Báo cáo khoa học: The interaction of the Escherichia coli protein SlyD with nickel ions illuminates the mechanism of regulation of its peptidyl-prolyl isomerase activity ppt

The interaction of the Escherichia coli protein SlyD with nickel ions illuminates the mechanism of regulation of its peptidyl-prolyl isomerase activity Luigi Martino1,2, Yangzi He1,*, Katherine L D Hands-Taylor1, Elizabeth R Valentine1, Geoff Kelly3, Concetta Giancola2 and Maria R Conte1 Randall Division of Cell and Molecular Biophysics, King’s College London, London, UK Department of Chemistry ‘P Corradini’, University of Naples ‘Federico II’, Italy MRC Biomedical NMR Centre, National Institute for Medical Research, London, UK Keywords FK506-binding protein (FKBP); nickel; peptidyl-prolyl cis-trans isomerase (PPIase); SlyD; structure Correspondence M R Conte, Randall Division of Cell and Molecular Biophysics, King’s College London, Guy’s Campus, London SE1 1UL, UK Fax: +44 2078486435 Tel: +44 2078486194 E-mail: sasi.conte@kcl.ac.uk *Present address Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000, Aarhus C, Denmark Database Structural data are available in the Protein Data Bank under the accession number 2KFW The sensitive to lysis D (SlyD) protein from Escherichia coli is related to the FK506-binding protein family, and it harbours both peptidyl-prolyl cis–trans isomerase (PPIase) and chaperone-like activity, preventing aggregation and promoting the correct folding of other proteins Whereas a functional role of SlyD as a protein-folding catalyst in vivo remains unclear, SlyD has been shown to be an essential component for [Ni–Fe]hydrogenase metallocentre assembly in bacteria Interestingly, the isomerase activity of SlyD is uniquely modulated by nickel ions, which possibly regulate its functions in response to external stimuli In this work, we investigated the solution structure of SlyD and its interaction with nickel ions, enabling us to gain insights into the molecular mechanism of this regulation We have revealed that the PPIase module of SlyD contains an additional C-terminal a-helix packed against the catalytic site of the domain; unexpectedly, our results show that the interaction of SlyD with nickel ions entails participation of the novel structural features of the PPIase domain, eliciting structural alterations of the catalytic pocket We suggest that such conformational rearrangements upon metal binding underlie the ability of nickel ions to regulate the isomerase activity of SlyD (Received 14 May 2009, revised 15 June 2009, accepted 17 June 2009) doi:10.1111/j.1742-4658.2009.07159.x Introduction The Escherichia coli sensitive to lysis D (SlyD) protein was originally discovered as a host factor required for E-protein-mediated cell lysis upon infection with bacte- riophage /X174 [1] It was then found to be identical to wondrous histidine-rich protein, independently identified as a persistent contaminant of histidine-tagged Abbreviations FKBP, FK506-binding protein; HsFKBP12, Homo sapiens FK506-binding protein; IF, insert in flap; ITC, isothermal titration calorimetry; MtFKBP17, Methanococcus thermolithotrophicus FK506-binding protein; PPIase, peptidyl-prolyl cis–trans isomerase; SlyD, sensitive to lysis D FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS 4529 Structure and interactions of SlyD L Martino et al recombinant proteins purified by immobilized metal affinity chromatography [2] SlyD has been suggested to bind to divalent cations, including Ni2+, Zn2+ and Co2+, via its C-terminal domain, a stretch of approximately 50 amino acids containing several short clusters of potential metal-binding residues such as histidines, cysteines, aspartates, and glutamates [2–5] Whereas the N-terminal region of SlyD shares primary sequence homology with the ubiquitous FK506-binding protein (FKBP) family of peptidyl-prolyl cis–trans isomerases (PPIases), this C-terminal tail appears to be a unique feature of SlyD bacterial proteins [3,5] (Fig 1) The functional profile of SlyD is rather intriguing As a member of the FKBP family, SlyD harbours prolyl isomerase activity, which is responsible for accelerating the rate-limiting trans-to-cis isomerization step in protein folding [6–9] More recent work, however, has shown that SlyD associates a PPIase function with a proficient chaperone-like activity, preventing aggregation and promoting the correct folding of other proteins [10–14] SlyD displays high affinity for unfolded proteins, irrespective of their proline content, in a manner evocative of the E coli trigger factor, which also combines PPIase and chaperone abilities [10,15,16] The chaperone-like activity of SlyD appears to reside in a characteristic insertion within the PPIase domain when compared to eukaryotic FKBPs, called the ‘insert-in-flap’ (IF) domain (Fig 1) [17] The IF domain is also a trait of the archaeal FKBP from Methanococcus thermolithotrophicus (MtFKBP17) [18,19], conferring, as in this case, chaperone-like competence to the protein [20] To date, the physiological role of SlyD as a chaperone assisting with protein folding in vivo has remained unclear [10,13,21] Nonetheless, a function for SlyD in the [Ni–Fe]-hydrogenase biosynthetic pathway has recently emerged, with the identification of SlyD as an essential component of the hydrogenase metallocentre assembly, probably serving as a nickel supplier for the formation of [Ni–Fe] clusters [22,23] Not only does this concur with the ability of SlyD to bind nickel ions, but, notably, nickel ion binding to SlyD provides the means to reversibly regulate its PPIase activity [9] Consistent with this, the PPIase ability of SlyD has been shown not to be critical for its role in hydrogenase biosynthesis [24] Further investigations have uncovered a key interaction between SlyD and the hydrogenase accessory factor HypB [23] E coli HypB contains two metalbinding sites – a high-affinity site in the N-terminal region and a low-affinity site within its GTPase domain – and both are required for hydrogenase maturation [25–27] However, in contrast to other bacterial HypBs, the E coli protein lacks additional storage capacity for nickel in the form of a histidine-rich stretch found in other organisms [28–30] It has been proposed that HypB interaction with SlyD may therefore circumvent this deficit, by recruiting extra metalbinding capacity to the system In support of this hypothesis, whereas the PPIase domain of SlyD is required to interact with an N-terminal proline-containing stretch of HypB, the putative metal-binding region, comprising residues 146–196, is strictly essential for the role in hydrogenase biosynthesis [23] The primary sequence homology of SlyD with other FKBP proteins terminates around residue 139, incorporating the IF domain, which is also found in archaea (Fig 1) The C-terminal tail is present in SlyD variants from other bacteria, with some degree of sequence conservation (Fig 1); this has been suggested to be unstructured and an easy target for proteolytic degradation [9,10] Fig Alignment of SlyD bacterial proteins and FKBP homologues The alignment was obtained using T-COFFEE (http://www.ebi.ac