(604c) Staphylococcal Protein a: Disordered in Full-Length and Ordered in Single Domains – a Computational and Experimental Study

Staphylococcus aureus, commonly known as “staph,” causes severe infections in humans, including pneumonia and sepsis. Its virulence is enhanced by Staphylococcal protein A (SpA), a surface protein that binds antibodies and suppresses immune responses. SpA consists of five homologous three-helix-bundle antibody-binding domains and a large disordered C-terminal anchor domain. Despite the fact that the structure and stability of individual domains are well studied, clinical attempts to target SpA have been largely ineffective, and an understanding of the conformational behavior of the full-length protein remains critically needed.

Here, we present the first comprehensive analysis of full-length SpA using all-atom molecular dynamics (MD) simulations applying enhanced sampling validated by experimental data. The AlphaFold-predicted structure displays all five folded domains linked by flexible segments and a disordered C-terminal tail. Lacking an experimental structure, we used this model as our folded state reference and projected the free energy surface along a reaction coordinate using this structure as a reference. Our results reveal that the AlphaFold-predicted structure is thermodynamically unstable relative to a compact, disordered state, with all domains losing stability in the full-length context.

To assess whether this instability reflects a methodological artifact, we performed simulations on individual domains with the same force fields, accurately reproducing their known thermal stabilities. We further calculated secondary structure content from both simulations and circular dichroism (CD) spectroscopy, which consistently support the lack of fully-folded structure in the full-length protein A. Differential scanning calorimetry (DSC) measurements confirmed the absence of cooperative unfolding transitions. Contact map analysis revealed numerous interactions not predicted in the AlphaFold model, suggesting that interdomain contacts may contribute to the instability of the folded full-length protein.

Our combined simulation and experimental results provide a new perspective on the conformational behavior of full-length SpA and challenge static structural assumptions, which may be crucial for understanding and ultimately targeting this virulence factor.