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

M Gabriela Bowden,Alejandro P Heuck,Karthe Ponnuraj,Elena Kolosova,Damon Choe,Sivashankarappa Gurusiddappa,Sthanam V.L Narayana,Arthur E Johnson,Magnus Höök

doi:10.1074/jbc.m706252200

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 beta-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 beta-strand complementation event.

Highlights

The blood plasma protein fibrinogen is targeted by many MSCRAMMs
SdrG-(273–597) Can Be Constrained into an Open or Closed Conformation—A key feature of the dock, lock and latch model of ligand binding is the transition of the MSCRAMM from an open apo-protein form to the closed form of the MSCRAMM peptide complex (Fig. 1B)
To explore if these proposed dynamics are critical for ligand binding, we decided to construct versions of the MSCRAMM that are constrained in one or the other conformation and test their ability to participate in the postulated conformational rearrangements

Summary

EXPERIMENTAL PROCEDURES

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). Four samples were prepared in parallel for each energy transfer experiment: microcells D (donor containing, 300 nM SdrG unlabeled) and DA (containing donor and acceptor, 300 nM SdrG labeled) received 3 ␮M (final concentration) of ligand peptide, whereas microcells B (blank) and P (3 ␮M peptide) contained HBS-EDTA buffer alone (B) or buffer plus peptide (P). For the peptide titration experiments, 500 nM SdrG was mixed with increasing concentrations of peptide (0 –10 ␮M), and the fluorescent emission of Trp (at 348 nm) and IAEDANS (at 515) is given by F ϭ Fcorr Ϫ Fpcorr, where Fscorr ϭ (Fs (V/V0) Ϫ Fb (V/V0)) and Fpcorr ϭ (Fp (V/V0) Ϫ Fb (V/V0)); V ϭ volume at the point of titration, V0 ϭ initial volume, F ϭ fluorescence intensity sample, Fp ϭ fluorescence intensity of the peptide, Fb ϭ fluorescence intensity of the buffer, and F ϭ fluorescence of the sample minus the contribution of the peptide in the buffered solution

RESULTS

Synthetic oligonucleotides used in this study

No of water molecules

DISCUSSION