uk/Tools/t-coffee/index.html) Invariant residues are boxed in black, and conserved residues in grey The secondary structure elements are superposed on the amino acid sequence The names of proteins of different species are as follows: SlyD_ECOL, E coli; SlyD_HAEIN, Haemophilus influenzae; SlyD_AERHY, Aeromonas hydrophila; SlyD_TREPA, Trepomena pallidum; SlyD_ HELPY, Helicobacter pylori; SlyD_HELPJ, He pylori J99; MtFKBP17, M thermolithotrophicus; HsFKBP12, H sapiens 4530 FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS L Martino et al In this study, we have investigated the solution structure of E coli full-length SlyD, and found an atypical PPIase domain containing an additional C-terminal a-helix packed against the rest of the domain This is in full agreement with the very recently published structure of the N-terminal part of SlyD, although a functional role for this extra structural element has not been established in this work [31] Intriguingly, our investigations reveal that this C-terminal helix, unprecedented for the FKBP family of proteins, is involved in nickel ion binding, causing conformational rearrangements in the PPIase domain and modulating its isomerase activity The basis of this molecular switch will be discussed Results Structure determination To characterize the structure of E coli SlyD, preliminary NMR analysis was applied to full-length SlyD (encompassing residues 1–196) and a truncated N-terminal fragment, homologous to other FKBP proteins, spanning residues 1–146 (SlyD1–146) Comparison of H-15N HSQC spectra of the two molecules (data not shown) indicated that, although many resonances were directly superimposable, a number of well-resolved signals appeared to be shifted in the context of the deletion mutant, suggesting potential intramolecular interactions involving the PPIase core domain and a number of residues beyond Glu146 As, in our hands, the purified recombinant full-length SlyD appeared to be stable in the conditions required for NMR analysis, structural determination of the wild-type protein was undertaken SlyD folds into two domains and a long, unstructured C-terminal tail The three-dimensional structure of E coli SlyD was determined using standard heteronuclear multidimensional NMR techniques as described in Experimental procedures In solution, SlyD folds into two globular domains, namely the PPIase domain and the IF domain, bisected by a deep cleft The PPIase domain consists of two polypeptide segments, spanning residues 1–69 and 129–154, and the insert fragment, comprising residues 76–120, constitutes the IF domain (Fig 2) The partitioning of the polypeptide chain creates a pair of antiparallel strands at the base of the cleft linking the two domains These connecting segments span residues 70–75 and 121–128, respectively, and act as a flexible hinge for a bending motion Structure and interactions of SlyD between the domains (see below) These fragments are not well defined, and few long-range NOE contacts to the other domains could be unambiguously assigned, although there is evidence of local structure in the turns spanning regions 71–75 and 122–126 The relative orientation of the PPIase and IF domains is also undefined Because both domains establish contacts with residues located within the connecting segments, they not tumble fully independently of each other in solution Nonetheless, no unambiguous contacts between them could be detected, and a fixed orientation could not be found in our investigation (Fig S1) This is in agreement with previous structural studies of the archaeal homologue MtFKBP17 [32] and with our backbone relaxation analysis (Fig 2c); in fact, estimates of the rotational correlation times for the two domains, based on analysis of T1 ⁄ T2 ratios, gave significantly different values for the PPIase and IF domains, 13.6 and 11.2 ns respectively Furthermore, 1DNH residual dipolar couplings for SlyD were measured in liquid crystalline media; however, attempts to find a single value for the magnitude and rhombicity of the alignment tensor using the maximum likelihood method [33] failed, suggesting that the two domains could not align to a single external axis Therefore, each domain was superimposed separately to calculate the rmsd A final family of 20 superimposed structures for the PPIase domain and the IF domain is shown in Fig 2; the overall values of rmsd between the family and the mean coordinate position ˚ are 0.749 and 0.828 A for backbone atoms in secondary structure regions, respectively The structure calculation statistics are given in Table 1, and a representative structure is reported in Fig The structural quality, in terms of restraints violation and deviation from the ideal geometry, was checked with the program procheck-nmr (Table 1) Our structure was also compared with the very recent structure of the N-terminal fragment of SlyD, encompassing residues 1–165 [31] The rmsd values for the PPIase and the IF domains are, respectively, 1.51 ˚ and 1.67 A over structured regions (defined in Table 1), underscoring the fact that the structure of the domains remains largely unaffected in the context of the intact full-length protein This is consistent with the finding that the region encompassing residues 153– 196 of SlyD appears to be largely unstructured in our study Severe spectral overlap prevented us from obtaining unequivocal sequence-specific assignment for the majority of the residues in this stretch; however, a comparative analysis of the HSQC spectra of fulllength SlyD and SlyD1–146 positively identified the FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS 4531 Structure and interactions of SlyD A B L Martino et al Fig Analysis of structure and backbone dynamics of SlyD Superimposition of the backbone traces for the 20 lowest-energy structures of SlyD (A) for the IF domain (traces showing residues 75–121) and (B) for the PPIase domain (residues 1–70 and 127–152) The N-termini and C-termini and secondary structure elements are indicated The relative orientation of the two domains with respect to each other is undefined; although they are not fully mobile with respect to each other, long-range contacts could not be unambiguously detected in this study (Fig S1) (C) Backbone relaxation analysis showing T1, T2 and {1H}-15N NOE values for SlyD measured at 18.8 T and 298 K resonances arising from the C-terminal tail as a cluster of signals around 8–8.5 p.p.m characterized by reduced {1H}-15N NOE values (data not shown) Structure of the PPIase domain The PPIase domain of SlyD possesses a b4–b5a–b5b– a1–b2–b3–a4 topology, and folds to generate a twisted four-stranded antiparallel b-sheet wrapped around the a1-helix and flanked by the a4-helix (Figs and 3) The numbering of the secondary structure elements adopted here reflects the convention used for other FKBP proteins (see below and Fig 3) The a1-helix displays a marked amphipathic character and sits on Table Summary of structural statistics for SlyD C NMR restraints Total distance restraints (inter-residue) Short–medium range (residue i to I + j, j = 1–4) Long range (residue i to I + j, j > 4) Hydrogen bonds Total dihedral