doi:10.1016/j.jmb.2011.04.036 J. Mol. Biol. (2011) 409, 630–641

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Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

The X-ray Structure of the Zinc Transporter ZnuAfrom Salmonella enterica Discloses a UniqueTriad of Zinc-Coordinating Histidines

Andrea Ilari1†, Flaminia Alaleona1†, Patrizia Petrarca2,Andrea Battistoni2 and Emilia Chiancone1⁎1CNR Institute of Molecular Biology and Pathology and Department of Biochemical Sciences,“Sapienza” University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy2Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, 00133 Rome, Italy

Received 8 February 2011;received in revised form11 April 2011;accepted 12 April 2011Available online21 April 2011

Edited by R. Huber

Keywords:ZnuA;ZnuABC zinc transporter;Salmonella enterica;X-ray structure;periplasmic protein

*Corresponding author. E-mail addemilia.chiancone@uniroma1.it.† A.I. and F.A. contributed equallThis paper is dedicated to the mem

Tommaso Scarascia Mugnozza.Abbreviations used: PBP, periplasmprotein; EcZnuA, E. coli ZnuA; SynZZnuA; SeZnuA, S. enterica ZnuA; PDRCSB, Research Collaboratory for SBioinformatics.

0022-2836/$ - see front matter © 2011 E

ZnuA is the soluble component of the high-affinity ZnuABC zinctransporter belonging to the cluster 9 group of ATP-binding cassette-typeperiplasmic Zn- and Mn-binding proteins. In Gram-negative bacteria, theZnuABC system is essential for zinc uptake and homeostasis and is animportant determinant of bacterial resistance to the host defensemechanisms. The cluster 9 members share a two (α/β)4 domainarchitecture with a long α-helix connecting the two domains. In the Zn-specific proteins, the so-called α3c and the α4 helices are separated by aninsert of variable length, rich in histidine and negatively charged residues.This distinctive His-rich loop is proposed to play a role in the managementof zinc also due to its location at the entrance of the metal binding sitelocated at the domain interface. The known Synechocystis 6803 andEscherichia coli ZnuA structures show the same metal coordinationinvolving three conserved histidines and a glutamic acid or a watermolecule as fourth ligand. The structures of Salmonella enterica ZnuA, with apartially or fully occupied zinc binding site, and of a deletion mutantmissing a large part of the His-rich loop revealed unexpected differences inthe metal-coordinating ligands, as histidine 140 from the mobile (at theC-terminal) part of the loop substitutes the conserved histidine 60. Thisunforeseen coordination is rendered possible by the “open conformation”of the two domains. The possible structural determinants of thesepeculiarities and their functional relevance are discussed.

© 2011 Elsevier Ltd. All rights reserved.

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lsevier Ltd. All rights reserve

Introduction

Zinc, like other transition metals, notably iron andcopper, is required by all living organisms to ensurecell growth but is potentially toxic to a number ofcell functions. Therefore, zinc homeostasis requiresto be controlled strictly by the concerted action ofspecific proteins.In bacteria, zinc is acquired from the growth

medium by different metal transporters so as toreach total zinc concentrations in the submillimolarrange, while the “free” metal is buffered at

d.

631The X-ray Structure of the Salmonella Zinc Transporter ZnuA

femtomolar to nanomolar levels by means oftranscriptionally controlled zinc uptake and effluxsystems.1–3 The relevant Zn-trafficking proteins arecharacterized by tight binding of the metal ion, toprevent its release and adventitious reactions, and bya coordination environment that allows the easytransfer of the metal to the next partner protein.4 Inthis respect, the ZnuABC uptake system, whichcomes into play when the zinc concentration in themedium is low and is used by Gram-negativebacteria to transport zinc from the periplasmicspace to the cytosol, is paradigmatic. It is a high-affinity ATP-binding cassette-type transporter and,like all such systems, is composed of three proteins: asoluble periplasmic component (ZnuA) that capturesZn(II) and delivers it to the membrane permease(ZnuB), whereas the ATPase component (ZnuC)provides the energy necessary for ion transportthrough the inner membrane.5,6 ZnuA is the mostextensively studied component also in view of theobservation that inactivation of the znuA-encodinggenes in several pathogenic bacteria, includingSalmonella enterica, results in decreased growth ratesin environments poor of zinc and in significantlyreduced virulence in different animal models.7

Phylogenetic analyses placed ZnuA in a group ofperiplasmic solute-binding proteins (PBPs) namedcluster 9, which includes also manganese-specificPBPs. The available structural information showsthat Zn- and Mn-specific PBPs share a two-(α/β)4-domain architecture with a long α-helix connectingthe two domains. These consist of a parallel four-stranded β-sheet with strand sequence 2-3-1-4. Thecentral β-sheet is lined by four α-helices, two oneach side, that alternate with the β-strands in theprimary sequence (Figs. 1 and 2). The metal ion isbound at the interface between the two domains andis coordinated tetrahedrally. Mn-specific PBPs coor-dinate the metal via two histidines and two acidicresidues (MntC and PsaA)10,11 or three histidinesand a glutamic acid (TroA).12 In contrast, in Zn-specific PBPs, the metal is coordinated by threeconserved histidines, while a fourth ligand isprovided by a glutamic acid, close to a histidineligand, or by a water molecule.13–17 It has beensuggested that differences in coordination geometrycontribute to metal specificity.15

