In the present study we examine the thermodynamics of binding of two related pyrazine-derived ligands to the major urinary protein, MUP-I, using a combination of isothermal titration calorimetry (ITC), X-ray crystallography, and NMR backbone (15)N and methyl side-chain (2)H relaxation measurements. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than the classical entropy-driven hydrophobic effect. Unfavorable entropic contributions from the protein backbone and side-chain residues in the vicinity of the binding pocket are partially offset by favorable entropic contributions at adjacent positions, suggesting a "conformational relay" mechanism whereby increased rigidity of residues on ligand binding are accompanied by increased conformational freedom of side chains in adjacent positions. The principal driving force governing ligand affinity and specificity can be attributed to solvent-driven enthalpic effects from desolvation of the protein binding pocket.

Staphylococcus aureus can adhere to and invade endothelial cells by binding to the human protein fibronectin (Fn). FnBPA and FnBPB, cell wall-attached proteins from S. aureus, have multiple, intrinsically disordered, high-affinity binding repeats (FnBRs) for Fn. Here, 30 years after the first report of S. aureus/Fn interactions, we present four crystal structures that together comprise the structures of two complete FnBRs, each in complex with four of the N-terminal modules of Fn. Each Ϸ40-residue FnBR forms antiparallel strands along the triple-stranded ␤-sheets of four sequential F1 modules ( 2-5 F1) with each FnBR/ 2-5 F1 interface burying a total surface area of Ϸ4,300 Å 2 . The structures reveal the roles of residues conserved between S. aureus and Streptococcus pyogenes FnBRs and show that there are few linker residues between FnBRs. The ability to form large intermolecular interfaces with relatively few residues has been proposed to be a feature of disordered proteins, and S. aureus/Fn interactions provide an unusual illustration of this efficiency.intrinsic disorder ͉ tandem ␤-zipper ͉ host-pathogen interaction S taphylococcus aureus is a dangerous human pathogen that causes a wide range of debilitating and life-threatening infections (1). Incidence of S. aureus resistance to antibiotics (2) makes the understanding of its mechanisms of pathogenesis imperative. S. aureus/Fn interactions were first reported 30 years ago, and an S. aureus Fn-binding protein was isolated and characterized Ϸ20 years ago (3). Our recent work has dissected the 363-residue C-terminal region of FnBPA into 11 FnBRs (4) (FnBPA1-11; Fig. 1 A and B), six of which bind the NTD (N-terminal domain) of Fn (comprising modules 1-5 F1) with dissociation constants in the nanomolar range (5). The Cterminal region of FnBPB, a second S. aureus Fn-binding protein, is very similar to FnBPA but lacks one of the shorter FnBRs (5). In FnBPA, which also binds fibrinogen, the fibrinogen-and Fn-binding regions (Fig. 1 A) appear to cooperate in disease progression, with the FnBR region being particularly associated with persistence of infection (6). FnBPA/Fn interactions both mediate S. aureus invasion of (7) and activate endothelial cells, evoking both the proinflammatory and procoagulant responses typical of infective endocarditis (8). FnBPAs ability to mediate platelet activation, a key step in thrombus formation, is also likely to play a role in cardiovascular disease (9) and FnBPA has been implicated in cardiac device infections through its ability to mediate S. aureus attachment to implanted prosthetic materials (10). We previously predicted that in Fn-BPA each FnBR binds a string of three or four F1 modules in the NTD of Fn through a longer version of the tandem ␤-zipper mechanism that we discovered in Streptococcus dysgalactiae interactions with 1 F1 2 F1 (4). Results and DiscussionCrystal Structure of FnBPA-1/ 2-5 F1. Fig. 1C shows two F1 module pair/peptide structures that together comprise the structure of the most N-terminal S. aureus FnBR (...

In the present study we examine the enthalpy of binding of 2-methoxy-3-isobutylpyrazine (IBMP) to the mouse major urinary protein (MUP), using a combination of isothermal titration calorimetry (ITC), NMR, X-ray crystallography, all-atom molecular dynamics simulations, and site-directed mutagenesis. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than a classical entropy-driven signature that might be expected given that the binding pocket of MUP-1 is very hydrophobic. The only ligand-protein hydrogen bond is formed between the side-chain hydroxyl of Tyr120 and the ring nitrogen of the ligand in the wild-type protein. ITC measurements on the binding of IBMP to the Y120F mutant demonstrate a reduced enthalpy of binding, but nonetheless binding is still enthalpy dominated. A combination of solvent isotopic substitution ITC measurements and all-atom molecular dynamics simulations with explicit inclusion of solvent water suggests that solvation is not a major contributor to the overall binding enthalpy. Moreover, hydrogen/deuterium exchange measurements suggest that there is no significant contribution to the enthalpy of binding derived from "tightening" of the protein structure. Data are consistent with binding thermodynamics dominated by favorable dispersion interactions, arising from the inequality of solvent-solute dispersion interactions before complexation versus solute-solute dispersion interactions after complexation, by virtue of poor solvation of the binding pocket.