Evidence for the “Dock, Lock, and Latch” Ligand Binding Mechanism of the Staphylococcal Microbial Surface Component Recognizing Adhesive Matrix Molecules (MSCRAMM) SdrG

Abstract

Staphylococcus epidermidis is an opportunistic pathogen and a major cause of foreign body infections. The S. epidermidis fibrinogen (Fg)-binding adhesin SdrG is necessary and sufficient for the attachment of this pathogen to Fg-coated materials. Based largely on structural analyses of the ligand binding domain of SdrG as an apo-protein and in complex with a Fg-like peptide, we proposed that SdrG follows a “dock, lock, and latch” mechanism to bind to Fg. This binding mechanism involves the docking of the ligand in a pocket formed between two SdrG subdomains followed by the movement of a C-terminal extension of one subdomain to cover the ligand and to insert and complement a β-sheet in a neighboring subdomain. These proposed events result in a greatly stabilized closed conformation of the MSCRAMM-ligand complex. In this report, we describe a biochemical analysis of the proposed conformational changes that SdrG undergoes upon binding to its ligand. We have introduced disulfide bonds into SdrG to stabilize the open and closed forms of the apo-form of the MSCRAMM. We show that the stabilized closed form does not bind to the ligand and that binding can be restored in the presence of reducing agents such as dithiothreitol. We have also used Förster resonance energy transfer to dynamically show the conformational changes of SdrG upon binding to its ligand. Finally, we have used isothermic calorimetry to determine that hydrophobic interactions between the ligand and the protein are responsible for re-directing the C-terminal extension of the second subdomain required for triggering the β-strand complementation event. Staphylococcus epidermidis is an opportunistic pathogen and a major cause of foreign body infections. The S. epidermidis fibrinogen (Fg)-binding adhesin SdrG is necessary and sufficient for the attachment of this pathogen to Fg-coated materials. Based largely on structural analyses of the ligand binding domain of SdrG as an apo-protein and in complex with a Fg-like peptide, we proposed that SdrG follows a “dock, lock, and latch” mechanism to bind to Fg. This binding mechanism involves the docking of the ligand in a pocket formed between two SdrG subdomains followed by the movement of a C-terminal extension of one subdomain to cover the ligand and to insert and complement a β-sheet in a neighboring subdomain. These proposed events result in a greatly stabilized closed conformation of the MSCRAMM-ligand complex. In this report, we describe a biochemical analysis of the proposed conformational changes that SdrG undergoes upon binding to its ligand. We have introduced disulfide bonds into SdrG to stabilize the open and closed forms of the apo-form of the MSCRAMM. We show that the stabilized closed form does not bind to the ligand and that binding can be restored in the presence of reducing agents such as dithiothreitol. We have also used Förster resonance energy transfer to dynamically show the conformational changes of SdrG upon binding to its ligand. Finally, we have used isothermic calorimetry to determine that hydrophobic interactions between the ligand and the protein are responsible for re-directing the C-terminal extension of the second subdomain required for triggering the β-strand complementation event. The attachment of microbes to host tissues represents the initial step in the pathogenesis of most infections, with specificity and tissue tropism being defined by precise adhesin-ligand interactions. For bacteria that are not obligatory intracellular pathogens, extracellular matrix proteins appear to be preferred targets for bacterial adhesion. The adhesins mediating these interactions have been termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). 2The abbreviations used are:MSCRAMMsmicrobial surface components recognizing adhesive matrix moleculesMES4-morpholineethanesulfonic acidPIPES1,4-piperazinediethanesulfonic acidDTTdithiothreitolFRETFörster resonance energy transferFgfibrinogen Multiple MSCRAMMs have been found in many organisms. On Gram-positive bacteria, a family of MSCRAMMs might have arisen from a common ancestor, as indicated by their amino acid sequence relatedness, similar modular design, and common binding domain organization. In general, these structurally related MSCRAMMs (Fig. 1A) contain an N-terminal ∼40 amino acid long signal sequence (S), and C-terminal features that are required for sorting the proteins to the cell wall including a proline-rich wall-spanning region (W), the wall anchoring LPXTG motif, a hydrophobic transmembrane region (M), and a positively charged cytoplasmic tail (C). The ligand binding activity of most of these MSCRAMMs is localized to the N-terminal A regions, which in the staphylococcal proteins are ∼500 amino acids long. microbial surface components recognizing adhesive matrix molecules 4-morpholineethanesulfonic acid 1,4-piperazinediethanesulfonic acid dithiothreitol Förster resonance energy transfer fibrinogen The blood plasma protein fibrinogen is targeted by many MSCRAMMs. In the cases of Staphylococcus epidermidis and Staphylococcus aureus, fibrinogen-binding proteins that share a common structure have been reported to mediate bacterial attachment to fibrinogen-covered surfaces (1McDevitt D. Francois P. Vaudaux P. Foster T.J. Mol. Microbiol. 