The ZnuA sequences display a further, mostremarkable difference with respect to those of Mn-specific transporters, that is, only the Zn-specificproteins are characterized by a flexible insert rich inhistidine and negatively charged residues (His-richloop) located between the α3c and α4 helices (ZnuAtopology). This exclusive presence and the location ofthe His-rich loop at the entrance of the metal bindingsite apparent from the known Escherichia coli13,14,16

and Synechocystis 680315 ZnuA structures led to theproposal that it may serve to facilitate zinc bindingwithin the periplasmic space. Recent studies carried

out on S. enterica support this hypothesis as theypoint to amajor role of theHis-rich loop in facilitatingZn(II) recruitment under conditions of metalstarvation.17 Alternatively, the His-rich loop mayserve to transfer the metal to ZnuB13–15 or to regulatethemetal import process by acting as sensor of the Zn(II) level.17 Determination of the structure and zincbinding properties of a Synechocystis 6803 ZnuAHis-rich loop deletionmutant14 did not provide a definiteanswer to this question but suggested that the loophas long-range structural effects that can influencethe binding of zinc. Thus, deletion of the His-richloop alters the relative position of the N- and C-terminal domains and leads to a 10-fold decreasedaffinity of the canonical Zn(II) binding site. It shouldalso be mentioned that a second Zn(II) binding sitewas identified in one of the available E. coli ZnuA(EcZnuA) structures. In this secondary site, Zn(II) isbound on the external surface of the protein by twohistidine residues, one belonging to the His-richloop.16 Calorimetric studies carried out on Synecho-cystis ZnuA (SynZnuA) likewise pointed to thepresence of two classes of Zn(II) binding sites.18

The data just reviewed in turn imply that thenatural variability in length that characterizes theHis-rich loop in the various bacterial species may impacton themanagement of Zn by the protein. To establishthe validity of this contention, we determined thestructure of S. enterica ZnuA (SeZnuA). SeZnuA ischaracterized by a His-rich loop of intermediatelength relative to SynZnuA and EcZnuA. Moreover,the sequences ofSeZnuAandEcZnuAdisplay only 17differences apart from those in the His-rich loopregion over a total of 310–314 amino acids. Impor-tantly, the residues in the immediate vicinity of thethree canonical Zn-ligating histidines (His60, His143and His207 in EcZnuA) are conserved (Fig. 1).The high-resolution structures of SeZnuA with the

Zn(II) binding site, either partially or fully occupied,and of a deletion mutant lacking a large part of theloop (SeZnuA Δ118–141), where the site is empty,unveiledZn(II)-induced conformational changes thatare likely of functional relevance in metal sequestra-tion from the periplasm and/or its delivery to ZnuB.Importantly, they disclosed for the first time a uniquetriad of Zn(II)-binding histidines that comprisesHis140, a residue placed on the C-terminal part ofthe His-rich loop that substitutes His60 (EcZnuAnumbering), one of the otherwise conserved metal-binding histidines in Zn- and Mn-specific PBPs.

Results and Discussion

SeZnuA: Overall fold

The crystals of SeZnuA as purified (SeZnuA) andof SeZnuA crystals soaked with zinc acetate [Zn(II)–

Fig. 1. Sequence alignment of SeZnuA, SynZnuA and EcZnuA. The sequence alignment was performed with theprogram CLUSTALVIEW-MULTIALIGN (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=clustalw-multialign). Thesecondary structure elements are indicated. The His-rich loop is colored red. The residues coordinating Zn(II) areunderlined. Identical residues between EcZnuA and SeZnuA are colored lime green, while asterisks indicate identicalresidues in the three sequences. “.” and “:”, respectively, indicate “similar” and “more similar” residues as defined by theCLUSTALVIEW program.8

632 The X-ray Structure of the Salmonella Zinc Transporter ZnuA

SeZnuA] are both hexagonal (P63) and contain onlyone molecule in the asymmetric unit. The structureof SeZnuA is characterized by a partially occupied

zinc binding site (occupancy=0.7) and has beensolved at 1.7 Å resolution, while the Zn(II)–SeZnuAstructure, having a fully occupied Zn(II) binding

Fig. 2. Comparison between the structures of SeZnuA, Zn–SeZnuA and the Δ118–141 SeZnuA deletion mutant. Thestructures of SeZnuA (light magenta), Zn–SeZnuA (yellow) andΔ118–141 SeZnuA (green) have been superimposed usingthe program Coot. The different conformations assumed by the 57–61 region are shown in the blow-up. The picture hasbeen generated using PyMOL.9

