Studies of interactions of staphylococcal and streptococcal peptides with a type-I module pair of human fibronectin

Thesis

A number of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes, can adhere to the protein fibronectin (Fn) in the human extracellular matrix. These host/pathogen interactions are likely to play an important role in the process of infection and have therefore been identified as targets for new therapeutics. Numerous proteins from grampositive bacteria have been shown to bind to the N-terminal part of Fn, which contains five F1 modules (^1-5F1). Fn-binding proteins have several homologous binding units, which exist in an unfolded state under physiological conditions. The primary binding site for the D3 repeat from S. aureus Fn-binding protein A (FnbpA) is the ⁴F1⁵F1 module pair (Huff et al., 1994).

The high-resolution structure of ⁴F1⁵F1 has been calculated de novo and the structure of ⁴F1⁵F1 in complex with D3 has been determined using nuclear magnetic resonance (NMR). Long-range orientational restraints derived from ¹⁵N relaxation and H^N-N residual dipolar couplings have been added to conventional NOE and J-coupling data. The ensemble of the final structures of bound ⁴F1⁵F1 was used as a model in molecular replacement in attempts to solve the crystal structure of the ⁴F1⁵F1/D3 complex.

A new alignment of Fn-binding repeats (U Schwarz-Linek - manuscript in preparation) suggests the presence of conserved short motifs that bind specifically to individual ^1-5F1 modules. This model was tested using residue-specific NMR binding studies; the ¹H¹⁵N chemical shift changes induced by ligands from S. aureus and S. pyogenes were mapped on the structure. 'Dissecting' the D3 repeat has shown that it consists of smaller units that exhibit different binding specificities. Ligands from two different regions of the FnbpA protein from S. aureus were shown to induce similar chemical shift changes in Fn modules. Isothermal titration calorimetry and NMR were used to determine the binding constants of the module pair/ligand interactions. Peptides consisting of several binding motifs were shown to bind stronger than ones containing a single binding site. These studies lead to a better understanding of FnBP-Fn interactions.

Authors: Pilka, E

• DOI: 10.5287/ora-bxod10mgx
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford