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J Biol Inorg Chem (2016) 21:185–196DOI 10.1007/s00775-015-1319-6

ORIGINAL PAPER

Structural characterization of zinc‑bound Zmp1, a zinc‑dependent metalloprotease secreted by Clostridium difficile

Jeffrey T. Rubino1 · Manuele Martinelli2 · Francesca Cantini1,3 · Andrea Castagnetti4 · Rosanna Leuzzi2 · Lucia Banci1,3 · Maria Scarselli2

Received: 2 September 2015 / Accepted: 28 November 2015 / Published online: 28 December 2015 © SBIC 2015

substrate-free Zmp1, highlighting similarities and differ-ences. We also combined the structural characterization to biochemical assays and site-directed mutagenesis, to pro-vide new insights into the catalytic site and on the residues responsible for substrate specificity. The Zmp1 structure showed similarity to the catalytic domain of Anthrax Lethal Factor of Bacillus anthracis. Analogies and differences in the catalytic and in the substrate-binding sites of the two proteins are discussed.

Keywords Protease · NMR · Solution structure · NMR relaxation · Clostridium difficile · Structural vaccinology

AbbreviationsEDTA Ethylenediaminetetraacetic acidNMR Nuclear magnetic resonanceDSF Differential scanning fluorimetrySDS Sodium dodecyl sulfatePAGE Polyacrylamide gel electrophoresisO/N OvernightSOFAST Band-selective optimized flip angle short

transientHMQC Heteronuclear multiple quantum coherenceBMRB Biological magnetic resonance data bankNOESY Nuclear overhauser effect spectroscopyWT Wild typedi-GMP Diguanosine monophosphate

Introduction

Zinc-containing metalloproteases are proteolytic enzymes widely distributed from prokaryotes to eukaryotes and usually classified on the basis of conserved sequence sig-natures. Human pathogenic microorganisms produce a

Abstract Proteases are commonly secreted by micro-organisms. In some pathogens, they can play a series of functional roles during infection, including maturation of cell surface or extracellular virulence factors, interference with host cell signaling, massive host tissue destruction, and dissolution of infection-limiting clots through degra-dation of the host proteins devoted to the coagulation cas-cade. We previously reported the identification and char-acterization of Zmp1, a zinc-dependent metalloprotease secreted by Clostridium difficile, demonstrated that Zmp1 is able to degrade fibrinogen in vitro, and identified two residues necessary to the catalytic activity. In the present work, we solved the solution structure of Zmp1 by Nuclear Magnetic Resonance (NMR) and compared it with the recently solved X-ray structures of substrate-bound and

J. T. Rubino and M. Martinelli contributed equally to this work.

An Interactive 3D Complement page in Proteopedia is available at: http://proteopedia.org/wiki/index.php/Journal:JBIC:33.

Electronic supplementary material The online version of this article (doi:10.1007/s00775-015-1319-6) contains supplementary material, which is available to authorized users.

* Lucia Banci banci@cerm.unifi.it

* Maria Scarselli maria.x.scarselli@gsk.com

1 Magnetic Resonance Center, University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy

2 GSK Vaccines SrL, Via Fiorentina, 1, 53100 Siena, Italy3 Department of Chemistry, University of Florence, Sesto

Fiorentino, Italy4 Department of Biotechnology, Chemistry and Pharmacy,

University of Siena, Siena, Italy

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subset of metalloproteases, called zincins, characterized by a consensus amino acid sequence HExxH, where two his-tidine side chains serve as zinc ligands and the glutamate as a catalytic base. A third zinc ligand is provided by the side chain of a histidine, a glutamic acid or an aspartic acid usually located downstream to this motif. Depending on the location of the third zinc ligand, zincins can be classified into three subfamilies: thermolysins, serralysins and neuro-toxins [1].

Bacterial zincins have often been related to pathogene-sis, like in the cases of serralysin from Serratia marcescens [2], elastase from Pseudomonas aeruginosa [3, 4], neuro-toxins from Clostridium botulinum and Clostridium tetani, enterotoxin from Bacteroides fragilis and anthrax lethal factor (ALF). ALF is a major virulence factor of Bacillus anthracis, which was demonstrated to enter into the cells and cleave the mitogen-activated protein kinase (MAPK) [5]. Recently, polylysine, a new member of the zincin superfamily involved in fibrinogen degradation has been described in Treponema pallidum [6]. It has been proposed that its protease activity plays a fundamental role for bacte-rial dissemination during the course of infection.

Clostridium difficile is a Gram-positive spore-forming anaerobic bacterium which emerged in the last decades as a major cause of antibiotic-associated diarrhea world-wide [7]. Tendency of infection to recur and increase in antibiotic-resistant strains pose new challenges to C. dif-ficile treatment. New-generation small molecules [8], use of pro-biotics, fecal microbiota transplantation (FMT) and vaccination are all emerging options for prevention and cure of C. difficile infection. Although FMT provided in the last years to be an effective treatment for recurrent C. difficile infection, reaching is some cases more than 90 % of cure rates [9], still some questions have to be solved regarding safety, costs and patients acceptance. Beside the development of new antibiotics, virulence targeting factors and vaccines still represent therefore promising strategies, which stimulate the research of new potential antigens and targets for drug design [10].

