It was then found to be identical to wondrous histidine-rich protein, independently iden-tified as a persistent contaminant of histidine-tagged Keywords FK506-binding protein FKBP; nickel
Trang 1nickel 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
1 Randall Division of Cell and Molecular Biophysics, King’s College London, London, UK
2 Department of Chemistry ‘P Corradini’, University of Naples ‘Federico II’, Italy
3 MRC Biomedical NMR Centre, National Institute for Medical Research, London, UK
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 iden-tified as a persistent contaminant of histidine-tagged
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 0 2078486435
Tel: +44 0 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
(Received 14 May 2009, revised 15 June
2009, accepted 17 June 2009)
doi:10.1111/j.1742-4658.2009.07159.x
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 aggre-gation 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 isomer-ase activity of SlyD is uniquely modulated by nickel ions, which possibly regulate its functions in response to external stimuli In this work, we inves-tigated the solution structure of SlyD and its interaction with nickel ions, enabling us to gain insights into the molecular mechanism of this regula-tion We have revealed that the PPIase module of SlyD contains an addi-tional 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
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.
Trang 2recombinant 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
approxi-mately 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,
how-ever, 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
Met-hanococcus 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
chaper-one 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 hydroge-nase biosynthesis [24]
Further investigations have uncovered a key interac-tion between SlyD and the hydrogenase accessory factor HypB [23] E coli HypB contains two metal-binding 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 matu-ration [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 there-fore circumvent this deficit, by recruiting extra metal-binding capacity to the system In support of this hypothesis, whereas the PPIase domain of SlyD is required to interact with an N-terminal proline-con-taining 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, incor-porating 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 1 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 differ-ent species are as follows: SlyD_ECOL,
E coli; SlyD_HAEIN, Haemophilus influen-zae; SlyD_AERHY, Aeromonas hydrophila; SlyD_TREPA, Trepomena pallidum; SlyD_ HELPY, Helicobacter pylori; SlyD_HELPJ,
He pylori J99; MtFKBP17, M thermolitho-trophicus; HsFKBP12, H sapiens.
Trang 3In this study, we have investigated the solution
structure of E coli full-length SlyD, and found an
atypical PPIase domain containing an additional
C-ter-minal 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
ele-ment has not been established in this work [31]
Intriguingly, our investigations reveal that this
C-ter-minal helix, unprecedented for the FKBP family of
proteins, is involved in nickel ion binding, causing
con-formational 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,
prelimin-ary NMR analysis was applied to full-length SlyD
(encompassing residues 1–196) and a truncated
N-ter-minal fragment, homologous to other FKBP proteins,
spanning residues 1–146 (SlyD1–146) Comparison of
1H-15N HSQC spectra of the two molecules (data not
shown) indicated that, although many resonances were
directly superimposable, a number of well-resolved
sig-nals appeared to be shifted in the context of the
dele-tion 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
multidimen-sional 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
resi-dues 1–69 and 129–154, and the insert fragment,
com-prising residues 76–120, constitutes the IF domain
(Fig 2) The partitioning of the polypeptide chain
cre-ates a pair of antiparallel strands at the base of the
cleft linking the two domains These connecting
seg-ments span residues 70–75 and 121–128, respectively,
and act as a flexible hinge for a bending motion
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 con-necting segments, they do not tumble fully indepen-dently 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 analy-sis 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 cou-plings 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 sepa-rately to calculate the rmsd A final family of 20 super-imposed 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 second-ary structure regions, respectively The structure calcu-lation statistics are given in Table 1, and a representative structure is reported in Fig 3 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 full-length SlyD and SlyD1–146 positively identified the
Trang 4resonances 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 2 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
A
B
C
Fig 2 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 resi-dues 75–121) and (B) for the PPIase domain (resiresi-dues 1–70 and 127–152) The N-termini and C-termini and secondary structure ele-ments 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 { 1 H}- 15 N NOE values for SlyD measured at 18.8 T and 298 K.
Table 1 Summary of structural statistics for SlyD.
Total distance restraints (inter-residue) Short–medium range
(residue i to I + j, j = 1–4)
728 Long range (residue i to I + j, j > 4) 474
Total dihedral angle restraints 230
Restraint violations Distance restraint violation > 0.2 A˚ None Dihedral restraint violation > 5 None Average rmsd (A ˚ ) among the 20 refined structures
129–149
IF 76–121 Backbone of structured regionsa 0.749 0.828 Heavy atoms of structured regions 1.481 1.674
Ramachandran statistics of 20 structures Percentage residues in
Additional allowed regions 8.9 Generously allowed regions 2.2
a Residues selected on the basis of 15 N backbone dynamics PPI-ase domain: 1–38, 41–68, and 129–148; IF domain: 76–83, 90–96, and 105–120.