angle restraints / w Restraint violations ˚ Distance restraint violation > 0.2 A SlyD 728 474 23 230 115 115 None Dihedral restraint violation > 5° None ˚ Average rmsd (A) among the 20 refined structures Residues PPIase 1–69, 129–149 Backbone of structured regionsa 0.749 Heavy atoms of structured regions 1.481 Backbone of all residues 0.778 Heavy atoms of all residues 1.501 Ramachandran statistics of 20 structures Percentage residues in Most favoured regions 87.8 Additional allowed regions 8.9 Generously allowed regions 2.2 Disallowed regions 1.1 IF 76–121 0.828 1.674 1.228 1.899 a Residues selected on the basis of 15N backbone dynamics PPIase domain: 1–38, 41–68, and 129–148; IF domain: 76–83, 90–96, and 105–120 4532 FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS L Martino et al Structure and interactions of SlyD A B C D E F Fig Structural comparison of HsFKBP12, MtFKBP17, and SlyD Top panel: cartoon representations of the representative structures for HsFKBP12 (A), MtFKBP17 (B), and SlyD (C) The flexible tail of SlyD has been truncated at residue 153 Lower panel: topological comparison of HsFKBP12 (D), MtFKBP17 (E), and SlyD (F) The N-termini and C-termini are indicated The secondary structure elements are labelled according to the convention adopted for HsFKBP12 one portion of the b-sheet surface, with its polar flank largely solvent-exposed and the apolar face making hydrophobic contacts with several residues of the b-sheet The shorter a4-helix is packed against one edge of the b-sheet and terminates in a sharp turn, after which the unstructured C-terminal tail begins It was annotated as the a4-helix to avoid confusion with the a2-helix found in archaeal proteins (see below and Fig 3) A reverse turn, comprising residues 64–69, follows on from the b2-strand, and is found in most FKBP structures, including Homo sapiens FKPB (HsFKBP12) and MtFKBP17 (Figs and 3) This is stabilized by hydrophobic interactions with the a1-helix, the b2-strand and the b3-strand, but it also establishes a few contacts with the interconnecting segments The structure of the PPIase module of SlyD closely resembles the structure of the PPIase domain of FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS 4533 Structure and interactions of SlyD L Martino et al FKBPs, as expected from primary sequence analysis; however, there are interesting differences One of the closest structural homologues is the PPIase domain of HsFKBP12 [34–36], although SlyD lacks the N-terminal b1-strand that lies antiparallel to the b2-strand in the human protein (Fig 3) The b1-strand is also missing in MtFKBP17 [32], and in both proteins the short structured N-terminal segment makes hydrophobic contacts with the a1-helix and the b-sheet The b5-strand of SlyD is split into two halves, namely b5a and b5b, separated by a five-residue bulge bearing a close resemblance to the structure of HsFKBP12 Conversely, the helix insertion observed in MtFKBP17 (a2-helix; Fig 3) appears to be confined to the archaeal kingdom, and is not conserved in bacterial SlyD One of the most interesting features of the SlyD structure is the presence of a novel helical extension to the PPIase fold, termed the a4-helix This elaboration of the PPIase domain structure is thus far unique to SlyD; it spans residues 144–149, and is almost entirely missing in the truncated version (SlyD1–146) This helix connects to the rest of the domain through a well-defined segment extending from the b3-strand, and is positioned at the convex side of the b-sheet near to the ends of the b4-strand and b5b-strand Residues in the a4-helix establish a network of contacts with Asp6, His38, Leu35, and Ala142, and undergo additional interactions with residues 151–153, which create a tight turn following the a4-helix Although longrange NOE contacts could also be assigned between Gly150, His151, Val152 and His153 in this turn and Leu35 and Ala142 in the core domain, the dynamic backbone analysis indicates that residues beyond Ala149 experience intrinsic mobility on the nanosecond to picosecond timescale The position of this turn in the structure as obtained from the structure calculation therefore has to be considered as one of the possible conformations This novel C-terminal extension of SlyD PPIase does not obscure the putative peptidyl-prolyl binding side; however, our results implicate it in the molecular switch triggered by nickel ions (see below) Furthermore, it appears to be conserved in all of the SlyD variants on the basis of primary sequence conservation (Fig 1) Structure of the IF domain The IF domain of SlyD displays a b6–a3–b9–b8–b7 topology, and folds to generate a four-stranded antiparallel b-sheet bordered by a short a-helix (a3-helix) (Figs and 3) This helix connects the b6-strand with 4534 a partially flexible loop leading to the b9-strand Phe84 and Val87 on the a3-helix engage in interactions with Val112, Ile109, Val117 and Phe96 on the b-sheet, generating a hydrophobicity-stabilizing cluster that is the core of the domain The IF domain of SlyD is aligned with the IF domain of the archaeal homologous MtFKBP17 [32] (Fig 3) The main difference between these two domains is the longer loop connecting the b9-strand and b8-strand in SlyD In our structure, several loops are not as well defined as in the archaeal protein – this is supported by relaxation analysis, although spectral overlap prevented us from obtaining a complete set of assignments and parameters for residues in this domain Inspection of the backbone relaxation, especially the {1H}-15N heteronuclear NOE values (Fig 2C), suggests a higher degree of intrinsic disorder for the entire IF domain than for the PPIase module This picture agrees with the recently reported observation that the IF domain in isolation was unable to adopt a stable fold in solution, and, when present in the intact SlyD protein, it was found to destabilize the PPIase domain [37] The structural flexibility and plasticity of the IF domain may constitute a necessary feature for an efficient chaperone-like activity Consistent with its chaperone-like role and in line with the archaeal counterpart, the SlyD IF domain exhibits a large exposed hydrophobic surface with potentially high affinity for unfolded or partially folded proteins (Fig S2) The IF domain might therefore perform a double activity: preventing aggregation of unfolded substrates, and orientating them to facilitate their insertion within the PPIase domain This is in agreement with the degree of relative flexibility observed in the structure Conformational changes involving bending motions of the hinge between the two domains might in fact modulate access to and from the PPIase active site The presence of the IF domain, which is unique in archaeal FKPBs and bacterial SlyD, coupled with the hinge-bending motion between the two domains, could enable the protein to sample the surrounding space for potential ligands and aid their interaction with the PPIase active site Comparison of PPIase domains and the FK506–rapamycin interaction The PPIase domain fold is highly conserved within the large family of FKBP and FKBP-like proteins, and it has also