633The X-ray Structure of the Salmonella Zinc Transporter ZnuA

site, has been solved at 1.9 Å resolution. The twoforms share the same overall fold, except for theshort 57–61 region that assumes a different confor-mation in the two structures as will be describedbelow in connection with the Zn(II) binding site (Fig.2). Accordingly, the superposition of the Cα traces ofSeZnuA and Zn(II)–SeZnuA yields a root-mean-square deviation (rmsd) of 0.356 Å.SeZnuA, like all Zn- and Mn-specific PBPs and

irrespective of the occupancy of the active site withzinc, is constructed by a pair of (α/β)4 sandwichdomains (comprising residues 27–173 and residues204–313, respectively), which are connected by along α-helix (residues 174–203) that is tightly packedagainst them (Figs. 1 and 2). As in the other ATP-binding cassette-type PBPs of the cluster 9 family,the two domains are related by pseudo-2-foldsymmetry. Moreover, residues 118–139, belongingto the long His-rich loop between the secondarystructural elements β4 and α4, are not visible in thestructure, an indication of a high degree of flexibil-

ity. In contrast, the short His-rich loop segments114–117 and 140–147 are clearly visible. Moreover,quite unexpectedly on the basis of sequencealignment and homology, His140 coordinates zinc(see below).The structure of SeZnuA has been compared with

the known ZnuA structures, namely, those ofEcZnuA and SynZnuA. The sequence identity ofSeZnuA with EcZnuA is very high (85%), as alludedto above. That with SynZnuA amounts to 27% (Fig.1). At variance with the structural alignmentpresented by Li and Jogl14 that assigns to EcZnuA17 secondary structure elements (namely, 9 α-helicesand 8 β-strands), the comparative analysis pre-sented in Fig. 1 points to the existence of threefurther helices: the short α1b helix is observed onlyin SeZnuA, whereas helices α3b and α3c are presentin SeZnuA and in the EcZnuA structures with a fullyoccupied Zn(II) binding site (2OSV and 2OGW). Inprevious comparative structural analyses on EcZ-nuA and SynZnuA, the entire β6–α7–β7–α8 region

Fig. 3 (legend on next page)

634 The X-ray Structure of the Salmonella Zinc Transporter ZnuA

635The X-ray Structure of the Salmonella Zinc Transporter ZnuA

was shown to adopt two different conformations. Itwas established that the β6–α7 loop has an “open”conformation and is packed against the α8 helix inapo-EcZnuA and in EcZnuA bound to one Co(II)[Protein Data Bank (PDB) codes 2PSE and 2PS9,respectively] (Fig. 3a). In contrast, this loop is moreshifted toward solvent and assumes a “closed”conformation in SynZnuA (1PQ4), in EcZnuAwith afully occupied Zn(II) binding site (2OSV) and inEcZnuA with an additional Zn(II) bound on theexternal surface by His228 and other unidentifiedresidues from the His-rich loop (2PRS).The superposition of the Cα traces (232 residues

out of 255) of SeZnuA and SynZnuA (PDB code1PQ4) yields an rmsd of 2.39 Å, as expected basedon the lower degree of sequence similarity betweenthe two proteins. Several structural differences areapparent. A completely different conformation isdisplayed by the long regions 80–117 and 228–273(SeZnuA numbering), the latter corresponding tothe β6–α7–β7–α8 subdomain. In SeZnuA, the 80–117 region is more structured due to the presence ofthe additional α3c helix. Moreover, as shown in Fig.3b, the α7 helix moves toward solvent and dragsthe β6–α7 loop (residues 228–238), which packsagainst the α8 helix and adopts the so-called“open” conformation. Significantly, this conforma-tion renders the zinc binding site accessible to theHis-rich loop (114–147) and allows Zn(II) bindingby His140, the first residue on the C-terminal partof the loop with a clearly defined electron density(Fig. 3a). In SynZnuA, the α7 helix is rotated by∼20° relative to the position in SeZnuA such thatthe β6–α7 loop assumes a “closed” conformation,as mentioned above.In the comparison between the SeZnuA and

EcZnuA structures, several superpositions of the Cα

traces have been performed, involving EcZnuA in theapo-form (2PS3), EcZnuAwith a fully occupied Zn(II)binding site (2OSVand2OGW) andEcZnuAwith twobound zinc ions (2PRS). The respective rmsd values of0.654 Å, 0.490 Å and 0.462 Å indicate that the highdegree of sequence identity carries over to structure.Despite these relative low rmsdvalues between theCα

traces, four structural segments assume significantlydifferent conformations in the two macromolecules,namely, the β6–α7 loop and the α2, α7 and α8 helices(Fig. 3c). In SeZnuA, the β6–α7 loop (228–238) ispacked against the C-terminal part of the α8 helix and

