Hermia Ip
These findings demonstrate that equivalent bulk conditions can mask distinct molecular states and identify chemical pathways as an important determinant of AcrA structural dynamics.
Adaptor protein AcrA plays a central role in the assembly and function of tripartite multidrug efflux pumps in Gram-negative bacteria, yet how its structural organization responds to coupled chemical perturbations rather than solely to equilibrium conditions remains unclear. Residues near His285 define a hinge microenvironment linking the lipoyl and β-barrel domains, suggesting a site for chemically sensitive structural modulation. Here, site-directed spin labeling combined with continuous-wave electron paramagnetic resonance spectroscopy was used to examine AcrA under an Mg2+-driven perturbation that simultaneously alters proton availability. Mg2+ addition produced spectral broadening at residue 62 that was fully reversed by spin dilution, indicating increased interspin proximity without changes in intrinsic side-chain dynamics. In contrast, direct acidification to a comparable bulk pH in the absence of Mg2+ did not reproduce this behavior. Structural mapping places residue 62 in proximity to the His285-centered hinge region, suggesting that coupled changes in protonation and metal coordination bias local interaction networks and modulate interdomain organization. These findings demonstrate that equivalent bulk conditions can mask distinct molecular states and identify chemical pathways as an important determinant of AcrA structural dynamics.