Reshaping globular dynamics of S. aureus pyruvate kinase via bond restraints to allosteric sites

The global dynamics of pyruvate kinase were examined using molecular dynamics (MD) simulations to investigate the effects of allosteric inhibition through bond restraints applied at two key allosteric sites. The study employed the experimentally resolved structure of the enzyme complexed with the allosteric inhibitor IS-130 at the small C-C interface, serving as a reference for analyzing an additional, computationally predicted allosteric site at the large A-A interface. Simulations identified the B and CT domains as the most mobile regions, with bond restraints at either interface significantly reducing CT domain flexibility up to 9 Å across all chains. Restraints at the C-C interface limited minimal global conformational sampling, whereas restraints at the A-A interface altered the dynamic profile without narrowing the sampled conformational space, suggesting distinct regulatory roles for each interface. Distance fluctuation analyses revealed enhanced interchain communication and reduced mobility near restrained sites, suggesting that these restraints reinforce allosteric inhibition by stabilizing otherwise flexible domains. Cross-correlation analysis showed a marked reduction in long-range residue-residue correspondence, especially under C-C restraints, indicating disrupted dynamic coordination essential for catalytic activity. Mutual information analysis, capturing both linear and non-linear dependencies, further supported these findings by showing a widespread loss of dynamic correspondence in positional fluctuations across the receptor upon restraint application. Notably, although the C-C interface has been experimentally linked to inhibition, these results suggest that the computationally predicted large A-A interface may also contribute to allosteric regulation. Together, these findings highlight the distributed and cooperative nature of allosteric control in pyruvate kinase.