We previously reported the identification of Zmp1, a zincin released in the culture supernatant of Clostridium difficile strains 630 and R20291, showing sequence similar-ity to the C-terminal domain of ALF [11]. We demonstrated that His146 was one of the zinc ligands and that Zmp1 was able to degrade in vitro fibronectin and fibrinogen in a zinc-dependent manner, identifying Glu143 and His146 as resi-dues necessary for such catalytic activity. Moreover, Zmp1 has been successively demonstrated by Hensbergen and colleagues to be involved in the cleavage of two putative adhesins of C. difficile, namely CD2831 and CD3246 [12]. Crystal structures of substrate-free and substrate-bound Zmp1 have been recently solved [13], confirming that Zmp1 has fold similarity to known members of the zincins

family, such as bacterial thermolysin, elastase, clostridial collagenase and neurotoxins.

In the present study, we report the tridimensional solu-tion structure of the zinc-bound Zmp1 (hereafter Zmp1) in the substrate-free form solved by nuclear magnetic resonance (NMR) and compare it with substrate- free and -bound crystal structures [13]. In particular, we investigated the dynamic properties of the molecule, deepening the characterization of the regions relevant for substrate rec-ognition and specificity. Finally, to enhance our knowledge about Zmp1 specificity, the activity of Zmp1 against a large set of peptides is tested. The present study contributes to shed light on mechanisms used by the bacterium to inter-act with human host and provides in perspective new tools to fight C. difficile infection using appropriate protease inhibitors.

Materials and methods

Cloning, expression and protein purification

All the proteins were expressed in T7 Express E. coli cells (New England Biolabs). Site-directed mutagenesis to generate the mutants H142A; E143A; H142A/H146A; H150A; Y178F and E185A or the Y92-P114 deleted form (Zmp1 ΔL92–114) from the pet15/TEv-Zmp1 vector [11] was performed using the PIPE cloning system [14]. To produce all the unlabeled and 15N labeled His-tagged pro-tein samples, the cells were cultured overnight (O/N) at 30 °C, respectively, in EnPresso B or Enpresso nitrogen-free media (Biosilta-Oulu, Finland) following the manu-facturer’s instructions. Protein expression was induced with 1 mM IPTG for 8 h at 30 °C. To produce 13C, 15N labeled samples, the cells were grown at 37 °C in M9 mini-mal medium containing 1 mg/ml of (15NH4)2SO4 and 3 g/l of 13C labeled glucose until OD600 ~0.7 and then induced with 1 mM IPTG for 3 h at 37 °C. The harvested cells were resuspended in binding buffer (20 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8.0), lysed by sonication and centrifuged. Protein purification was carried out as previ-ously reported [11]. The final purity of the proteins was checked by SDS-PAGE. To obtain the recombinant Zmp1 apo samples, the protein was incubated in 50 mM sodium acetate, 20 mM EDTA, pH 5.0, at 4 °C O/N. The buffer was then exchanged with Hepes 20 mM at pH 7.2 using a PD-10 desalting column (GE Healthcare, NJ, USA). The zinc-bound form of wild-type Zmp1 was obtained by titrat-ing the apo protein with a stock solution of 10 mM ZnCl2 and monitored by 1H-15N SOFAST HMQC or by incubat-ing the apo forms with a slight excess of ZnCl2. The excess of Zinc(II) was sub-sequentially removed using a desalting column. The final protein concentration in all samples for

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NMR experiments was ~0.2 mM. NMR samples contained 10 % v/v D2O for NMR spectrometer lock.

In vitro fibrinogen cleavage assay

The ability by Zmp1 wild-type and the mutants to digest fibrinogen from human plasma was monitored by incuba-tion of 1 µM fibrinogen with 2 µM of Zmp1 or mutants at 37 °C in Tris buffer at pH 8.0 up to 12 h in the presence of 0.5 mM ZnCl2 following a protocol already described [11]. Reactions were loaded on 4–12 % SDS-PAGE gels followed by staining with Problue Safe stain (Giotto Biotech).

In vitro peptides cleavage assay

The activity of Zmp1 (25 µM) against a collection of 360 internally quenched Dabcyl/EDANS peptides (Protease substrate set- JPT Peptide Technologies GmbH) was tested according to the manufacturer’s instructions, to character-ize the activity of the enzyme versus specific substrates. Screened peptides are listed on the manufacturer’s home-page (http://www.jpt.com). To evaluate peptide cleavage, fluorescence (Ex: 336 nm/Em: 490 nm) was quantified for every peptide after 2-h incubation at 37 °C.

