Dynamics of the homotrimeric tolc transmembrane protein

Efflux-mediated β-lactam resistance poses a significant challenge to public health,undermining the effectiveness of various β-lactam antibiotics against a multitude of clinicallyrelevant pathogenic bacteria. Structural and functional analyses have elucidated the role ofthe efflux protein TolC in several Gram-negative bacteria as a conduit for antibiotics,bacteriocins, and phages, impacting bacterial susceptibility and virulence. This studyundertook a comprehensive examination of β-lactam drug efflux mediated by TolC,employing extensive experimental and computational analyses. Our computationalinvestigations delved into the molecular dynamics of drug-free TolC, revealing criticalunidirectional movements of the trimeric TolC and identifying residues significantlyinvolved in TolC opening. To validate these findings, a whole-gene-saturation mutagenesisassay was conducted, systematically mutating each residue of TolC to 19 other amino acidsand assessing the fitness effects of these mutations under β-lactam-induced selection.Oxacillin, piperacillin, and carbenicillin were selected for study due to their varyingefficiencies in being effluxed by the AcrAB-TolC complex. This approach elucidatedsimilarities and differences in the efflux processes of the three β-lactam antibiotics throughtrimeric TolC. Further analysis via steered molecular dynamics simulations unveiled generaland drug-specific efflux mechanisms employed by TolC. Key positions at the periplasmicentry of TolC were identified, whose altered dynamics influence long-range efflux motionsas allosteric modulators. These findings offer valuable insights into the structural dynamicsof TolC, laying a foundation for understanding multidrug resistance mechanisms andinforming the design of novel antibiotics capable of circumventing bacterial effluxmechanisms.