Transport Dynamics of MtrD: An RND Multidrug Efflux Pump from Neisseria gonorrhoeae

Antibiotic-resistant gonorrheal infections are an urgent health concern. The MtrCDE system confers multidrug resistance to Neisseria gonorrhoeae, an obligate human pathogen, and the causative agent of the sexually-transmitted infection gonorrhea. The inner membrane pump MtrD effluxes a variety of hydrophobic and amphiphilic substrates and thereby confers resistance to a multitude of antibiotics. Using a combination of free and directed Molecular Dynamics (MD) simulations, we analyzed the interactions of MtrD with Azithromycin, an MtrD substrate and one of the last remaining courses of treatment for multidrug resistant gonorrhea. We also simulated the interactions between MtrD and Streptomycin, a non-substrate of MtrD. Using targeted MD (TMD) techniques and known conformations of MtrD homologues, we guided MtrD through the conformational changes of a putative transport cycle by applying small forces to α-carbons of the protein backbone; forces were not applied to Azithromycin or to Streptomycin. In our TMD experiments, we observed the transport of Azithromycin (in three possible protonation states) and the rejection of Streptomycin. To supplement our findings, we then demonstrate the spontaneous diffusion of Azithromycin through the periplasmic cleft in long time-scale, unbiased MD simulations. Our findings support the hypothesis that the transition from Binding to Extrusion is an energy requiring step in the transport process. Our data also suggest that multiple binding modes, and potentially multiple residue contact pathways, exist within the periplasmic cleft of MtrD, even for bulky substrates. To our knowledge, this is the first computational demonstration of substrate transport, and non-substrate rejection, by MtrD.