The capability by Zmp1 and the mutants to digest the Edans/Dabcyl fluorescently self-quenched peptide (named Fib-1 peptide) containing the EEAPSLRPAPPPISGG sequence were also analyzed. The hydrolysis of the pep-tides by Zmp1 results in an enhanced fluorescence signal as the Edans group is separated from the quencher Dabcyl group. Activity tests were performed in triplicate in Tris 20 mM, NaCl 150 mM, pH=8, 25 µM enzyme and 125 µM of peptides. Reactions were started by the addition of the enzyme and were monitored by measuring the increase in fluorescence (Ex: 336 nm/Em: 490 nm) every 10 min at 37 °C on an Infinite M200 Spectrophotometer microplate reader (TECAN). As controls, the peptides were incubated without the enzyme and the RFU (Relative Fluorescence Units) values were normalized against the control.

NMR experiments and structure calculations of zinc‑bound Zmp1

NMR spectra were acquired at 298 K on Avance 950, 900, 800, 700, and 500 Bruker spectrometers equipped with triple resonance cryoprobes. The NMR experiments used for the assignment of backbone and aliphatic side chain resonances are summarized in supplementary Table S1. Chemical shifts of the assigned NMR resonances are listed in Table S2. Resonance assignments of Zmp1 have been deposited to the BMRB database (RCSB ID code rcsb 25766).

Manual NOE assignment of the three NOESY experi-ments (2D NOESY, 3D 13C-resolved NOESY and 3D 15N-resolved NOESY, all recorded at 950 MHz with a mix-ing time of 110 ms), was combined with structure calcu-lation using CYANA-2.1 [15]. 114 φ and 114 ψ dihedral angle constraints were derived from 15N, 13C’, 13Cα, 13Cβ, and Hα chemical shifts, using TALOS+ [16]. The zinc ion was included in the calculations, following a procedure already reported for other metals, which does not impose any fixed orientation of the ligands with respect to the zinc [17]. The conformers with the lowest residual target func-tion values were subjected to restrained energy minimiza-tion with AMBER 12.0 [18] implemented in the web portal AMPS-NMR (https://www.wenmr.eu/) [19]. NOE and tor-sion angle constraints were applied with force constants of 50 kcal mol−1 Å−2 and 32 kcal mol−1 rad−2, respectively. AMBER parm98 force field parameters and charge distri-bution for the zinc coordination sphere followed the proce-dure described in Hong et al for the ATLF active site [20].

The final family of Zmp1 conformers (Fig. 1) has an average total target function of 0.96 ± 0.09 Å. The aver-age backbone and heavy atoms RMSD values (to the mean structure, over residues 29–220) is 0.80 ± 0.12Å and 1.11 ± 0.10Å, respectively (Figure S1 of Supplemen-tary Material). The quality of the family of conformers was evaluated using the PSVS [21] and iCING validation programs. Table S3 reports some statistics on constraint violations together with selected quality parameters. The programs CHIMERA [22] and Molmol [23] were subse-quently used for structure analysis.

The atomic coordinates of final family of Zmp1 in its substrate-free form (PDB code 2n6j) have been deposited in the Protein Data Bank (http://www.rcsb.org/). These conformers contain the zinc-bound water molecule.

Heteronuclear relaxation data

The dynamic properties of Zmp1 were experimentally characterized through 15N relaxation measurements. 15N longitudinal and transverse relaxation rates (40) and 15N{1H}-NOEs (41) were recorded at 298 K both at 500 and 600 MHz, using a protein concentration of 0.2 mM.

The average backbone 15N longitudinal R1 and trans-versal R2 relaxation rates and 15N{1H}-NOEs values are 1.51 ± 0.08 s−1, 14.9 ± 0.7 s−1 and 0.78 ± 0.05 at the 500 MHz and 1.29 ± 0.06 s−1, 15.2 ± 0.5 s−1 and 0.77 ± 0.04 at the 600 MHz, respectively (Figure S2).

They are essentially homogeneous along the entire polypeptide sequence, with the exception of few residues located at the N-terminus and in some loop regions (Figure S2). The correlation time for the molecule tumbling (τc), as estimated from the R2/R1 ratio, is 11.4 ± 0.7 ns, consist-ent with the molecular weight of the monomeric protein

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and in agreement with the value of 12.1 ns predicted by the HYDRONMR program [24]. In this analysis, care was taken to remove from the input relaxation data those NHs having an exchange contribution to the R2 value or exhibit-ing large-amplitude internal motions on a time scale longer than a few hundred picoseconds as inclusion of these data would bias the calculated τc value [25, 26].

The relaxation data were analyzed according to the model-free approach of Lipari and Szabo [27, 28] using the program Relax [29, 30] (Fig. 2). The “Model-Free” formalism parameterizes intramolecular dynamics in terms of an overall tumbling correlation time τc, general-ized order parameters S2, and of the correlation time for internal motions, which can be considered as arising from two components, one describing faster (τf) and one slower (τs) motions (collectively called τe), but always faster than τc. Motions on intermediate time scales (ms to μs), char-acteristic of exchange processes (Rex), may also contrib-ute to transverse relaxation through the fluctuation of the

chemical environment of a nucleus. The average general-ized S2 value, calculated over residues 29–220 of Zmp1, was 0.86 ± 0.04.

