Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation

Nadia N Casillas-Ituarte,Carlos H.B Cruz,Roberto D Lins,Alex C Dibartola,Jessica Howard,Xiaowen Liang,Magnus Höök,Isabelle F.T Viana,M Roxana Sierra-Hernández,Steven K Lower

doi:10.1074/jbc.m117.786012

Abstract

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2-0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections.

Highlights

We focused on the portion of Fn-binding repeats (FnBRs)-9 that overlaps with the binding site in Fn, two F1 domains, 3F1 and 2F1 (Fig. 1B)
Two residues within this region, corresponding to amino acids 782 and 786 in fibronectin-binding protein A (FnBPA) in S. aureus 8325, were changed to match the nonsynonymous single-nucleotide polymorphisms (SNPs) associated with endovascular disease in humans (Table 1)
Metadynamics simulations were performed where the peptide and F1 modules were progressively decoupled by applying a bias that increases their relative distance but does not impose a predetermined pathway

Summary

To whom correspondence may be addressed

Strate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. This microorganism is an opportunistic pathogen and can cause serious endovascular infections if bacteria access the bloodstream [4, 5] These kinds of infections have been linked, at least in part, to interactions between host fibronectin (Fn) and Fn-binding protein (e.g. FnBPA encoded by fnbA) anchored to the outer cell wall of S. aureus (6 –9). Amino acid substitutions H782Q and K786N sig-

The abbreviations used are

Results

Discussion

Experimental procedures