(368bl) Unexpected Folding Instabilities in Full-Length Staphylococcal Protein a: Insights from Advanced Molecular Dynamics Simulation

Staphylococcus aureus, notorious for causing severe infections in humans, owes much of its virulence to the Ig-binding surface protein Staphylococcal protein A (SpA). This protein, while aiding the pathogen in evading the immune response, has also emerged as a pivotal tool in purifying pharmaceutical antibodies. SpA, with its five homologous three-helix-bundle antibody-binding domains and a disordered C-terminal anchor domain, facilitates the purification of numerous monoclonal antibody medicines, including many of the top-selling drugs worldwide. SpA also serves as a therapeutic, being used in treating autoimmune diseases. Despite numerous studies on SpA as a multifunctional protein, the exact molecular mechanism of SpA’s role as a virulence factor has remained elusive. This challenge is compounded by the fact that most studies are conducted on monodomain SpA due to the difficulties associated with studying full-length SpA.

In this study, we explored the folding thermodynamics of full-length SpA and compared its behavior with that of its individual domains. We employed advanced molecular dynamics simulations that allowed us to reliably obtain the folding free energy surface of full-length SpA. Our analysis revealed that full length SpA is does not have a stable folded configuration unlike its individual domains. Our investigations revealed a strong intramolecular contact formation between SpA's structural elements, illuminating its inherent instability in the context of the full-length protein. Notably, our predictions for the thermal stability of individual functional domains in isolation match experimental observations, validating our methodology.

The projected free energy for dihedral angles indicates that turns within the protein cannot form in the context of the full-length protein. Additionally, calculating inter-residue interactions revealed steric clashes between domain termini and other domains. These clashes prevent contacts within that domain from forming in the full-length protein, as inferred from the compact structure of SpA observed in our simulations.

Our findings reveal the stable domains of SpA become unstable when they come together in a full-length SpA. Besides implications for theoretical protein folding, this study provides valuable insights into the fundamental mechanisms governing SpA's behavior, which may contribute to developing effective approaches to tackle Staphylococcal infections.