Sequence analysis of Zmp1

Homologous proteins of Zmp1 from Clostridium difficile were identified by performing BLAST searches on bac-terial sequences [31]. Only not redundant proteins with a threshold (E value) calculated by BLAST <10−5 were selected. Multiple sequence alignment was generated using ClustalW [32].

Results

Solution structure and dynamic characterization of Zmp1

The three-dimensional solution structure of Zmp1 (21.6 kDa) consists of a nine-helix bundle packed against one face of a four mixed stranded β-sheet (Fig. 1) and a long loop, between the second and third β-strands, encom-passing residues 83–114 (L83–114) (Fig. 1). The latter loop contains three short 310 helices. The structure is well defined (the average backbone RMSD value over residues 29–220 is 0.80 ± 0.12Å) with the exception of the stretch 101–108, which presents average backbone RMSD val-ues to the mean structure of 1.54 ± 0.25Å (Figure S1). 15N heteronuclear relaxation measurements revealed that such residues are affected by local internal motions occur-ring on a fast timescale with respect to the overall re-ori-entational correlation time (τc) of the molecule. For some of these residues, a correlation time for internal motions (τe) can be fitted according to the model-free formalism (Fig. 2). Backbone NH of Gly105 is even not observed, likely as a consequence of an increased local mobility or solvent exchange (Table S2). Fast local internal motions were also observed for several residues located in the loop between β4 and α4 (L125–140) and for the first 5 residues at the N-terminal region. The analysis of 15N relaxation meas-urements reveals conformational exchange processes on the ms–μs timescale which affect residues located in L125–140. Such residues are fitted, within the Model-Free analysis, with a Rex contribution to the transversal R2 relaxation rate (Fig. 2).

The core of the protein is characterized by several hydrophobic interactions which involve aromatic and ali-phatic residues. Stacking contacts are present among the side chains of Phe162(α5), Phe166(α5) and Phe186(α7), which are clustered within the helix bundle. Residue Ile64 establishes hydrophobic interaction with aromatic rings of Phe191(α7) and Tyr195(α7), while stacking contacts are

Fig. 1 a Solution structure of the zinc-bound Zmp1 shown as tube whose radius is proportional to the backbone RMSD of each residue. The β-strands are shown in cyan and α-helices in red. The second-ary structure elements comprise residues 29–39(α1), 42–43(β1), 51–62(α2), 66–74(α3), 79–82(β2), 115–116(β3), 121–124(β4), 139–151(α4), 160–169(α5), 176–179(α6), 183–195(α7), 198–207(α8), 209–219(α9). b Ribbon diagram of zinc-bound Zmp1. The side chains of the zinc ligands and of the catalytic Glu143 are also shown. c Zoom of the zinc coordination site. The amino acids in the active site are labeled. The zinc atom is shown as orange sphere

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present between Tyr194(α7), Tyr212(α9) and Tyr214(α9) that are also clustered within the helix bundle. Finally, hydrophobic interactions involving residues Ile31(α1), Leu38(α1), Val42(β1), Val58(α2) and Ala62(α2) and stack-ing contact between Tyr49 and Phe44, both in loop L41–50, determine the spatial vicinity of helix α1 to beta β1 and helix α2.

We previously demonstrated, through mutagenesis stud-ies, that His146 (α4) was required for zinc binding. On the contrary, the mutation of Glu143, which is required for the catalytic activity, to alanine left the affinity for zinc sub-stantially unaffected [11].

To further characterize the Zmp1 catalytic site, we performed additional point mutations (His142Ala, His-150Ala, Tyr178Phe and Glu185Ala) on conserved residues located in proximity of the catalytic site. Protein stability and zinc-binding activity of each mutant were evaluated by Differential Scanning Fluorimetry (DSF) and NMR spectroscopy, respectively. In the DSF assay (Figure S3),

Zmp1 mutants showed a melting temperature higher or comparable to the wild type, indicating that the muta-tions do not disrupt the protein stability. Tyr178Phe and Glu185Ala mutants exhibited an increase of 5 °C in melt-ing temperature, suggesting a modest increase of the pro-tein stability. Given the spatial proximity of Tyr178 and Glu185 to hydrophobic side chains (Leu129 and the pair Leu179 and Val181, respectively), it is possible to argue that both Tyr178Phe and Glu185Ala substitutions can enforce the hydrophobic interactions, stabilizing the pro-teins compared to the wild type.

NMR titrations of apo His142Ala and Glu185Ala Zmp1 mutants with Zn2+ showed that these mutants completely lose the ability to bind the metal ion (Figure S4). On the contrary, the mutations His150Ala and Tys178Phe did not affect zinc binding, excluding their role in the metal coordi-nation (Figure S4).