Fig. 3. Comparison between SeZnuA and the EcZnuA structthe β6–α7–β7–α8 region. The structures of SeZnuA andrespectively. EcZnuA with two bound Zn(II) (2PRS) is colorcolored deep-teal cyan; Co(II)-bound EcZnuA (2PS9) andrespectively. (b) Comparison between the SeZnuA (light magethe last residue of the His-rich loop, Gly117 and His139, and thComparison between the SeZnuA (light magenta) and the EcZThe first residue and the last residue of the His-rich loop, Gly228–238 are labeled. The picture has been generated using Py

adopts an “open” conformation, at variance withEcZnuA where the α8 helix is rotated by ∼20°. TheSeZnuA α7 helix is rotated toward solvent withrespect to the position adopted in all zinc boundEcZnuA structures, and the α2 helix is likewisecharacterized by a rotation toward solvent withrespect to the other zinc bound ZnuA structures.13–18

The detailed comparison of the SeZnuA andEcZnuA structures carried out thereafter revealedfurther distinctive differences in the 57–61 regionand in the C-terminal part of the His-rich loop thatcomprise two residues that coordinate Zn(II) at theprimary binding site. Differences in these regions,therefore, are apparent also when the structures ofSeZnuA with a partially or a fully occupied Zn(II)binding site are compared (see below). An interest-ing distinction is related to the substitution ofGlu143 in SeZnuA by Asp in EcZnuA, sinceGlu143 gives rise to a salt bridge with Lys116(NZ–OE1 distance=2.7 Å). This additional interac-tion between two residues placed respectively at theC- and N-terminal ends of the His-rich loop anchorsthe loop on the protein scaffold and therefore islikely to influence the protein metal affinity (Fig. 4).The other changes in the sequences of SeZnuA and

EcZnuA, apart from those in the His-rich loopregion, were mapped on the structures. The corre-sponding residues appear to be locatedmostly on thesurface of the N-terminal region of the protein(Fig. 4). It is not known whether they are offunctional importance, for example, are involved inthe interaction of ZnuA with its molecular partners.

SeZnuA: The Zn(II) binding site

The primary Zn(II) binding site was identified bya strong peak in an anomalous Fourier map. Itsposition is shifted by about 1.0 Å from the metalbinding site in EcZnuA and by about 1.5 Å from thatin SynZnuA (Fig. 5).In Zn(II)–SeZnuA, where the occupancy of this

binding site is 1.0, Zn(II) is coordinated tetrahe-drally by the canonical, highly conserved His147and His211 residues [NE2–Zn(II) distances of 2.2 Åand 2.0 Å, respectively], by Glu59 [OE1–Zn(II)distance of 1.7 Å] and by His140 [NE2–Zn(II)distance of 2.1 Å] placed on the C-terminal part ofthe long His-rich loop (Fig. 6a). Participation ofHis140 in metal ligation was fully unexpected, as in

ures. (a) Close-up view of the conformational differences inZn(II)–SeZnuA are colored light magenta and yellow,ed slate blue; EcZnuA with one bound Zn(II) (2OSV) isapo EcZnuA (2PS3) are colored magenta and orange,nta) and SynZnuA (gray) structures. The first residue ande β6–α7 loop comprising residues 228–238 are labeled. (c)nuA (cyan) structures. Zn(II) is indicated by a gray sphere.117 and His139, and the β6–α7 loop comprising residuesMOL.9

Fig. 4. Differences between the Zn(II)–SeZnuA and Zn(II)–EcZnuA structures. The two structures are superimposedusing Coot.19 Zn(II)–SeZnuA is colored yellow, Zn(II)–EcZnuA is colored cyan, the residues differing in the two structuresare indicated in red. The visible part of the His-rich loop in the two structures is shown in the blow-ups. The distinctivesalt bridge between Lys116 and Glu143 in Zn(II)–EcZnuA is indicated. The picture has been generated using PyMOL.9

636 The X-ray Structure of the Salmonella Zinc Transporter ZnuA

all the other known ZnuA X-ray structures, the Zn(II)-coordinating ligands belong to the proteinscaffold (see Table 1).TheZn(II) ligandGlu59 is located in the short 57–61

region that adopts a different conformation depend-ing on the occupancy of the Zn(II) binding site withmetal. Thus, in SeZnuA, where the metal occupancyis 0.7, the map of the 57–61 region lacks detail, anindication of high mobility in accordance with the B-factor that is higher than the overall one (45 Å2 versus27 Å2). After assigning an occupancy of 0.7 to thewhole 57–61 region, a better map was obtained. Theside chains of Ser58 and Asp61 appear to be rotatedby ∼180° and that of His60 by ∼90° around the Cβ

atom with respect to the position adopted by thesame residues in Zn(II)–SeZnuA (Fig. 2).The conformational differences in the 57–61

segment, which comprises the Zn(II) ligand Glu59,affect coordination of the metal ion as follows. InSeZnuA [where the Zn(II) occupancy is lower than1.0], the metal is not tetra-coordinated as in Zn(II)–SeZnuA, but is penta-coordinated, with a watermolecule as fifth ligand [HOH–Zn(II) = 2.1 Å]

