RESOLVE35 and CNS.30 The molecular coordinateswere constructed using the computer program O36 andrefined with CNS30 or REFMAC.37 Shake and wARP38

was used to minimize model bias.


NMR spectroscopy

All NMR spectra were acquired on Varian Unity Inova500-MHz and 600-MHz spectrometers in the Biomolecu-lar NMR Laboratory at Texas A and M University.Typical solution conditions were 0.6–2 mM 15N-labeledor 15N/13C-labeled protein, pH 6.0, 10 mM d18-Hepes,100 mM NaCl and 10% (v/v) 2H2O, 40 8C. Samples in.99.9% (v/v) 2H2O were obtained by buffer exchangeover a G-25 column (Sigma). Chemical shift referencingis relative to 2,2-dimethyl-2-silapentane-5-sulfonic acid(DSS).39 All spectra were processed and analyzed usingNMRPipe40 and Sparky 3.† 1H–15N HSQC spectra41

were recorded as 128 £ 512 two-dimensional matrices inthe t1 and t2 dimensions, respectively. The three-dimen-sional 15N-separated nuclear Overhauser effect spec-troscopy (NOESY) spectrum42 ðtm ¼ 100 msÞ wasrecorded as a 12 £ 256 £ 741 three-dimensional matrixwith acquisition times of 49.4 ms (t1;

15N), 21.3 ms (t2;1H), and 61.8 ms (t3;

1HN), at a decoupler offsetpositioned at 168 ppm. The 2D 1H–15N heteronuclearmultiple bond correlation (HMBC)43 spectrum of Zn2

CzrA was acquired in 2H2O with the 15N spectral widthset to 70 ppm, centered at 195 ppm. The 2D [13C,1H]-transverse relaxation optimized spectroscopy (TROSY)-H(CDCG)CB experiment44 was acquired in 1H2O at40 8C with a 13C spectral width of 32.5 ppm centered onCb resonances. Complete resonance assignments of apoand Zn2 CzrA will be reported elsewhere.

Samples for hydrogen–deuterium (H– 2H) exchangewere prepared by exchanging protein samples into2H2O in 10 mM d18 Hepes (pH 6.0), 100 mM NaClusing a G-25 spin column equilibrated in the finalNMR buffer in 2H2O.45 The sample was placedimmediately into the NMR spectrometer and 15N– 1HHSQC spectra acquired continuously every 20–30minutes over 11 hours immediately followingexchange and then at various exchange times ðtexÞfor five to six weeks thereafter (apo-CzrA, tex ¼ 456hours and 575 hours; Zn2 CzrA, tex ¼ 35; 45, 100, 113and 912 hours). Cross-peak intensities of the 15N– 1HHSQC spectra for each non-overlapping peak of apoand Zn(II) CzrA were measured by volume inte-gration using Sparky 3. Peak intensities were then fitto a single-exponential function:

A ¼ A0½eð2ktÞ� þ C

where C is baseline noise of a particular sample.46,47

Protein Data Bank accession codes

The atomic coordinates for apo-SmtB, Zn2 a5-SmtB,apo-CzrA, Zn2 CzrA and Zn1 SmtB have been depos-ited in the Protein Data Bank, Research Collaboratoryfor Structural Bioinformatics, Rutgers University, NewBrunswick, NJ (http://www.rcsb.org/) underaccession codes 1R1T, 1R22, 1R1U, 1R1V and 1R23,respectively.

Acknowledgements

The authors thank the APS beamline operators

for assistance at beamline 14-BM-C and D. Theresearch was supported by grants from theNational Institutes of Health, the National ScienceFoundation, the Texas Higher Education Coordi-nating Board Advanced Technology Program, andthe Robert A. Welch Foundation. M.A.P. andM.L.V. were supported, in part, by an NIH Chem-istry-Biology Interface Training Grant (T32GM08523).

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Edited by M. F. Summers

(Received 12 August 2003; accepted 4 September 2003)

Note Added in Proof: During the review of this manuscript, the crystallographic structures of two metallo-regulatory MerR family proteins, Cu(I)-CueR and Zn(II)-ZntR, appeared (Changela, A., Chen, K., Xue, Y.,Holschen, J., Outten, C. E., O’Halloran, T. V., & Mondragon, A. (2003) Science 301, 1383–1387). The zinc-sensing sites of Zn-ZntR are structurally distinct from those of SmtB and CzrA characterized here.


A Metal–Ligand-mediated Intersubunit Allosteric Switch in Related SmtB/ArsR Zinc Sensor Proteins

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Transcript of A Metal–Ligand-mediated Intersubunit Allosteric Switch in Related SmtB/ArsR Zinc Sensor Proteins