These results are in agreement with the typical zinc coordination observed within the zincin superfamily,

Fig. 2 Parameters characterizing the overall and internal mobility of Zmp1 within the Lipari-Szabo model. The “Model-Free” formal-ism parameterizes intramolecular dynamics in terms of an overall tumbling correlation time τc, generalized order parameters S2, and of the correlation time for internal motions, which can be considered as arising from two components, one describing faster (τf) and one slower (τs) motions (collectively called τe), but always faster than τc. Motions on intermediate time scales (ms to μs), characteristic of

exchange processes (Rex), may also contribute to transverse relaxation through the fluctuation of the chemical environment of a nucleus. The line reported in the graph of Rex represents the average value. The secondary structure elements are reported at the top. Residues which experience local mobility (Rex or τe) are also mapped into the Zmp1 structure in orange and blue, respectively. The radius of the atom bonds is proportional to the magnitude of these values. Zinc is repre-sented as orange sphere, binding residues as sticks

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where the metal ion is tetrahedrally bound by the side chains of the histidine residues forming the HExxH motif, as well as by an additional glutamate and a water molecule that complete the first coordination shell [33]. Accord-ingly, the crystal structure of Zmp1 showed that His146 is a zinc ligand together with His142, Glu185 and a water molecule [13].

To investigate the presence of a water molecule as a possible fourth zinc ligand in the solution structure, molecular dynamics calculations were conducted with and without the presence of a H2O molecule in the first coordination sphere of the zinc ion. The H2O molecule was placed near the active site without any restraints between the zinc ion and the oxygen atom of water. In 36 of the 40 structures examined, the water molecule remained within the bond distance to zinc, while in 3 structures it was replaced by another water molecule of the solvation shell. Water was absent from the first coordination sphere in only one structure, where it was replaced by the hydroxyl oxygen of Tyr178. The substi-tution of this residue however, as described above, had no evident effects on zinc coordination, suggesting the transient character of this interaction. These results sug-gested that the used force field parameters for molecular dynamic calculations, derived from a similar zinc coor-dination site [20], were appropriate to describe the posi-tion of water molecules in proximity of the zinc coor-dination site and support the hypothesis that one water molecule can be accommodated in the catalytic pocket. Analysis of the conformers of this protein family showed that the side chain of Glu143 was well positioned to act as a general base to activate the zinc-bound water dur-ing catalysis [34], lying 3.5 ± 0.1Å from the water mol-ecule (distance between Cδ of the glutamate and the oxygen atom of the water), while it was less ordered in the ensemble calculated without the presence of a water molecule in the first coordination sphere of the zinc ion. This is in agreement with its catalytic role, as muta-tion Glu143Ala compromised the proteolytic activity of zinc-bound Zmp1, without affecting zinc binding [11]. In half of the conformers, the hydroxyl group of Tyr178 formed a hydrogen bond with the water molecule, on the opposite side of Glu143. This position is in agreement with the function of Tyr178 suggested in other metallo-proteases, where a tyrosine in the same spatial orienta-tion acts as a general acid to protonate the amine leaving group [35].

To highlight which residues located in proximity of the catalytic site, beside those involved in zinc binding, could have a key role in the substrate cleavage in solution, fibrinogenolytic activity of the His150Ala and Tyr178Phe mutants was compared to that of WT Zmp1. The zinc-bound form of Tyr178Phe showed abolished proteolytic

activity on fibrinogen, while His150Ala substitution had only a minor effect (Fig. 3a). Time-dependent proteolytic activity of WT Zmp1, His150Ala and Tyr178Phe was also monitored by fluorimetry using an Edans/Dabcyl fluores-cently self-quenched peptide (Fib-1 peptide) containing the EEAPSLRPAPPPISGG sequence that mimics the region of the fibrinogen digested by Zmp1 (Fig. 3b). His150Ala sub-stitution had no effect on the proteolytic activity, while Tyr-178Phe mutant showed a reduced activity (after 100 min less than 50 % of the peptide was cleaved by Y178F com-pared to WT Zmp1), suggesting that this residue affects the catalytic activity of Zmp1 without abolishing it completely. The same experiment was performed also on His142Ala, Glu185Ala and the His142Ala/His146Ala mutants,

Fig. 3 a Time-dependent proteolytic activity of recombinant zinc-bound wild-type (WT) and mutants Zmp1 on fibrinogen, monitored by SDS-PAGE. b) Time-dependent proteolytic activity of recombi-nant zinc-bound WT (red diamond) and Zmp1 mutants H150A and Y178F, blue squares and yellow spheres, respectively, on Fib-1 pep-tide monitored by fluorimetry (excitation: 336 nm/emission: 510 nm)

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showing, as expected, that the abolition of zinc binding dis-rupts also the protein proteolytic activity (Figure S5).