been found in parvulins, another group of cis–trans prolyl isomerases [38,39] The conserved moiety of this fold appears to constitute the minimum structural frame for PPIase activity, and includes the FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS L Martino et al b4-strand, b5-strand, b2-strand and b3-strand, and the loop–helix–loop that forms the a1-helix [32,40,41] The active site of the extensively studied HsFKBP12 has been mapped to a hydrophobic cavity delineated by the concave surface of the b-sheet, the a1-helix, and several loops [36] The immunosuppressive agents FK506 and rapamycin, which act as potent inhibitors of the PPIase activity of the FKBPs, have been shown to be lodged within the active site crevice of HsFKBP12, cushioned by Tyr26, Phe36, Asp37, Phe46, Phe48, Val55, Ile56, Trp59, Tyr82, Ile90, Ile91, and Phe99 [36] The corresponding binding pocket in SlyD is structurally similar (Fig 4), with the hydrophobic residues Tyr13, Val23, Asp24, Leu32, Tyr34, Leu41, Ile42, Leu45, Tyr68 and Phe132 in analogous positions, respectively, to those of the residues in the conserved side chains in the human protein (Fig 4) The main difference lies in the position of Tyr82 ⁄ 68, located in the reverse turn following the b2-strand, also observed by Weininger et al [31] Furthermore, Ile90 and Ile91, which reside in the loop between the b2-strand and b3-strand of HsFKBP12, not have direct equivalents in SlyD Nonetheless, because of the relative mobility of the PPIase and IF domains, hydrophobic residues, such as Met124 and Leu125 in the interconnecting segments, might be able to relocate in the close vicinity of the crevice and undergo transient interactions with the ligand Also, importantly, the additional a4-helix of SlyD does not affect the shape of the cavity or obscure its entrance, as underscored by comparable values of solvent-accessible surface areas for the binding pockets of HsFKBP12 (Protein Data Bank ID: 1FKF) and SlyD (460 ± 20 and ˚ 490 ± 80 A, respectively) Notably, the exact mechanism of the PPIase catalytic process uncertain, as is the role of the conserved hydrophobic residues within the common domain fold [8,39,41] Unexpectedly, parvulins and a number of FKBP-like proteins, such as the trigger factor, not bind FK506, despite the high structural homology with genuine FKBPs, adding conviction to the view that the shape of the cavity as well as its charge distribution might determine substrate specificity [39,41] The issue of whether FK506 influences the PPIase activity of SlyD has been somewhat unclear in the past [9] Scholz et al [10] have recently demonstrated that FK506 is capable of inhibiting the refolding activity of SlyD, with an apparent binding affinity for the protein estimated to be about three orders of magnitude weaker than that for HsFKBP12 To characterize further the interaction between FK506 and SlyD and, most importantly, to assess which regions of the protein make contact with the ligand, we carried out a series of Structure and interactions of SlyD A turn B C Fig Structural alignment between the PPIase domains of SlyD and HsFKBP12 (A) Ribbon representation of the superimposed structures (HsFKBP12 in green and SlyD in yellow) The superimposition of the PPIase domains was performed using DALI (http:// ˚ ekhidna.biocenter.helsinki.fi/dali_server/) (Z 9.7, rmsd $ 1.8 A over 107 residues; identity 22%) (B, C) Magnification of the active site crevices of HsFKBP12 and SlyD, respectively (in grey) Selected key catalytic residues showing a direct correspondence between the two proteins are shown in stick representation and labelled H-15N HSQC NMR experiments, monitoring the backbone amide chemical shift changes in SlyD upon titration with FK506 This sensitive method allows the detection of amide chemical shift perturbations caused by direct binding or conformational changes induced by ligand interaction, and can therefore demarcate the regions directly affected by complex formation Upon addition of FK506, several SlyD resonances belonging FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS 4535 Structure and interactions of SlyD L Martino et al to the PPIase domain, on and around the active cleft, disappeared, whereas others on the IF domain experienced a chemical shift variation on the fast equilibrium timescale (Fig S3) Similar results were obtained with rapamycin (Fig S3) In both cases, the titration was terminated at a protein ⁄ drug ratio of : 1, as further additions of the largely water-insoluble agents caused the formation of a white precipitate, and no further change in the spectra was observed Although these data are not conclusive, they indicate that both FK506 and rapamycin interact weakly with SlyD Although it cannot be excluded that the canonical PPIase site is implicated in the interaction, our results show that the IF domain is clearly perturbed by the presence of the ligand, on and around the exposed hydrophobic patch Interaction of SlyD with nickel ions SlyD is a unique FKBP protein, as its PPIase activity is modulated by the presence of nickel ions [9] The nickel ions might therefore exert an important switchlike regulatory control over the different functions of SlyD, but the molecular basis of this attractive mechanism remains uncertain Because the PPIase activity of the truncated SlyD1–146 was unaffected by nickel ions, it was proposed that the C-terminal tail could be responsible for binding metal ions and for the resultant conformational change observed upon nickel ion binding [9]; however, this hypothesis leaves the question open as to how this structural effect would be sensed by the PPIase domain To achieve a deeper understanding of this regulatory mechanism, the interaction between SlyD and nickel ions was investigated using an array of biophysical techniques NMR titrations were employed to map the binding site of the nickel ion on SlyD, and around a : nickel ion ⁄ protein ratio, several signals of the protein disappeared in the 1H-15N HSQC spectra (Fig S4) Intriguingly, the resonances perturbed by the presence of nickel ions can all be mapped within the PPIase domain, involving mainly, but not exclusively, residues in the novel extension of the PPIase fold (Figs and S4) Moreover, a section of the PPIase core domain, at or near the catalytic pocket, was also affected A more detailed mapping analysis of the extent of the chemical shift variation upon nickel ion binding was impeded by the loss of the perturbed resonances, which could be attributable to either an intermediate equilibrium of the complex and ⁄ or the paramagnetic effect of the nickel ion (see below) Nonetheless, our results clearly indicate that the ability of SlyD to interact with nickel ions is not confined to 4536 the unstructured C-terminal tail, as previously suspected, but that the binding of at least one nickel ion per molecule occurs on the PPIase domain, probably affecting the conformation of the PPIase binding site (see below) These results therefore provide the first molecular explanation of the modulation of PPIase activity of SlyD by nickel ions These findings are not in conflict with previously reported