1995; 16: 895-907Crossref PubMed Scopus (141) Google Scholar, 2Deivanayagam C.C. Wann E.R. Chen W. Carson M. Rajashankar K.R. Höök M. Narayana S.V. EMBO J. 2002; 21: 6660-6672Crossref PubMed Scopus (140) Google Scholar, 3Wann E.R. Gurusiddappa S. Höök M. J. Biol. Chem. 2000; 275: 13863-13871Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). SdrG from S. epidermidis is necessary and sufficient for the attachment of this bacterium to surfaces coated with fibrinogen (4Hartford O.M. O'Brien L. Schofield K. Wells J. Foster T.J. Microbiology. 2001; 147: 2545-2552Crossref PubMed Scopus (108) Google Scholar). Furthermore, SdrG is expressed on the surface of S. epidermidis during infection, as indicated by an increase of anti-SdrG titers in sera from patients infected with S. epidermidis (5McCrea K.W. Hartford O. Davis S. Eidhin D.N. Lina G. Speziale P. Höök M. Microbiology. 2000; 146: 1535-1546Crossref PubMed Scopus (133) Google Scholar). SdrG binds to a site at the N terminus of the fibrinogen β-chain that covers the thrombin cleavage site and the MSCRAMM has anticoagulant properties (6Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Hook M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). ClfA appears to be the major fibrinogen binding MSCRAMM on S. aureus; this adhesin has been shown to act as a virulence factor in animal models of staphylococcal-induced endocarditis (7Que Y.-A. Haefliger J.-A. Piroth L. Francois P. Widmer E. Entenza J.M. Sinha B. Herrmann M. Francioli P. Vaudaux P. Moreillon P. J. Exp. Med. 2005; 201: 1627-1635Crossref PubMed Scopus (243) Google Scholar), septicemia, and arthritis (8Josefsson E. Hartford O. O'Brien L. Patti J.M. Foster T. J. Infect. Dis. 2001; 184: 1572-1580Crossref PubMed Scopus (243) Google Scholar, 9Palmqvist N. Foster T. Fitzgerald J.R. Josefsson E. Tarkowski A. J. Infect. Dis. 2005; 191: 791-798Crossref PubMed Scopus (49) Google Scholar). In addition to ClfA, the S. aureus MSCRAMMs ClfB and FnbpA also bind to fibrinogen (3Wann E.R. Gurusiddappa S. Höök M. J. Biol. Chem. 2000; 275: 13863-13871Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 10Ní Eidhin D. Perkins S. Francois P. Vaudaux P. Höök M. Foster T.J. Mol. Microbiol. 1998; 30: 245-257Crossref PubMed Scopus (321) Google Scholar). ClfA and FnbpA bind to similar sites located at the C terminus of the fibrinogen γ chain (3Wann E.R. Gurusiddappa S. Höök M. J. Biol. Chem. 2000; 275: 13863-13871Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), whereas ClfB has a more complex binding specificity (11Perkins S. Walsh E.J. Deivanayagam C.C. Narayana S.V. Foster T.J. Höök M. J. Biol. Chem. 2001; 276: 44721-44728Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The A-regions of the four fibrinogen-binding staphylococcal MSCRAMMs are very similar. Each A-region is predicted to contain three subdomains with IgG-like folds (12Ponnuraj K.B. Bowden M.G. Davis S. Gurusiddappa S. Moore D. Xu Y. Hook M. Narayana S.V. Cell. 2003; 115: 217-228Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Fibrinogen binding involves subdomains two and three. For SdrG, ClfA, and FnbpA, synthetic peptides have been described corresponding to the respective binding sites in fibrinogen; these peptides bind to the respective MSCRAMMs with relatively high affinity. We reported the crystal structures of the ligand binding region of SdrG as an apo-protein and in complex with a synthetic peptide ligand (12Ponnuraj K.B. Bowden M.G. Davis S. Gurusiddappa S. Moore D. Xu Y. Hook M. Narayana S.V. Cell. 2003; 115: 217-228Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) (Fig. 1, A and B). The apo-protein folds as an open structure where a ligand binding trench formed between two subdomains (N2 and N3) is accessible for the docking of the peptide. The protein-peptide complex structure has a closed conformation where the ligand binding trench is no longer exposed. Based largely on these structures we proposed a dynamic multistep ligand binding mechanism that we called “dock, lock and latch.” We envisioned that the ligand peptide would first dock in the binding trench exposed in the open conformation. The peptide-MSCRAMM interaction would then induce a redirection of the C-terminal extension