Structural comparison with crystal structures of Zmp1

The superimposition of the present NMR solution structure, in its substrate-free form, with the crystal structure of Zmp1, in the free and bound to the octapeptide Ac-EVNPPVPD-NH2 form [13], shows that the solution structure is nearly identical to the crystal unbound structure (RMSD 1.07 Å calculated over secondary structural elements) (Fig. 4). The region comprising residues involved in the zinc binding has identical conformation in the two structures. The same 3D arrangement of the zinc-binding site is also maintained in the substrate-bound Zmp1. Even the conformation of resi-dues Glu189 and Asp149, which contribute to the second zinc coordination sphere, is maintained in the three struc-tures. On the contrary, meaningful structural differences are present between the unbound form (solution and crystal) and the substrate-bound one. In particular, the most signifi-cant difference, i.e., outside the uncertainty of the solution structure is observed in correspondence of the loop L83–114, which is named S loop by Tallant et al [35] (Fig. 4). Many residues in this region change sizably in their backbone and side chain conformations. A RMSD of 3.6 Å ± 1.0

(standard deviation) for amino acids 99–115 was detected between solution substrate-free structure and substrate-bound crystal form, while this region has no meaningful structural variation between solution and crystal unbound forms. In particular, the substrate-bound crystal structure assumes a more compact arrangement with residues Lys101 and Trp103 experiencing the most extensive change. Their side chains move indeed towards the binding site in the sub-strate-bound crystal structure while they point towards the solution in the NMR substrate-free structure. This behavior indicates that the zinc ion organizes the catalytic site in one conformation also in the absence of substrate, while the sub-strate binding induces a significant conformational change of L83–114. This allows the establishment of interactions between specific residues of Zmp1 and the substrate, which is in turn locked in the proper conformation to be efficiently cleaved.

Structural comparison with Anthrax toxin lethal factor

The closest structural homolog to Zmp1 is the C-terminal domain of Anthrax Lethal Factor of Bacillus anthracis (ATLF domain, PDB ID 1j7n) The two proteins share 23 %

Fig. 4 Structural comparison of solution substrate-free Zmp1 (cyan) with X-ray substrate-free Zmp1 (PDB ID 5A0P) (green) and X-ray substrate-bound E143A Zmp1 (gray) (PDB ID 5A0R). The zinc ligands are shown as sticks. Zinc-binding water molecule is shown as gray sphere and the zinc ion is shown as orange sphere Fig. 5 a Structural comparison of the solution zinc-bound Zmp1

(blue) with ATLF domain from Bacillus anthracis (green). b Overlay of the structures of the solution zinc-bound Zmp1 (blue) with ATLF domain (green), highlighting only the regions of structural variation

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of sequence identity and the overall RMSD value between the zinc-bound Zmp1 structure and ATLF, calculated on the Cα atoms of aligned residues excluding gaps, is 3.1 Å. Although contacts of ATLF with the other domains of Lethal Factor and the insertion and deletions occurring within the loops make the two proteins quite different in specific regions, their comparison reveals that part of the β-sheet and the alpha helices at the C-terminal are well superimposable, with an RMSD of about 1.04 Å. Major structural differences are indeed located in regions of the ATLF domain having contacts with the other structural domains of Lethal Factor not present in Zmp1 (Fig. 5). This is the case of helix α1 which has a different orientation in

Zmp1 with respect to ATLF, where it constitutes the linker between the third and the fourth domain. Similarly, the N-terminal part of α2 and loop L125–138, located within the same region of Zmp1 and having in ATLF domain contacts with the third domain of Lethal Factor, shows different conformations in the two proteins. Moreover, α4 οf ATLF is two helical turns longer than in Zmp1, corresponding to an eight amino acid long insertion in the ATLF sequence (Fig. 6).

The most striking difference between Zmp1 and ALF resides in the extended loop encompassing residues 83–114 of Zmp1 (L83–114), which protrudes to form a cap at the top of the active site in Zmp1 (Fig. 5). With the exception of

Fig. 6 a Structure-based align-ment of the sequences of Zmp1 and ATLF domain, obtained by ClustalW from UCSF Chimera/MultAlignviewer program [23]. Amino acid numbering is reported according to the sequence of Zmp1. Secondary structure elements are indicated for Zmp1. Identical or con-servative substituted residues are indicated by the symbols asterisks and colon, respec-tively, below the sequences. The orange boxes highlight the zinc-binding site, while the cyan box highlights the loop 83–114. b Identical residues between Zmp1 and ATLF domain are mapped in red on the ribbon diagram of the solution Zmp1

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the tip (residues 101–108), L83–114 resulted well-ordered, with hydrophobic residues stratified at the interior face and polar side chains well exposed to the solvent. Multi-ple sequence alignment of Zmp1 from Clostridium difficile with its homologous proteins identified conserved residues in correspondence of L83–114 (Figure S6), suggesting that this region might have some influence on the enzymatic activity of Zmp1. To test this hypothesis, a mutant where residues 92–114 were deleted (ΔL92–114) has been produced and analyzed by DSF and NMR. DSF profile showed that deletion did not significantly alter the thermal stability of the protein (Figure S7a). The 1H-15N HSQC NMR spec-trum of ΔL92–114 indicated a well-folded polypeptide chain with dispersed amide signals whose number matched the expected one (Figure S7b). Many NHs signals overlapped those of the WT apo Zmp1 indicating that deletion of the stretch 92–114 does not alter the architecture of the rest of the protein. Accordingly, NMR titration with Zn2+ showed that the ΔL92-114 construct maintains its ability to coordi-nate the metal ion (Figure S7b).