data, indicating that the PPIase domain in isolation did not bind nickel ions, because the putative PPIase fragment used in this study terminated at residue 146, and so did not encompass the full domain and lacked the key C-terminal helical extension [9] The NMR titration was conducted up to a final nickel ion ⁄ SlyD ratio of : 1; nonetheless, further nickel ion additions beyond the : point caused only general line-broadening and protein precipitation The issue of the stoichiometry of this interaction, however, deserved further attention, as previous reports suggested : 1, : or even higher nickel ion ⁄ protein ratios [2,9,24] To address this key point and to further characterize such interaction events, we employed isothermal titration calorimetry (ITC) and CD techniques ITC is largely used to investigate binding reactions by measuring the heat generated or absorbed in the binding event and thereby providing the binding constant, the stoichiometry and the enthalpy change (DH°) of the interaction [42,43] For the nickel ion–SlyD interaction, carried out at 298 K and pH 7.25, the integrated heat data showed that the process of nickel ion binding to the protein is composed of one clear binding event (Fig 6) The binding isotherm corresponding to this reaction has been obtained using an independent-site model [43], revealing a stoichiometry of one nickel ion per protein, an association constant of 4.16 · 105 m)1, and enthalpic (DH°) and entropic (TDS°) contributions of )166 and )134 kJỈmol)1 respectively (Table 2) These negative values of enthalphy and entropy are typical of a thermodynamic process describing metal coordination by a protein molecule, with specific amino acid side chains adopting a rigid conformation around the metal ion [44,45] The binding event is enthalpically driven, suggesting that the formation of new interactions between the nickel ion and the protein is the key feature of the binding process Further evidence that SlyD interacts with nickel ions with a : stoichiometry is provided by the analysis of the changes in the far-UV CD spectra of the protein upon titration with nickel ions (Fig 6) Dramatic changes in the molar ellipticity of SlyD were in fact observed up to a : nickel ion ⁄ protein molar ratio, and further additions of the ligand caused only minor alterations in the CD spectra This behaviour is in FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS L Martino et al Structure and interactions of SlyD A B Fig Structure mapping of the chemical shift perturbations for the PPIase domain of SlyD upon nickel ion binding The positions of residues that disappear in the 15 H- N HSQC spectra upon complex formation are indicated in red on the protein secondary structure (A) and surface (B) Selected perturbed residues are labelled The novel a4-helix appears to be significantly involved in the interaction, and residues on the b5b-strand are suggested to undergo conformational rearrangements in the nickel ion-bound protein (see text) C 12 10 Power (µJ·s–1) [θ] 10–3 (deg·dmol–1·cm2) A –1 200 210 220 230 λ (nm) 240 –2 250 2500 5000 7500 Time (s) 10 000 D –0.8 –20 –0.9 –40 –60 KJ·mol–1 [θ]215 nm 10–3 (deg·dmol–1·cm2) B –1.0 –1.1 –1.2 –1.3 –80 –100 –120 –140 –160 [Ni2+]/[SlyD] 0.0 0.5 1.0 1.5 [Ni2+]/[SlyD] 2.0 2.5 Fig Analysis of SlyD–nickel ion interaction (A) Far-UV CD spectra of apo-SlyD (straight line) and SlyD in the presence of NiCl2 (dotted line) at a protein ⁄ nickel ion molar ratio of : The secondary structure content estimated by CD spectral analysis gave the following values: apo-SlyD: 5% a, 47% b, 22% turn, and 25% irregular; SlyD–nickel ion: 5% a, 38% b, 20% turn, and 36% irregular) (B) Plot of the molar ellipticity at 215 nm as function of the nickel ion ⁄ SlyD molar ratio Interpolation of the experimental data (filled squares) with an equation (dotted line) based on an independent binding sites model gives a stoichiometry of one nickel ion per protein molecule and a binding constant of · 105 M)1 (C) Raw titration data show the thermal effect of 10 lL injections of 400 lM NiCl2 solution into a colorimetric cell filled with 40 lM SlyD solution at pH 7.25; the heat effect reveals an exothermic effect during the interaction (D) Normalized heat of interaction: data were obtained by integrating the raw data and subtracting the heat of ligand dilution into the buffer The dashed line represents the best fit obtained by a nonlinear least squares procedure based on an independent binding sites model FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS 4537 Structure and interactions of SlyD L Martino et al Table Results of the interpolation analysis for the binding of SlyD to nickel ions determined using ITC and CD n DH° (kJỈmol)1) TDS° (kJỈmol)1) DG°298 K (kJỈmol)1) Kb, 1.0 ± 0.1 )166 ± 15 )134 ± 10 )32 ± (4.1 ± 0.8) · 105 agreement with the NMR and ITC results, indicating that a major binding event occurs with a : stoichiometry, accompanied by distinctive conformational rearrangements in the protein The far-UV CD spectrum of free SlyD, in the range 190–250 nm, shows a well-defined minimum at 215 nm and a shoulder centred at 230 nm Although the shapes of the curves are very similar overall, the molar ellipticity is appreciably less negative in the CD spectrum of the protein in a : complex with nickel ions than for the apo-protein, indicating a decrease in the secondary structure content in the protein upon nickel ion binding The hyperbolic curve obtained by plotting the intensity of the CD signal at 215 nm versus the nickel ion ⁄ protein concentration molar ratio (Fig 6) fits well with an equation describing a simple model of a : interaction (see Experimental procedures) Most importantly, the association constant derived in this analysis is in excellent agreement with the binding constant obtained by ITC (Table 2), indicating that both techniques are following the same process The conformational changes associated with the SlyD–nickel ion interaction could therefore explain the higher values obtained for the enthalpic and entropic contributions when compared to other protein–nickel ion systems studied [44,45], on the basis that the ITC phenomenon measured here is the result of both a molecular association event and a concurrent conformational rearrangement To examine further such a conformational change, deconvolution analysis of the far-UV CD spectra was performed using dichroweb (see Experimental procedures) For the apo-protein, the assessed a ⁄ b content is, overall, consistent with its solution structure (Fig 6), but a marked decrease in the b-strand content was estimated for the protein in the complex (without appreciable changes in the a-helical regions) This is particularly interesting, as it might suggest that nickel ion binding promotes the disruption of the b-sheet catalytic core of the PPIase domain; this agrees well with the results of the NMR titration experiments, where Leu32, Asp33, Tyr34, Leu35 and His36 on the b-sheet appeared to be perturbed by the metal ion interaction (Figs and S4) Discussion In this work, we have investigated the solution structure and molecular interactions of SlyD, a bacterial protein