of the second subdomain (N3). This segment covers the bound peptide, effectively locking the ligand in place. We proposed a final latching event, where a short segment C-terminal of the locking residues would insert through a β-strand complementation in a trench on the surface of the N2 subdomain (Fig. 1B). In the present study we have experimentally examined the dock, lock and latch model. Construction and Purification of SdrG Point Mutants—Recombinant SdrG-(273-597) point mutants were generated with hexahistidine tags at their N termini using the expression vector pQE30 (Qiagen). Each construct was amplified by PCR using SdrG-(273-597) DNA as the template and Pfu (Stratagene) as the polymerase. To obtain the desired point mutations, the primers listed in Table 1 were used. The PCR fragments were purified and cloned into pQE30 at the sites indicated for each primer in Table 1, and the resulting plasmids were transformed into XL1-Blue supercompetent cells (Stratagene). Large-scale expression and preparation of recombinant proteins using HiTrap nickel chelating chromatography were described previously (6Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Hook M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Protein concentrations were determined from the absorbance at 280 nm as measured on a Beckman Du-70 UV-visible spectrophotometer. The molar extinction coefficient of the proteins was calculated using the method of Pace et al. (13Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3452) Google Scholar). The structural integrity of each SdrG-(273-597) point mutant was verified using circular dichroism (14Sillanpaa J. Xu Y. Nallapareddy S.R. Murray B.E. Hook M. Microbiology. 2004; 150: 2069-2078Crossref PubMed Scopus (73) Google Scholar).TABLE 1Synthetic oligonucleotides used in this studyPrimerSequenceE273F5′-CCCGGATCCGAACAAGGTTCGAATGTTAATE381C in a and were by the method using and and between and The to the and to The were at using an The W. 276: PubMed Scopus Google Scholar) was used for The structure of the disulfide closed conformation of SdrG was by using J. Biol. 2001; PubMed Scopus Google Scholar), with P. J. M. T. Biol. Sci. 1998; PubMed Scopus Google Scholar), and the with The crystal structure of the complex the bound (12Ponnuraj K.B. Bowden M.G. Davis S. Gurusiddappa S. Moore D. Xu Y. Hook M. Narayana S.V. Cell. 2003; 115: 217-228Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) was used as the model. and of the peptides were described previously (6Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Hook M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). For the peptides the amino acid are and the sequence peptide peptide and a of peptide calorimetry were with a calorimetry at In a the cell and the peptide. were in and were at for The was as one of and of with an of The was and the between the was To into the of two were one peptide into and into the The of were from the were using and to a binding site model. was used to determine the for the interaction of (6Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Hook M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) with peptide The peptide was with and concentrations of in were with nm peptide for in the at The were to were with a using The binding were by used to a binding as defined by the where to the in is the in and is the of the A binding site was in this SdrG and were with dithiothreitol final and in 1 at The proteins were then through a to the The protein was and was then with a of in for 1 at The was to a final of and was by through a with The protein was the SdrG absorbance at 280 nm coefficient and the of the was determined the absorbance of the at nm were of of The was to bind to the in and this was by the to the of SdrG, which has no C residues in its amino acid resonance energy transfer was measured using an and in a were examined at in The and were and nm for and and nm for with were closed during to of the The and were determined as described P. L. PubMed Scopus Google Scholar). were in for each energy transfer nm SdrG and and nm SdrG of ligand peptide, whereas and peptide The of a was by the signal of the from and peptide from and then for the protein the addition of peptide. The is by where 1 and for as a and as an is of of and of the site by the The between residues was calculated using the a For the peptide nm SdrG was with concentrations of peptide and the of and is by where and at the point of initial of the peptide, of the and of the the of the peptide in the SdrG-(273-597) into an of the dock, lock and latch of ligand binding is the of the MSCRAMM from an open apo-protein form to the closed form of the MSCRAMM peptide complex (Fig. 1B). To these proposed are for ligand we to construct of the MSCRAMM that are in one the conformation and their to in the conformational The we for this was to through two residues that form a disulfide and lock the C-terminal extension of on of the the extension from in the lock and latch This disulfide would the in an open conformation. the in the C-terminal be with a introduced at the of the N2 locking the latch segment in the latching trench and a closed conformation. of the open and closed crystal structures to residues to be with and form disulfide and are predicted to form an open and a closed stabilized by their introduced disulfide bonds (Fig. Table The forms of SdrG were and of the two mutants and proteins by that the protein were with as a reducing the proteins as a with very similar (Fig. their In the of reducing the protein as a at the for the protein a major of and protein the protein This that the of disulfide bonds a form of the Furthermore, protein mutant contain these and These forms were to be SdrG in using anti-SdrG We purified the form of and were to The structure of was by using the structure of the of the complex as a model. The structure was at a with molecules in an of a cell is very similar to the previously reported structure of in complex with the ligand peptide, with the of the of the peptide and the presence of the which are shown to form a disulfide (Fig. we that a of the C-terminal extension of and of the segment into the latching trench that we proposed ligand can also in the of ligand residues and to form a disulfide and of molecules in the with of protein of in a The to the that the mutant proteins appear as a of and in resulting from disulfide indicated these forms can be by The forms can be to the proteins are with reducing agents such as (Fig. We the of these forms to bind to the proteins are a shown in the ligand in the and present in the mutants preparation bind whereas the not to the Fg. to the proteins the and disulfide the and to bind to To the of the to bind to was necessary to obtain a of To the from the we purified the of each mutant using this the preparation a of protein that has not formed the predicted disulfide as the protein in This was by the through a with where the proteins that were in the and the forms were were their to bind to and the peptide was The to of the SdrG to was measured in forms of SdrG not bind to of these are in an open closed conformation. the of the proteins to bind to was restored by the SdrG with To the that on a surface the of the SdrG to the we examined the of a peptide to bind to forms of where the protein and the ligand peptide are in the stabilized closed and open not bind to the peptide (Fig. The of these to bind to the ligand can be using and structural The open conformation binding similar to in a C-terminal where the ligand the binding is not On the the closed conformation does not bind the peptide the ligand binding trench is by the amino acids that the locking region of the peptide to the binding the proteins were with their to bind to the peptide was similar to that of the protein (Fig. These that the to from open to closed conformation of the MSCRAMM the of SdrG for the and that locking SdrG in conformation its to bind to Fg. upon the of the C terminus between the apo-protein and the protein-peptide complex in we used J. Full Text Full Text PDF PubMed Google Scholar). In this the C-terminal was to which can act as an energy SdrG does not contain was with a and its was with a these two are localized a short energy transfer from the to the in The structural integrity of the point mutants was by circular dichroism (14Sillanpaa J. Xu Y. Nallapareddy S.R. Murray B.E. Hook M. Microbiology. 2004; 150: 2069-2078Crossref PubMed Scopus (73) Google Scholar), the of the mutant proteins to of the whereas the binding of the proteins was in that the binding properties of the protein were not by the the properties of the and the were and the addition of the peptide. the at the of the latching trench the domain and the at the C terminus to in the presence of the ligand. These can be as changes in the of the C terminus to the N2 the present at the of the N2 latching trench is in of the at the C terminus we can energy transfer (Fig. In this the peptide is to a in from can be and at the an increase in is The ligand binding was calculated an of for this The movement of the C terminus can also be from the changes upon the addition of concentrations of ligand peptide (Fig. concentrations of peptide are to SdrG, the is and the These show that binds to SdrG with an of which is similar to the determined for the protein (Fig. a we show the of the protein in the and presence of the ligand (Fig. A in is also in the of peptide, to a of the Y. 1998; PubMed Scopus Google Scholar) by the C residues We a similar using a protein with a at located a of the N2 a region predicted not to with the C terminus addition of the ligand peptide, a in was (Fig. to