Cooperative Binding and Activation of Fibronectin by a Bacterial Surface Protein

Abstract

Integrin-dependent cell invasion of some pathogenic bacteria is mediated by surface proteins targeting the extracellular matrix protein fibronectin (FN). Although the structural basis for bacterial FN recognition is well understood, it has been unclear why proteins such as streptococcal SfbI contain several FN-binding sites. We used microcalorimetry to reveal cooperative binding of FN fragments to arrays of binding sites in SfbI. In combination with thermodynamic analyses, functional cellbased assays show that SfbI induces conformational changes in the N-terminal 100-kDa region of FN (FN100kDa), most likely by competition with intramolecular interactions defining an inactive state of FN100kDa. This study provides insights into how long range conformational changes resulting in FN activation may be triggered by bacterial pathogens.Invasion of nonprofessional phagocytes is a common virulence mechanism among bacterial pathogens. It involves the subversion of cellular uptake mechanisms, cell signaling and cytoskeletal dynamics (1, 2). For Staphylococcus aureus and Streptococcus pyogenes, adhesion to, and internalization by, epithelial and endothelial cells is mediated by cell wall-attached fibronectin-binding proteins (FnBPs) 4 FnBPA and SfbI, respectively. FnBPs recruit fibronectin (FN) to the bacterial surface where it forms a molecular bridge between bacteria and host cell integrins (3, 4).FN occurs ubiquitously in vertebrate tissues either in its soluble form or as an insoluble component of fibrillar matrix networks. FN is a disulfide-linked homodimer consisting almost entirely of three types of domains or modules, FNI, FNII, and FNIII (Fig. 1A). FN interacts with other extracellular components and different integrin subfamilies, most notably ␣5␤1, ␣v␤3, and ␣4␤1 (5). The major integrin-binding site in FN, the Arg-Gly-Asp (RGD) motif, is located in the 10th FNIII module ( 10 FNIII). RGD may be cryptic in plasma FN and only exposed upon fibrillogenesis (6 -8). Understanding of FN activation is hampered by the lack of information on its in-solution conformation. Important progress has been made with the discovery of long range interactions between FNI modules and module 3 FNIII in an N-terminal 100-kDa FN fragment (FN100kDa) (Fig. 1A) (9). Although FN100kDa lacks the RGD motif, it is a soluble construct containing cryptic sites that are also observed in full-length FN. Neither FN nor FN100kDa stimulate fibroblast migration into collagen gels (9, 10), which depends on motogenic Ile-Gly-Asp (IGD) motifs (located on 7 FNI and 9 FNI) and ␣v␤3 integrins (11). In contrast, a truncated oncofetal FN isoform known as migrationstimulating factor and the very similar proteolytic N-terminal 70-kDa FN fragment (FN70kDa) (Fig. 1A) are efficient triggers of cell migration (10, 12). The IGD sites appear to be partially exposed in an FN100kDa mutant (FN100kDa-R222A). FN100kDa-R222A lacks a salt bridge between 4 FNI and 3 FNIII that stabilizes a closed (inactive) FN100kDa conformation and is proposed to be broken upon FN activati...

BsaB, a Novel Adherence Factor of Group B Streptococcus

“…4). Fibronectin is a large glycoprotein constituent of the ECM and blood plasma that has been shown to be a binding substrate for a variety of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes (41,(45)(46)(47)(48)(49)(50)(51)(52)(53)(54). At least one of two genes coding for closely related fibronectin-binding proteins is found in almost all clinical isolates of S. aureus (55).…”

Ligation of the Fibrin-binding Domain by β-Strand Addition Is Sufficient for Expansion of Soluble Fibronectin

“…Polypeptides based on individual FNBRs of F1 adhesin (e.g. SfbI-2 or -4) bind to N- 9 FNI with Ͼ10-fold looser affinity than FUD (34). We therefore designed, expressed, and purified HADD, which contains SfbI-4 and the downstream region of SfbI-5 (Fig.…”

Plasma fibronectin stabilizes Borrelia burgdorferi –endothelial interactions under vascular shear stress by a catch-bond mechanism

“…Preservation of the inert, nonadhesive properties of this molecule is important in blood, where constitutive exposure of pFn ligand-binding sites would affect blood flow, thrombosis, and movement and adhesion of circulating cells (11,21). However, upon activation or force-induced stretching or conformational change induced by certain FnBPs, pFn undergoes a conformational change that exposes ligand-binding sites (11,12,26,27,35).…”