COMPUTATIONAL CHARACTERIZATION OF THE LIPID A PROTEIN G INTERACTION: MOLECULAR DOCKING, MOLECULAR DYNAMICS SIMULATION, AND BINDING FREE ENERGY ANALYSIS REVEAL A NOVEL MOONLIGHTING FUNCTION RELEVANT TO POLYMICROBIAL INFECTION

Abstract

Sepsis is a life-threatening syndrome of immune dysregulation that drives multi-organ failure and remains one of the leading causes of death worldwide. Despite decades of mechanistic work, validated biomarkers and effective drugs are still scarce. Much of the underlying pathophysiology — aberrant signaling, failed inflammatory control, energy deficits at the cellular level — converges on one common thread: disrupted lipid metabolism. Specific lipid species are increasingly used as early sepsis biomarkers, and understanding how they act mechanistically could open new therapeutic avenues. This study focuses on Lipid A, the outer-membrane component of gram-negative bacteria that host immune receptors recognize to trigger sepsis. Using AutoDock Vina, we found that Lipid A docks onto Protein G at two distinct sites: Domain III (the B domain, residues 297–354) and the N-terminal helical segment. We then ran a 100-ns molecular dynamics simulation of the complex in GROMACS 2024 and calculated binding free energy with the MM/PBSA method (gmx_MMPBSA). RMSD and RMSF confirmed the complex remained structurally stable, while Rg and SASA showed the interface stayed compact and consistently buried over the trajectory. Hydrogen bonds persisted throughout the simulation, and MM/PBSA gave a favorable ΔGbinding, with per-residue decomposition identifying which residues drove the interaction. We also docked Lipid A against the canonical TLR4/MD2 complex as a benchmark. Together, these results show that Protein G — a Streptococcal surface protein already known to bind IgG and serum albumin — also binds Lipid A, at two separate sites, pointing to a previously undescribed moonlighting function. Because Gram positive and Gram-negative organisms frequently co-infect the same patient, this interaction could matter for how Lipid A is sequestered during mixed infection. That possibility now needs testing in vitro and in vivo.