was in the protein a in upon addition of the peptide (Fig. These that in SdrG undergoes the conformational changes predicted in the dock, lock and latch model. SdrG-(273-597) and the in the SdrG the dock, lock and latch we that upon peptide binding to the binding the C-terminal extension of the domain would the N2 the peptide, lock in the binding and insert in the latching trench in N2 (Fig. 1B). Based on the initial of the C-terminal was proposed to with the The extension then the binding bonds between the ligand peptide and the lock region These bonds hydrophobic interactions between and in the peptide and in the lock region of SdrG-(273-597) (Fig. To determine these hydrophobic interactions are in the initial of the C-terminal we calculated the binding of amino acid in the SdrG protein region using The show that with hydrophobic such as would the high energy interaction between the peptide and the protein that are necessary for peptide an such as at would the described interactions. We the of and to bind to fibrinogen in an and to the peptide using calorimetry (Fig. such as the changes in and were the not be from these were at the were with that binds to the ligand as SdrG, whereas binds to the peptide with a affinity. These that the hydrophobic interactions between the peptide and the lock residues the necessary energy to the extension and the MSCRAMM from an open conformation to a closed interactions between SdrG and amino acid the amino acid in SdrG, by in the amino acid residues are as the amino acid in SdrG, by in the amino acid residues are as the amino acid in SdrG, by in the amino acid residues are as the amino acid in SdrG, by in the amino acid residues are as in a The of the of SdrG for to that upon ligand the SdrG C terminus the N2 the ligand peptide and a β-sheet in This proposed SdrG would with a in the C-terminal This of movement would the region to be for the described To the proposed we and with a a to to We the of the and to bind to the peptide in a (Fig. that with a the of SdrG-(273-597) to bind to the peptide. This that the of the is necessary for the conformational events that in SdrG upon ligand In the two S. epidermidis has as an opportunistic pathogen with and in infections. The to for the of these has in of the increase in S. epidermidis is to the by which this and in the The interaction of SdrG with fibrinogen the attachment of S. epidermidis to which a step in the of SdrG is a for the of to S. epidermidis infections. the interaction between SdrG and its ligand the initial the of such The the dock, lock and latch using We show that locking SdrG into the open closed conformation the MSCRAMM binding to the that movement of the extension is required for ligand This that in SdrG in an between the open and a closed This dynamic was by the of a synthetic peptide to the to bind the latching trench not the peptide represents an of the that into the latching trench (Fig. we were to binding of the peptide to the protein in These that the sequence that represents the binds to the latching trench and that this interaction is to in the of the ligand. we that in the of the and are to with the N2 domain SdrG in an of between open and In (12Ponnuraj K.B. Bowden M.G. Davis S. Gurusiddappa S. Moore D. Xu Y. Hook M. Narayana S.V. Cell. 2003; 115: 217-228Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), we that the ligand peptide binds to SdrG, the protein into a closed conformation. we show SdrG changes dynamically from an open to a closed form upon ligand To the of events during this dynamic we the interactions of the peptide and the extension We show that the is necessary for such as the initial that the the ligand. The hydrophobic interactions between the peptide and the protein extension most a event, the peptide in place. The final the β-strand complementation of the N2 a step using the the of events proposed in the dock, lock and latch binding model. This binding the for the interaction of IgG-like MSCRAMMs with conformational changes the the described events to be in the of the MSCRAMMs to are proposed to be of IgG-like This is the also for the S. aureus MSCRAMM which binds by a of the dock, lock and latch mechanism Y. Xu Y. Höök A. Gurusiddappa S. Höök M. Narayana S.V. EMBO J. 2005; PubMed Scopus Google Scholar, K. Xu Y. J. Murray B.E. Narayana Höök M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The region of be from the cell surface by the of the C.C. Carson M. S. T. Hook M. Narayana S.V. 2000; Full Text Full Text PDF Scopus Google Scholar). is to that the in SdrG, which are predicted to in a E. D. Foster T.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), also in a and and from the bacterial cell in the of the region from the cell We the ligand is the ligand region folds effectively the ligand in and the structure of the complex (Fig.