Trang 5one 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 2 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
Fig 3 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.
Trang 6FKBPs, 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-termi-nal b1-strand that lies antiparallel to the b2-strand in
the human protein (Fig 3) The b1-strand is also
miss-ing 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
bac-terial 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
addi-tional interactions with residues 151–153, which create
a tight turn following the a4-helix Although
long-range 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)
Further-more, 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
anti-parallel b-sheet bordered by a short a-helix (a3-helix)
(Figs 2 and 3) This helix connects the b6-strand with
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, gen-erating 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, espe-cially 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 observa-tion that the IF domain in isolaobserva-tion 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 plas-ticity 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 there-fore perform a double activity: preventing aggregation
of unfolded substrates, and orientating them to facili-tate 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 bacte-rial 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 moi-ety of this fold appears to constitute the minimum structural frame for PPIase activity, and includes the
Trang 7b4-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
hydro-phobic 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, do not have
direct equivalents in SlyD Nonetheless, because of the
relative mobility of the PPIase and IF domains,
hydro-phobic 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
cata-lytic 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, do 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
esti-mated to be about three orders of magnitude weaker
than that for HsFKBP12 To characterize further the
interaction between FK506 and SlyD and, most
impor-tantly, to assess which regions of the protein make
contact with the ligand, we carried out a series of
1H-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
A
B
C
turn
Fig 4 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 superimpo-sition 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.
Trang 8to the PPIase domain, on and around the active cleft,
disappeared, whereas others on the IF domain
experi-enced 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 : 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
canoni-cal 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
switch-like regulatory control over the different functions of
SlyD, but the molecular basis of this attractive
mecha-nism 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
bind-ing [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
1 : 1 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 5 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
the unstructured C-terminal tail, as previously sus-pected, 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 encom-pass the full domain and lacked the key C-terminal helical extension [9] The NMR titration was con-ducted up to a final nickel ion⁄ SlyD ratio of 3 : 1; nonetheless, further nickel ion additions beyond the
1 : 1 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 : 1, 3 : 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 inves-tigate binding reactions by measuring the heat gener-ated 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· 105m)1, and enthal-pic (DH) and entroenthal-pic (TDS) contributions of )166 and )134 kJÆmol)1 respectively (Table 2) These nega-tive 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 1 : 1 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 1 : 1 nickel ion⁄ protein molar ratio, and further additions of the ligand caused only minor alterations in the CD spectra This behaviour is in
Trang 9A
B
Fig 5 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
1 H- 15 N HSQC spectra upon complex
forma-tion are indicated in red on the protein
sec-ondary structure (A) and surface (B).
Selected perturbed residues are labelled.
The novel a4-helix appears to be
signifi-cantly involved in the interaction, and
resi-dues on the b5b-strand are suggested to
undergo conformational rearrangements in
the nickel ion-bound protein (see text).
[Ni 2+ ]/[SlyD]
–20 –40 –60 –80 –100 –120 –140 –160 0.0 0.5 1.0 1.5 2.0 2.5
0
2 )
[Ni 2+ ]/[SlyD]
–0.8 –0.9 –1.0 –1.1 –1.2 –1.3
0 1 2 3 4 5 6
4 3 2 1 0 –1
2 )
λ (nm)
200 210 220 230 240 250
–2 0 2 4 6 8 10 12
0 2500 5000 7500
Time (s)
10 000
A
B
C
D
Fig 6 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 1 : 1 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 con-stant of 2 · 10 5
M )1 (C) Raw titration data show the thermal effect of 10 lL injections of 400 lMNiCl
2 solution into a colorimetric cell filled with 40 l M 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.
Trang 10agreement with the NMR and ITC results, indicating
that a major binding event occurs with a 1 : 1
stoichi-ometry, accompanied by distinctive conformational
rearrangements in the protein The far-UV CD
spec-trum of free SlyD, in the range 190–250 nm, shows a
well-defined minimum at 215 nm and a shoulder
cen-tred 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
1 : 1 complex with nickel ions than for the
apo-pro-tein, 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 1 : 1
interac-tion (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
interac-tion 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
mea-sured 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
proce-dures) 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
esti-mated for the protein in the complex (without
apprecia-ble 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 5 and S4)
Discussion
In this work, we have investigated the solution
structure and molecular interactions of SlyD, a
bacte-rial protein related to the FKBP family of prolyl
isomerases As for many members of the PPIase super-family, 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 struc-ture 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 resi-due 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 counter-part [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 flexi-bility 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-Table 2 Results of the interpolation analysis for the binding of SlyD to nickel ions determined using ITC and CD.
b, CD ( M )1)