| Summary: | Staphylococci form micro-communities, known as biofilms, on the surface of inserted medical devices leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. Bacteria in the biofilm are enclosed within an endogenously-produced exopolymeric matrix, which protects them from antimicrobial agents and host immune responses. Prolonged antibiotic therapy and device removal are often required to resolve such infections. Thus, there is a need for new therapeutics but the structural mechanisms of biofilm formation that could assist their development remain unknown. SasG and Aap are highly homologous proteins from Staphylococcus aureus and Staphylococcus epidermidis, respectively, that promote cell-to-cell accumulation during biofilm formation. They are cova:lently attached to the cell-wall and form extended fibrils on the bacterial surface. Both proteins consist of an N-terminal A domain followed by aB region, composed of tandemly arrayed 128 residue repeats. The repeats, which show very high levels of sequence identity, have been proposed to mediate intercellular accumulation through Zn2+ -dependent homo-dimerisation. The present work is a biochemical, biophysical and structural dissection of the biofilm-forming protein SasG from S. aureus that addresses these issues. Zn2+ was found not to cause SasG to dimerise suggesting other components of the biofilm matrix, such as teichoic acids, might eo-mediate the intercellular aggregation. NMR spectroscopy in combination with in vitro folding studies demonstrated that each 128-residue sequence repeat is in fact comprised of a G5 domain of 78 amino acids and a smaller sub-domain of 50 amino acids (herein called E), which is unstable in isolation but folds cooperatively in the context of a C-terminal G5. X-ray crystallography revealed that G5 and E domains are arranged head-to-tail and share the same fold (two single-layer ~- sheets connected via a collagen-like triple-helical motif), which lacks a compact hydrophobic core. This domain arrangement, coupled with interlocking G5-E and E-G5 inter-domain interfaces, results in a contiguous, elongated, apparently rigid, monomeric structure, which explains the fibrillar nature of SasG at the bacterial cell surface. Despite all these unusual structural features, multi-domain SasG constructs exhibit thermodynamic stabilities comparable with other globular proteins of similar size. This work provides the first structural insight into staphylococcal biofilm-associated proteins and a paradigm for the formation of fibrils on the 100 nrn scale from a single polypeptide chain. It presents a novel protein fold, which results in elongated yet thermodynamically stable domains via a 'distributive hydrophobicity' mechanism, distinct to the familiar compact hydrophobic core of globular proteins. Finally, formation of two domains by conserved sequence repeats might provide a simple solution to avoid protein misfolding when a tandem arrangement of highly similar sequences is biologically advantageous.
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