(Fig. 6b). Binding of the water molecule by Zn(II)is favored by a slight shift of Glu59 with respect tothe position observed in Zn(II)–SeZnuA. Accord-ingly, Glu59 coordinates Zn(II) with OE2 [OE2–Zn(II)=2.1 Å] (Fig. 6c). The slight displacements of theother Zn(II)-binding residues, His140, His147 andHis211, do not affect their metal coordination.The data just presented call for a detailed

comparison of the Zn(II) binding site in all knownZnuA structures as they point to an unforeseenvariability inmetal ligation. The typical coordinationof Zn(II) in structural zinc binding sites istetrahedral,20 but can be four or five in enzymeswhere it is frequently distorted—tetrahedral ortrigonal—bipyramidal in the absence of the enzymesubstrate.20,21 In all members of the ZnuA familycrystallized to date, the metal is coordinated with atetrahedral geometry by the ligands detailed in Table1. The first two coordination positions are occupiedby the conserved His147 and His211 residues(SeZnuA numbering; Fig. 5). The third coordinationposition is occupied by Glu59 in SeZnuA and by awater molecule in SynZnuA since Glu59 is

Fig. 5. Stereo view of the zinc binding sites of Zn(II)–SeZnuA, SynZnuA and Zn(II)–EcZnuA. Zn(II)–SeZnuA (inyellow), SynZnuA (PDB accession code 1PQ4; in gray) and EcZnuA (PDB accession code 2OSV; in cyan). Zn(II) isindicated by a sphere of the same color as the structure; the red sphere indicates a water molecule. The structures weresuperimposed using the program Coot.19 The picture has been generated using PyMOL.9

637The X-ray Structure of the Salmonella Zinc Transporter ZnuA

substituted by a proline residue. Interestingly, inEcZnuA, this coordination position can be occupiedbyGlu5914,16 or a bywatermolecule.14 It follows thatalso in theE. coli structures,13,14,16 the short structuralmotif 57–61 may adopt different conformations. Thefourth coordinating ligand is His60 in all previouslysolved ZnuA structures. This highly conservedresiduemost likely coordinates Zn(II) in all membersof the family. In SeZnuA, His60 is brought at toolarge a distance to coordinate the metal by theconformation of the short 57–61 region (Fig. 1), andthe fourth coordination position is occupied byHis140, the first residue in the C-terminal part ofthe His-rich loop (114–147) whose electron density isclearly defined (Fig. 6a and b). The electron densitymap of the preceding residue in the sequence,His139, is difficult to interpret and does not allowthe assignment of a unique conformation. Theelectronic density map of the other residues in theloop, 118–138, is not visible as in the other ZnuAstructures deposited in the PDB, indicating a highdegree of flexibility.

Structure of the His-rich loop deletion mutantSeZnuA Δ118–141

The unexpected and unprecedented coordinationof Zn(II) by His140 belonging to the His-rich looplends support to the contention that this loopparticipates directly in the management of themetal.18,22 To shed light on this specific point, wesolved the structure of the SeZnuAΔ118–141 deletionmutant (SeZnuA Δ118–141) at 2.0 Å resolution.The superposition of the SeZnuA and SeZnuA

Δ118–141 Cα traces yields an rmsd of 0.273 Å,

indicating that the remaining part of the proteinscaffold is not affected by the deletion in accordancewith its rigidity, except for the 57–61 region.However, deletion of the His-rich loop bringsabout remarkable local structural changes. Theresidues of the loop that have not been deleted(114–117 and 142–147) form an additional two-strand antiparallel β-sheet. The primary binding sitedoes not contain Zn(II). However, inspection of theFo−Fc electron density map contoured at 4 σ revealsthe presence of a peak that was assigned to a sodiumion on this basis. This sodium ion is displaced by∼2 Å with respect to the metal in the SeZnuAstructure (Fig. 6c) and is hydrogen bonded to His188(His211; SeZnuA numbering) [NE2–Na(I)=3.5 Å],while His124 (His147; SeZnuA numbering) ishydrogen bonded to Glu59 [NE2–OE1=2.9 Å].Moreover, the conformation of the 57–61 regiondiffers relative to that adopted in SeZnuA andresembles that of the Zn(II)-soaked structure (Fig. 2).Therefore, removal of the long His-rich loop andbinding of Zn(II) have the same stabilizing effect onthe 57–61 region as shown clearly also by the valuesof the mean B-factor, which become similar to theoverall ones in the various structures analyzed.Thus, in SeZnuA, the overall B-factor is 27 Å2, whilethat of the 57–61 region is 45 Å2; in the SeZnuAΔ118–141 deletion mutant and in Zn(II)–SeZnuA,the overall B values are 33.8 Å2 and 30 Å2,respectively, while those of the 57–61 region are35 Å2 and 34 Å2.A different situation seemingly applies to SynZ-

nuA, where deletion of the 138–171 His-rich loopleads to structural differences in the β6–α7–β7–α8region that have been ascribed to the different

Fig. 6. Zinc binding site in the X-ray structures from SeZnuA. (a) Fully occupied zinc binding site in Zn(II)–SeZnuA.The residues binding Zn(II) are indicated as sticks. The 2Fo−Fc electron density map (contoured at 1 σ) for residues in theactive-site region is shown in blue. The anomalous difference Fourier map for data collected at the zinc peakwavelength isshown at a 5-σ contour level in green. (b) Partially occupied zinc binding site in SeZnuA. The Zn(II)-binding residues areindicated as sticks. 2Fo−Fc electron density map (contoured at 1 σ) for residues in the active-site region is shown in blue.The anomalous difference Fourier map for data collected at the zinc peak wavelength is shown at a 4-σ contour level ingreen. (c) Comparison among SeZnuA (light magenta), Zn–SeZnuA (yellow) and the Δ118–141 SeZnuA deletion mutant(green) zinc binding site. The pictures have been generated using PyMOL.9

638 The X-ray Structure of the Salmonella Zinc Transporter ZnuA

crystal packing. Moreover, the affinity constant forZn(II) decreases only by 1 order of magnitude.18

When the structures of the SeZnuA and SynZnuAdeletion mutants are compared by superimpositionof their Cα traces, rmsd values of 2.155 Å and2.055 Å, respectively, are obtained for SynZnuAΔ138–171 in the apo (2OV1) and Zn(II)-bound

Table 1. Zn(II)-coordinating amino acids in the solved ZnuA

Zn(II) ligandsGlu59

(OE1) (Å)Glu59

(OE2) (Å)His60

(NE2) (Å)

Zn(II)–SeZnuA 1.7SeZnuA 2.0 2.1EcZnuA 1.9 2.0SynZnuA 1.9

The SeZnuA numbering is used.

(2OV3) forms. The major difference concerns theentire β6–α7–β7–α8 region.Lastly, it should be mentioned that the attempts

to obtain X-ray quality crystals of Zn(II)-boundSeZnuA Δ118–141 by co-crystallization or in soak-ing experiments on preformed crystals had nosuccess.

X-ray crystal structures

His140(NE2) (Å)

His147(NE2) (Å)

His211(NE2) (Å)

H2O(Å)

2.1 2.2 2.02.1 2.1 2.1 2.1

2.1 2.02.1 2.1 2.3

639The X-ray Structure of the Salmonella Zinc Transporter ZnuA

Conclusions

The structures of SeZnuA with the primary zincbinding site, either partially occupied (SeZnuA) orfully occupied [Zn(II)–SeZnuA], and of a deletionmutant missing a large part of the His-rich loop(SeZnuA Δ118–141) unexpectedly revealed that themetal is coordinated by a unique set of histidines thatcan ligate Zn(II) with a distinctive geometry relativeto the known ZnuA structures.14–16,18 In fact, one ofthe canonical Zn(II)-binding residues, His60, thoughconserved in allmembers of the ZnuA family, is not ametal ligand in the abovementioned structures offull-length SeZnuA. However, its participation inmetal coordination during specific phases of theZn(II) binding/transfer processes cannot beexcluded as suggested by structural and dynamicalinvestigations18 and by the different conformationsthat the 57–61 region can adopt. The place of His60at the fourth Zn(II) coordination position is takenby His140, located on the C-terminal part of theHis-rich loop, while coordination at the otherpositions involves the conserved His147 andHis211 residues and Glu59 as in EcZnuA.14,16

The participation of His140 in the binding of Zn(II), on the one hand, demonstrates that the His-richloop can have a direct role in the management ofzinc [e.g., may contribute to the intramoleculartransfer of Zn(II) from a secondary to the primarymetal binding site or to metal transfer from ZnuA toZnuB] and, on the other hand, calls for the presenceof specific structural features, in the framework ofhighly similar overall structures such as those ofSeZnuA and EcZnuA, that bring His140 at therequired Zn(II)-coordinating distance. The uniqueZn(II) coordination displayed by SeZnuA appears tobe linked to the “open conformation” adopted bythe β6–α7–β7–α8 region, which opens the Zn(II)-binding cavity between the two (α/β)4 subdomains,thus allowing the metal to reach His140. A structuralrestraint on the position of this residue and of theHis-rich loop can be provided by the salt bridgeestablished between Lys116 and Glu143 that an-chors this loop to the protein scaffold (Fig. 4) and hasno counterpart in EcZnuA and SynZnuA. However,the alignment of 100 sequences selected usingBLAST and SeZnuA as query yields 30 sequencescontaining Lys and Glu in the positions correspond-ing to Lys116 and Glu143 in SeZnuA. It is temptingto predict that such transporters will adopt thealternative SeZnuA Zn binding mode rather thanthe “classical” one.The other feature to be highlighted in comparison

with the previously obtained ZnuA structuresconcerns the conformational changes induced byZn(II) binding. These appear to be local andrestricted to the vicinity of the metal binding site.A significant change, for example, involves the shortmobile 57–61 region that can assume different

conformations based on the comparison betweenthe SeZnuA and Zn(II)–SeZnuA structures (respec-tive metal occupancy of 0.7 and 1.0). In turn, theconformational changes linked to Zn(II) bindingaffect the coordination geometry. Thus, when the Zn(II) binding site is partially occupied, a change incoordination geometry takes place. The metalbecomes penta-coordinated, since Glu59 ligates Zn(II) with OE2 instead of OE1 and shifts in position,allowing coordination of the metal by a watermolecule (Fig. 6b and c).The rigidity of the ZnuA scaffold is evidenced also

by the structure of the SeZnuA Δ118–141 mutant.Thus, deletion of the His-rich loop does not causelarge conformational changes even though it affectsthe affinity for the metal dramatically. No Zn(II) isbound in the SeZnuA Δ118–141 active site, anindication that the missing His140 is of primaryimportance in determining the affinity for Zn(II). Inthis connection, it may be recalled that, in SynZnuA,the histidine-rich loop is believed to sequester Zn(II)from the medium and deliver it to the metal bindingsite.18 However, based on studies designed to unveilthe mechanisms of ZnuABC-mediated import inSalmonella,17 the role played by the His-rich loopappears to be more complex. Thus, the growth of aznuA mutant strain producing SeZnuA Δ118–141 inplace of the wild-type protein is not impaired in aLuria–Bertani medium containing ethylenediamine-tetraacetic acid, indicating that ZnuA can mediatezinc transport also in the absence of the His-richregion. However, a severe growth defect wasobserved in a mutant strain expressing SeZnuAΔ118–141 and unable to produce ZinT, anotherperiplasmic protein assisting ZnuA in the recruitmentof zinc within the periplasm, a sign that ZinT andZnuA cooperate in the process of zinc recruitment.

Materials and Methods

Protein crystallization, data collection anddata reduction

The purified proteins were prepared, according topublished procedures,17 in 20 mM Hepes, 10 mM NaCl atpH 7.0. All the crystallization experiments were carriedout at 298 K by the hanging-drop vapor diffusionmethod. The SeZnuA crystallization trials were per-formed using a protein sample concentrated to about25 mg/ml. Aliquots (1 μl) of the protein sample weremixed with an equal amount of reservoir solutioncontaining 1–2% (w/v) polyethylene glycol 400,2.3–2.5 M ammonium sulfate and 0.1 M Hepes atpH 7.5 and were allowed to equilibrate with a 500-μlvolume of reservoir solution. Crystals grew in 2 weeksand reached dimensions of 0.05 mm×0.05 mm×0.3 mm.Crystals of Zn(II)–SeZnuA were obtained by soaking

the crystals with 2 mM zinc acetate for 1 h. SeZnuAΔ118–141 crystals were obtained by mixing 1 μl of protein

Table 2. Crystal parameters, data collection statistics andrefinement statistics of SeZnuA in the free form and incomplex with Zn(II) and of the ZnuA Δ118–141 deletionmutant

SeZnuAwild type

SeZnuA+Zn

SeZnuAΔ118–141

Space group P63 P63 P63Unit cell parameters (Å)a 143.65 143.65 143.65b 143.65 143.65 143.65c 30.81 31.80 30.81No. of molecules in the

asymmetric unit1 1 1

⟨Bñ for atomic model (Å2) 27.6 30.0 33.8Resolution ranges (Å) 1.70–50.0

(1.70–1.76)1.93–50.0(1.93–2.0)

2.07–50.0(2.07–2.14)

Total observations 464,748 124,194 122,045Unique reflections 40,175

(3940)26,743(2626)

22,468(2204)

Completeness (%) 100 (99.7) 99.0 (99.9) 99.0 (100)Redundancy 11.6 (11.4) 4.6 (4.8) 5.4 (5.5)Rmerge

a 7.2 (40.5) 8 (43) 13 (43)χ2b 1.119 1.195 1.448

640 The X-ray Structure of the Salmonella Zinc Transporter ZnuA

solution, concentrated to about 15 mg/ml, with an equalamount of the same reservoir solution used for SeZnuA.For data collection, SeZnuA, Zn–SeZnuA and SeZnuA

Δ118–141 crystals were cryo-protected in a solutioncontaining 80% (v/v) of mother liquor and 20% (v/v)glycerol; the crystals soaked with zinc were transferred inthe same cryo-solution with the addition of 2 mM zincacetate.The crystals were mounted in nylon loops and flash

frozen by quick submersion into liquid nitrogen fortransport to the synchrotron-radiation source.Three single-wavelength data sets (λ=0.918 Å) were

collected from crystals of SeZnuA, Zn–SeZnuA andSeZnuA Δ118–141 at the BL-14.1 beamline of theSynchrotron Radiation Source BESSY (Berlin, Germany),using a CCD detector at a temperature of 100 K. The datasets were processed with Denzo23 and scaled withScalepack.23 The autoindexing procedure indicates thatthe crystals are hexagonal. All the measured crystalsbelong to the P63 space group. Crystal parameters anddata collection statistics for the measured crystals arelisted in Table 2.