Strikingly although the loop deletion did not desta-bilize the protein conformation, it completely impaired the enzyme’s proteolytic activity against both full-length fibrinogen and Fib- peptide (Fig. 7a, b). This result indi-cates that the loop plays a role in substrate recognition and modulates the substrate specificity.

Zmp1 shows high specificity versus proline‑rich regions

Analysis on the Zmp1 substrates indicates that proline-rich sequence motifs are typically found in proxim-ity of the scissile peptide bond, being proline required in P3′ and highly recurrent in P1 and P1′ positions [11, 12]. Amino acid residues in a substrate undergoing cleavage are

commonly designated P3, P2, P1, P1′, P2′, and P3′ where the two positions flanking the scissile bond are P1 and P1′.

To enhance our knowledge about Zmp1 specificity, the activity of zinc-bound Zmp1 against a large peptide collec-tion was tested. Interestingly, out of the 360 tested peptides, only 6 show a significant increment of fluorescence (fluo-rescence at least 10 % of the fluorescence of the peptide that shows the highest fluorescence). The common feature of these peptides is the presence of at least 3 clustered pro-lines with no more than 2 amino acid insertion between them (Table 1). No fluorescence increment was indeed detected for any of the 119 peptides that contain only 1 proline and 19 peptides which containing 2 prolines (five of which contain a ProPro motif). Moreover, out of the 3 tested peptides that contain 4 prolines (K21, L4 and L10), no fluorescence increment was detected for the peptide L4 (LPPKSQPP), which contains prolines spaced by 3 amino acids. Interestingly, the L10 peptide (PPPPLSGG), which showed the highest fluorescence increment, shares a high sequence identity (75 %) with the Fib-1 that mimic the fibrinogen digestion site (Fig. 8).

Fig. 7 a Time-dependent proteolytic activity of recombinant zinc-bound WT and ΔL92–114 Zmp1 on fibrinogen, monitored by SDS-PAGE. b Time-dependent proteolytic activity of recombinant

zinc-bound WT and ΔL92–114 Zmp1 on Fib-1 peptide monitored by fluorimetry (excitation: 336 nm/emission: 510 nm)

Table 1 Peptide cleavage rate vs proline residue recurrence in the set of synthetic peptides collection

Prolines Peptides

Total With ProPro motif

With ProAla motif

Digested %

0 211 0 0 0 0

1 119 0 7 0 0

2 19 5 2 0 0

3 8 4 4 4 50

4 3 3 1 2 66

194 J Biol Inorg Chem (2016) 21:185–196

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Discussion

C. difficile releases the zinc-dependent metalloprotease Zmp1 in the extracellular medium. Different possible tar-gets have been identified for this protein, including the C. difficile surface-anchored proteins CD2831 and CD3246 [7] and the human fibrinogen [11], leading to the hypoth-esis that it could play multiple roles during bacterial host infection. This protein can indeed contribute to several aspects of C. difficile pathogenesis ranging from the dam-age of host tissues, through hydrolysis of the components of extracellular matrix, to the release and cell wall attach-ment of virulence factors. Recently, a novel regulatory mechanism has been also demonstrated, where release from the bacterial surface of CD2831 and the Zmp1 expression are both regulated by intra-cellular concentrations of cyclic di-GMP [36].

The three-dimensional structure of Zmp1 shows the highest fold similarity with ATLF. However, the two pro-teins show differences in substrate specificity. Hensber-gen et al. [12] recently demonstrated that, for Zmp1, the presence of proline residues in the peptide substrate is an important feature of the cleavage site, and that Zmp1 toler-ates only proline or alanine residues on the P1 and P1′ posi-tions of the substrate.

Previously, we showed [8] that, differently than ATLF, Zmp1 cleaves human fibrinogen in vitro, while no proteo-lytic activity can be observed on MAPK2 peptide, one of