related to the FKBP family of prolyl 4538 ITC (M )1 ) Kb, CD (M )1 ) (2.0 ± 0.1) · 105 isomerases As for many members of the PPIase superfamily, an explicit function for SlyD in assisting with protein folding in vivo remains, as yet, uncertain [46] A number of the prolyl isomerases have been shown to be involved in many other cellular processes [47,48] and, likewise, SlyD has been identified as a key player in the [Ni–Fe]-hydrogenase biosynthetic pathway [22] SlyD consists of a long, unstructured C-terminal tail preceded by two independently folded modules, the PPIase domain and the IF domain, with isomerase and chaperone-like properties respectively (see above) In the final stages of this investigation, the solution structure of an N-terminal part of SlyD, SlyD(1–165), was also determined [31] A comparison of these reports shows that the structure of the individual domains is largely conserved within the context of the full-length protein, and that the C-terminal region beyond residue 157 is highly unstructured and independent of the rest of the molecule Given that the SlyD structure bears unmistakable similarities to that of MtFKBP17, it is conceivable that these two domains work synergistically in SlyD, in line with what has been suggested for the archaeal counterpart [32] This is corroborated by the finding that insertion of the IF domain of SlyD into HsFKBP12 considerably boosts the chaperone-like activity of the latter [17], and by the recent observation that the IF domain of SlyD is directly involved in the binding of unfolded proteins and peptides [31] The relative flexibility of the two domains revealed in the solution structure implies a degree of domain swivelling that might facilitate access to the PPIase catalytic pocket and thereby enhance the ability of SlyD to act as a folding catalyst Collectively, the observations from the NMR analyses and the existing literature point towards a stepwise mechanism of catalysis whereby the IF domain performs the initial docking of the peptide, perhaps ideally positioning it for insertion within the PPIase active site Notably, FK506 and rapamycin also appear to be transiently anchored to the IF domain of SlyD in our NMR chemical shift analysis, possibly mimicking the recognition process of target peptides, consistent with what has been suggested by Weininger et al [31] As expected by comparison with other FKBP proteins, key catalytic residues within the PPIase domain of SlyD also experience some perturbation in the NMR titrations upon ligand binding (see above) Nonethe- FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS L Martino et al less, we believe that our data are not conclusive, and whether SlyD is a genuine FK506-binding protein is yet to be established The PPIase domain of SlyD emerges as being distinctive and rather intriguing, as its catalytic activity is uniquely inhibited by the presence of nickel ions [9] The overall architecture of this PPIase domain shows a high degree of correspondence with other FKPB domains, despite low sequence identity Nonetheless, a significant difference stems from the occurrence of a novel helical extension to the PPIase module Previous structure predictions correctly anticipated the presence of an FKBP-like domain in SlyD, but did not detect the elaborations to the canonical fold revealed by our study, which add valuable information to the expanding databases of PPIase domains The novel a4-helix packs closely against one edge of the b-sheet in the FKBP domain, with no apparent detrimental effect on the shape and accessibility of the catalytic site Unexpectedly, our investigations have shown that this additional structural feature does confer on the PPIase domain the specific ability to bind to nickel ions, by a mechanism never revealed before The lack of structural information led to the suggestion that the putative unstructured C-terminal fragment, rich in histidines, cysteines, aspartates, and glutamates, was entirely responsible for the observed ability of SlyD to bind metal ions, on the assumption that its PPIase domain would terminate on or around residue 146 [9] The structural results combined with the biophysical characterization of the interaction with nickel ions presented here demonstrate unambiguously the existence of one clear binding event of micromolar affinity and : stoichiometry that occurs on the PPIase domain, in particular involving the novel structural features, thereby assigning a new function (metal ion binding) to this class of domains However, most importantly, our CD experiments have shown that nickel ion binding promotes loss and ⁄ or rearrangement of part of the b-sheet within the PPIase catalytic pocket; the NMR chemical shift analysis completes this picture by mapping such conformational changes to residues 32–36, the only b-strand stretch exhibiting chemical shift perturbation upon interaction Intriguingly, Leu32 and Tyr34 have been tentatively identified as key catalytic residues by comparison with HsFKBP12 (see above), prompting suggestions that this structural perturbation could be responsible for the reported loss of isomerase activity of SlyD in the presence of nickel ions As chemical shift mapping is unable to discriminate between residues directly in contact with the ligand and residues undergoing conformational changes upon binding, it is not straightforward at this stage to delin- Structure and interactions of SlyD eate the side chains in SlyD responsible for nickel ion coordination Potential candidates may include histidines (i.e His36, His38, His149, His151, and His153), glutamates (i.e Glu146) and ⁄ or aspartates (i.e Asp33) (Figs and S4) The detailed structural basis of the SlyD–nickel ion interaction therefore remains to be elucidated, and further studies are currently underway; nevertheless, this article provides the first molecular explanation of the observed modulation of the isomerase activity of SlyD by nickel ions, supporting the hypothesis of a switch mechanism for SlyD, possibly in response to environmental cues Our experiments could not detect further binding of SlyD to nickel ions beyond the : stoichiometry This, however, does not exclude the possibility that the C-terminal tail of SlyD beyond residue 156, rich in metal-binding residues, would also be capable of interacting with nickel ions In fact, our NMR titration experiments were hampered by line-broadening and precipitation past the : molar ratio and by the severe spectral overlap for amide protons beyond residue 156; CD experiments would be unable to detect binding events that not cause conformational changes; and ITC methodologies could be inadequate for systems with an association constant equal to or lower than 103, as the heat associated with such interactions would be negligible As a different stoichiometry was detected using different techniques that not rely on the same biophysical parameters [24], it is conceivable that the C-terminal stretch is also involved in nickel ion binding This would agree well with the observation that, in E coli, the polyhistidine stretchdeficient HypB is counterbalanced by the C-terminal extended