(0.683) (0.922) (0.788)⟨I/σ(I)⟩ 32.19

(5.30)18.42(3.49)

18.00(4.00)

Rcrys (%) 21.36 20.56 19.5Rfree (%) 24.55 22.78 22.51rms (angles) (°) 1.172 1.540 1.596rms (bonds) (Å) 0.0086 0.0161 0.0177Residues in core region of

Ramachandran plot (%)259 (98.5) 258 (98.9) 261 (99.6)

Residues in generouslyallowed region ofRamachandran plot (%)

3 (1.1) 3 (1.1) 1 (0.4)

Residues in disallowedregion of Ramachandranplot (%)

1 (0.4) 0 (0.0) 0 (0.0)

Values in parentheses are for the highest-resolution shell.a Rmerge=∑hkl∑i|Ii(hkl)− ⟨I(hkl)⟩|/∑hkl∑iIi(hkl), where Ii(hkl) is

the ith observation of the reflection (hkl) and ⟨I(hkl)⟩ is the meanintensity of the (hkl) reflection.

b χ2=∑ii(|Iij(hkl)− ⟨Ii(hkl)⟩|)2/(σi2N/(N−1)).

Structure solution and refinement

The three structures (SeZnuA, Zn–SeZnuA and SeZnuAΔ118–141) were determined by molecular replacementusing EcZnuA (PDB entry 2PRS) as search model, whichdisplays 86.94% sequence identity with SeZnuA (calculat-ed using CLUSTALVIEW). The rotational and transla-tional searches performed with the program MOLREP24

(CCP4 suite) in the resolution range 10.0–3.0 Å producedclear solutions, corresponding to a monomer in theasymmetric unit. Refinements were performed using themaximum-likelihood method with the programREFMAC25 and model building with the programCoot19 (see Table 2). The quality of the models wasassessed using the program PROCHECK;26 all theresidues of the three structures were within the allowedor generously allowed regions of the Ramachandran plot(see Table 2).The structure of SeZnuA was refined to 1.71 Å

resolution. The final Rcrys was 20.9%, and the free Rvalue, calculated using the test set reflections, was 24.4%.The final model contains 266 residues (residues 27–117and 139–314), 285 water molecules, 2 sulfate ions, 1polyethylene glycol molecule (C12H26O7) and 1 Zn(II) withan occupancy of 0.7. The residues 118–138 are not visiblein the structure. The residues 57–60 are disordered andassume different conformations.To identify the bound metal ion, we collected a single-

wavelength anomalous diffraction data set at the zincanomalous peak wavelength at 1.282 Å. A peak search inan anomalous difference Fourier map identified peak witha height of 18 σ located at the zinc binding site.The structure of Zn(II)–SeZnuA was refined to 1.71 Å

resolution. The final Rcrys was 20.6%, and the free R value,calculated using the test set reflections, was 22.8%. Thefinal model contains 265 residues (residues 27–117 and139–313), 243 water molecules, 2 sulfate ions and 1 Zn(II)with full occupancy. The 118–138 residues are not visiblein the structure.The structure of SeZnuA Δ118–141 was refined to

2.08 Å resolution. The final Rcrys was 19.5%, and the free R

value, calculated using the test set reflections, was 22.51%.The final model contains 263 residues (residues 27–290),148 water molecules and 2 sulfate ions.

PDB accession codes

The coordinates for SeZnuA have been deposited in theResearch Collaboratory for Structural Bioinformatics(RCSB) PDB with accession code 2XY4. The coordinatesfor Zn(II)–SeZnuA have been deposited in the RCSB PDBwith accession code 2XQV. The coordinates for theSeZnuA Δ118–141 deletion mutant have been depositedin the RCSB PDB with accession code 2XH8.

Acknowledgements

F.A. is a recipient of a Fondazione Roma fellow-ship. The authors thank Dr. Mattia Falconi for useful

641The X-ray Structure of the Salmonella Zinc Transporter ZnuA

discussions and gratefully acknowledge FondazioneRoma for a research grant and the Helmholtz-Zentrum Berlin electron storage ring BESSY II forproviding synchrotron radiation at beamline BL-14.1 and for funding from the European Commu-nity's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 226716.

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