the characterized substrates of ATLF, even if it contains a Pro-Ala cleavable bond. The screening of the 360 peptides here performed demonstrates that the presence of Pro-Pro or Pro-Ala alone is not enough to ensure the Zmp1 sub-strate digestion and that the most stringent requirement is the presence of 3 clustered prolines with no more than 2 aa insertion after the cleavage site (Table 1). The most remark-able structural differences between ATLF and Zmp1 are pre-sent in loop L83–114. The deletion of a region of such loop (ΔL92–114 mutant) completely abolishes the proteolytic activity of Zmp1. These results revealed that L83–114 con-tains important residues for substrate recognition, in agree-ment with the x-ray structure of the substrate-bound Zmp1 [13]. Multiple sequence alignment of bacterial homologous of Zmp1 (Figure S6) showed that the region removed in the ΔL92–114 mutant contains some conserved hydrophobic resi-dues (Figure S8), such as Pro100, Thr109, Trp110, Val113, Pro114 and Gly115. Residue Tyr92 and Trp103 are highly conserved, with the exception of few cases where they are replaced by other hydrophobic residues. With the exception of Tyr92, these residues are not conserved in ATLF domain, where this loop results to be shorter than L83–114 of Zmp1 and extremely variable (Fig. 6b). The sequence conservation profile in Zmp1 homologous sequences and the variability of L83–114 in the sequence of ATLF domain are in agreement with their role in substrate recognition and proteolytic activ-ity, as they are able in Zmp1 to form a hydrophobic pocket which accommodates the substrate. Heteronuclear NMR

Fig. 8 Zmp1 preference for multi-proline regions. a Substrate digestion profiling of zinc-bound Zmp1 using a set of synthetic peptides (protease substrate set; JPT Peptide Tech-nologies). Fluorescence (excita-tion: 360 nm/emission: 490 nm) was measured after incubation zinc-bound Zmp1 (25 μM) with each peptides for 40’ at 37 °C. b Sequence alignment of L10 peptide showing the highest fluorescence out of the 360 tested to Fib-1 peptide mimick-ing the fibrinogen sequence

195J Biol Inorg Chem (2016) 21:185–196

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relaxation studies reveal that residues located in such loop experience fast motions and for these residues a correlation time for internal motions (τe) can be fitted within the Model-Free analysis (Fig. 2). Such local flexibility is also present for several residues of the loop L125–140 and in particular for residues His134, Asp135 and Ala136 that, together with the side chains of residues Asn175, Tyr178 and Leu179 consti-tute the S2′ subsite of the substrate [13]. For many residues of L125–140, a Rex contribution to the transversal R2 relaxa-tion rate can be also fitted indicating the occurrence of con-formational exchange phenomena which lead also to line broadening of NMR signals.

The dynamic NMR characterization of the substrate-free Zmp1 in solution reveals flexibility in the regions involved in the substrate recognition. The dynamic properties of substrate-free Zmp1 suggest that the loop flexibility on the ns-ps timescale and conformational rearrangements on the ms timescale could have a functional role in the substrate binding to efficiently drive the catalytic activity. Several reviews have already summarized the functional role of dynamics in catalysis demonstrating that substrate specific-ity can be attributed to differences in the character of the motions at the binding pocket [37, 38].

The solved crystal structures of the substrate-free and -bound Zmp1 suggest that the protein achieves the final active state characterized by the right orientation of cata-lytic residues necessary for proteolytic activity, after changes in protein structure (i.e., opening and closure of L83–114) that are induced by the binding of the substrate. The existence of local backbone motions experienced by the conserved residues of L83–114 and residues in L125–140 in the substrate-free Zmp1 might have a role in the selection of the optimal conformation which facilitates the right sub-strate-protein contacts. It has been previously demonstrated that changes in protein dynamics can play an important energetic role in substrate binding and that the time scales and amplitudes of these motions can vary widely between ns-ps to ms-μs regimes [39–41].

The occurrence of local mobility in different regions of substrate-free Zmp1 suggests that in solution Zmp1 can exist in equilibrium between the major, open (substrate-free) conformation and a minor, closed (substrate-bound) conformation also in the absence of the substrate as reported for other apo- and substrate-bound enzymes [42, 43]. It is possible that, once the substrate is bound to Zmp1, the equilibrium is shifted towards the closed configuration, whilst a minority of Zmp1 molecules still assumes in solu-tion the open conformation.

Overall, the heteronuclear NMR studies here performed have revealed that two regions of the substrate-free Zmp1, which do not comprise residues directly involved in zinc binding and substrate cleavage, are affected by local mobil-ity. Such dynamic behavior could play a role in substrate

specificity and offers the possibility to investigate and develop specific inhibitors or mutants, that acting on the internal mobility, may impair the activity of C. difficile Zmp1 contributing to prevent the dissemination of this pathogen.

In the field of vaccine development against Clostridium difficile, the NMR assignment and the solution structure of Zmp1, together with its dynamic characterization, can be useful for mapping residues involved in important func-tions such as eliciting bactericidal antibodies and it will provide insight into improved vaccine antigen design with higher stability and improved epitope presentation.

Acknowledgments We thank Prof. Paolo Carloni and Dr.ssa Ales-sandra Magistrato for providing us with the force field parameters and charge distribution for the zinc coordination sphere for molecu-lar dynamic calculations. We are grateful to Giorgio Corsi for art-work. This work was supported by the BIOVAX grant funded by Piano Operativo Regionale (POR) of Regione Toscana and the grant PON01_00117 from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR).

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