SlyD, possibly to allow for extra nickel storage However, our experiments unambiguously show that the first nickel ion occupies the higher-affinity site on the PPIase domain, so any binding in the unstructured tail would be substantially (at least 100-fold) weaker than the first nickel binding and, contrary to previous suggestions, would not trigger conformational rearrangements in SlyD or the resultant modulation of its isomerase activity The physiological relevance of the nickel ion-binding site on the PPIase domain remains to be established SlyD has been shown to interact with a 77-mer N-terminal fragment of HypB, with a proline-rich stretch (encompassing residues 28–36) partaking in such recognition [23] Notably, this fragment is in the vicinity of the high-affinity nickel ion-binding motif in HypB [CXXCGC(2–7)] Whereas the PPIase activity of SlyD has been shown not to be required for hydrogenase assembly [24], it is unknown whether such activity may actually be detrimental to the process It may be FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS 4539 Structure and interactions of SlyD L Martino et al possible to envisage a mechanism in which the binding of nickel ions to SlyD would ensure that its isomerase activity is switched off, to prevent unwanted cis–trans isomerization of the prolines adjacent to the high-affinity metal-binding motif in HypB Another model may imply that the interaction between SlyD and HypB somehow weakens the nickel ion affinity of the latter; nickel ions thereby released by HypB may be readily sequestered by the PPIase site of SlyD Although coherent with the fact that SlyD appears to interact in the proximity of the nickel ion high-affinity motif of HypB, the exact mechanism of the SlyD–HypB interaction remains to be established, and this hypothesis awaits testing Little is known about the determinants of SlyD responsible for HypB interaction A role for the IF domain in establishing contacts with HypB is supported by the observation that the deletion mutant lacking stretch 107–111 does not retain its chaperone ability or its ability to crosslink HypB in vivo [23] The structure of SlyD maps fragment 107–111 to the b8-strand, arguing that such a deletion mutant would probably not retain its native conformation, and therefore substantiating the hypothesis that a fully functional IF domain is indeed essential for such recognition; moreover, the IF domain has been shown to be responsible for the binding of several unfolded protein and peptides substrates [31] Further experiments are needed to understand the molecular basis of the interaction between SlyD and HypB, and this could shed light on the role of the atypical nickel ion-binding PPIase domain of SlyD in the hydrogenase assembly In conclusion, the molecular view of the SlyD–nickel ion binding mode presented here provides a comprehensive structural context for the interpretation of in vitro and cellular binding studies Together, the structure of SlyD and its interactions suggest a plausible mechanism for a molecular switch between its different functions, and lay the groundwork for further experimentation Experimental procedures Plasmid construction and protein expression Full-length SlyD and the truncated SlyD1–146 mutant were subcloned from E coli strain BL21 chromosomal DNA into a pQE60 vector (Qiagen, Hilden, Germany) Each recombinant protein was cloned with and without a C-terminal hexahistidine tag, and all of the proteins were expressed in an M15 (pREP4) E coli strain (Qiagen) The untagged fulllength SlyD (SlyD) was used for the structural and biophysical studies reported here For NMR, cells were grown on minimal media enriched with 0.8 gỈL)1 [15N]ammonium 4540 chloride and gỈL)1 [13C]glucose, at 37 °C, until a D600 nm of 0.6 was reached, and then induced with mm isopropyl thiob-d-galactoside Cells were harvested h after induction, resuspended in 20 mm Tris ⁄ HCl, 100 mm KCl and 10 mm imidazole at pH 8, and lysed by sonication After centrifugation at 39 700 g for 40 min, the soluble fraction was purified by affinity chromatography on an Ni2+–nitrilotriacetic acid resin (Qiagen), following the manufacturer’s protocol The eluted protein was dialysed in 20 mm Tris ⁄ HCl, 100 mm KCl and mm dithiothreitol at pH 7.25, and loaded on a mL Hi-trap DEAE column (GE Healthcare, Uppsala, Sweden) The protein was eluted with a linear 0–2.0 m KCl gradient in buffer A [50 mm Tris ⁄ HCl, 0.1 mm EDTA, and 10% (v ⁄ v) glycerol, pH 7.25], and dialysed in 20 mm Tris ⁄ HCl, 100 mm KCl, and mm dithiothreitol (pH 7.25) (NMR buffer) NMR spectroscopy For NMR studies, pure SlyD was concentrated to 0.8 mm in a volume of 700 lL NMR spectra were recorded at 298 K on Varian Inova spectrometers operating at 14.1 and 18.8 T, and on Bruker Avance spectrometers at 14.1 and 16.4 T, equipped with triple resonance cryoprobes The 1H, 15 N and 13C resonance assignments for SlyD will be reported elsewhere All NMR data were processed using nmrpipe ⁄ nmrdraw [49] and analysed using xeasy [50] Distance restraints were obtained from 15N-edited and 13Cedited NOESY experiments; backbone dihedral angles were determined using talos software [51] Hydrogen-bonded amide protons were detected by performing a series of H-15N HSQC experiments up to 10 h after the protein was buffer-exchanged in D2O T1, T2 and {1H}-15N NOE experiments were performed using the pulse sequences adapted from standard schemes, and analysed using nmrpipe 1DNH residual dipolar couplings were measured at 298 K in a liquid crystalline phase composed of $ 5% alkyl-poly(ethylene glycol) C8E5 ⁄ n-octanol in NMR buffer [52] Precise measurements of 1JNH splittings were obtained from 1JNH-modulated 2D spectra [53] For NMR titration experiments FK506 (Sigma) and rapamycin (Caltag-MedSystems) were dissolved in 96% ethanol at a concentration of 22 mm and added in increasing amounts to 600 lL of 0.5 mm 15N-labelled protein in NMR buffer 1H-15N HSQC spectra were recorded at ligand ⁄ protein molar ratios of 0.33 : 1, 0.66 : 1, 0.99 : 1, 1.22 : and 1.55 : to follow the resonances perturbed by ligand binding to the protein The backbone amide assignments of SlyD were transferred in the complex to the resonances that would have the smallest DdAV The weighted average of 15 N and 1HN chemical shift variation was calculated as follows: DdAV ẳ f0:5ẵDd1 HN ị2 ỵ 0:2Dd15 Nị2 g1=2 Titrations were repeated with 96% ethanol alone to minimize the risk of buffer interference For the titration with nickel ions, a solution of 45 mm NiCl2 in NMR buffer was added to the 15N-labelled FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS L Martino et al protein, dissolved in the same buffer, at 0.6 : 1, 1.2 : 1, 1.8 : 1, 2.4 : and 3.0 : nickel ion ⁄ protein molar ratios Structure calculation The solution structure of SlyD was calculated using a combined torsion angle and Cartesian coordinates dynamics protocol executed in xplor [54] The structures were calculated from random starting coordinates on the basis of 1202 NOE distance restraints, including 728 shortrange connectivities (residue i to residue I + j, where < j £ 4), 474 long-range connectivities (residue i to residue I + j, where j > 4), 230 dihedral angle restraints, and 23 hydrogen bond distance restraints NOEs observed at 100 ms were classified as strong, medium or weak ˚ (< 2.8, 3.8, and 5.5 A, respectively) on the basis of peak intensities calibrated internally using known distances The structures were analysed using molmol [55], pymol [56], and procheck-nmr [57] Structures were displayed using molmol and pymol The final family comprises the 20 structures of the lowest total energy from a total of 100 calculated structures; structure statistics are shown in Table ITC experiments The nickel ion solutions were prepared by diluting concentrated stocks of metal ions (100 mm) in a 20 mm Tris ⁄ HCl buffer (pH 7.25) Metal ion titrations were performed at 298 K using a high-sensitivity CSC-4200 ITC microcalorimeter from Calorimetry Science (Lindon, UT, USA) Before each ITC experiment, the pH of each solution was checked, the reference cell was filled with deionized water and the protein solution was degassed for 2–5 to eliminate air bubbles Care was taken to start the first addition after baseline stability had been achieved Measurement from the first injection was discarded from the analysis of the integrated data, in order to avoid artefacts due to the diffusion through the injection port occurring during the long equilibration period, locally affecting the protein concentration near the syringe needle tip In each titration, 10 lL of a solution containing 200–600 lm NiCl2 was injected into a solution of SlyD (30–40 lm) in the same buffer, using a computer-controlled 250 lL microsyringe In order to allow the system to reach equilibrium, a spacing of 400 s was applied between each ligand injection Heat produced by nickel ion dilution was verified to be negligible by performing a control titration of NiCl2 into the buffer alone, under the same conditions Integrated heat data obtained for the titrations were fitted using a nonlinear least-squares minimization algorithm to a theoretical titration curve, using the software bindwork DH° (reaction enthalpy change in kJỈmol)1), Kb (binding constant in m)1) and n (number of binding sites) were the fitting parameters The Structure and interactions of SlyD reaction entropy was calculated using the relationships DG° = )RT lnKb (R = 8.314 JỈmol)1ỈK)1, T = 298 K), and DG° = DH°)TDS° The measurements were performed at two different final Ni(II) ⁄ protein molar ratios: in a first set of experiments, a final molar ratio of : was reached, whereas in the second set, the measurements were repeated up to a : molar ratio to obtain a better characterization of the binding event centred on a stoichiometry of : CD CD spectra were recorded with a Jasco J-715 spectropolarimeter equipped with a Peltier-type temperature control system (model PTC-348WI), and calibrated with an aqueous solution of 0.06% d-10-(1)-camphorsulfonic acid at 290 nm Experiments were conducted in the same buffer (20 mm Tris ⁄ HCl, 100 mm KCl, pH 7.25) and at same temperature (298 K) used for the ITC measurements The molar ellipticity per mean residue, [h] (degặcm2ặdmol)1), is calculated from the equation ẵh ẳ ẵhobs ðmÞ=10 l C, where [h]obs is the ellipticity (deg), m is the mean residue molecular weight (124 Da), C is the protein concentration (gỈmL)1), and l is the optical path length of the cell (cm) Cells with 0.1 cm path length and protein concentrations of about 0.2 mgỈmL)1 were used to record CD spectra between 205 and 250 nm, and cells with 0.01 cm path length and a protein concentration of mgỈmL)1 were used to measure CD spectra between 190 and 250 nm A time constant of 16 s, a nm bandwidth and a scan rate of nmỈmin)1 were used to acquire the data The spectra were signal-averaged over at least five scans, and baseline corrected by subtracting a buffer spectrum To estimate the secondary structure content, curve fitting was performed using dichroweb [58] The association between the protein and the nickel ions was analysed by following the changes in molar ellipticity at 215 nm ½hŠ215 upon nickel ion addition to a solution containing the protein The binding constant (Kb) was determined using the simplest binding model, assuming that one molecule of ligand binds to one molecule of protein The molar ellipticity at 215 nm could therefore be defined as the sum of the contributions of free and bound protein: ẵh215 ẳ ẵP ẵhP ỵ ẵPL ẵhPL 1ị where [P] and [PL] represent the concentration of the free and bound protein, respectively, and [h]P and [h]PL are the corresponding molar ellipticity signals at 215 nm The concentration of the bound protein could be written as the difference between the total concentration of ligand in solution [L]tot and the fraction of unbound ligand [L]: FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS ẵPL ẳ ẵLtot ẵL 2ị 4541 Structure and interactions of SlyD L Martino et al The constant of binding of the reaction Kb is defined as: Kb ẳ ẵPL ẵP ẵL 3ị The total concentrations of the protein [P]tot and the nickel ions [L]tot per each CD measurement is given by: ẵPtot ẳ ẵP ỵ Kb ẵLị 4ị ẵLtot ẳ ẵL ỵ Kb ẵPị 5ị If Eqn (4) is combined with Eqn (5), a quadratic expression is obtained that can be solved to express the concentration of the free ligand, [L], in terms of the total concentration of ligand and protein This can be substituted into Eqn (2) to give an analytical expression for [PL]: ẵPL ẳ ẵLtot Kb 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Brunger AT (1993) X-PLOR Manual, Version 3.1 Yale ă University, New Haven, CT 55 Koradi R, Billeter M & Wuthrich K (1996) MOLMOL: a program for display and analysis of macromolecular structures J Mol Graph 14, 51–55, 29–32 56 Merritt EA & Bacon DJ (1997) Raster3D: photorealistic molecular graphics Methods Enzymol 277, 505–524 57 Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R & Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR J Biomol NMR 8, 477–486 58 Whitmore L & Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data Nucleic Acids Res 32, W668–W673 Supporting information The following supplementary material is available: Fig S1 Structural traces of full-length SlyD Fig S2 Electrostatic surface potential for SlyD Fig S3 Interactions of SlyD with FK506 and rapamycin Fig S4 NMR titration experiments of SlyD with Ni(II) and table of chemical shift perturbations of SlyD upon interaction with Ni(II) This supplementary material can be found in the online article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 4529–4544 ª 2009 The Authors Journal compilation ª 2009 FEBS ... around the exposed hydrophobic patch Interaction of SlyD with nickel ions SlyD is a unique FKBP protein, as its PPIase activity is modulated by the presence of nickel ions [9] The nickel ions might... PPIase site of SlyD Although coherent with the fact that SlyD appears to interact in the proximity of the nickel ion high-affinity motif of HypB, the exact mechanism of the SlyD? ??HypB interaction. .. Structure and interactions of SlyD L Martino et al The constant of binding of the reaction Kb is defined as: Kb ẳ ẵPL ẵP ẵL 3ị The total concentrations of the protein [P]tot and the nickel ions [L]tot