Functional and structural characterization of H105Y mutation in MtrR protein of Neisseria gonorrhoeae

Abstract

MtrR is a negative regulator of MtrCDE efflux pump. Various N-Terminal and C-Terminal mutations have been reported in different multidrug resistant clinical isolates of Neisseria gonorrhoeae across the world. Mutations in N-terminal region of MtrR, are known to abrogate its binding with the promoter thereby providing an evidence that this region encodes for DNA binding domain. In contrast, mechanism of action of mutations in C-terminal, known to play a role in protein dimerization and has site for ligand binding, is left unexplored. In the present study, using in silico approach, we observed that H105Y mutation affects the conformation of the protein and its binding with penicillin. Purified, recombinant wild type and mutant MtrR were compared for their binding to promoter region and various antibiotics. Fluorescence spectroscopy, CD spectroscopy and dynamic light scattering assay with wild type and mutant MtrR suggested decreased binding of H105Y MtrR with its promoter without affecting protein dimerization, but due to altered conformation of mutant dimer. Our in silico results also suggest altered conformation of the mutant dimer protein leading to difference in the posture of homodimer formed and hence altered binding with DNA. Mutant protein also showed stronger binding with various antibiotics: penicillin, ceftriaxone and ofloxacin. Binding of drugs also leads to altered conformation of the protein which may lead to its decreased binding with the promoter DNA.Importance Mutations in MtrR, a transcriptional repressor of MtrCDE efflux pump have been reported in various multi-drug resistant Neisseria gonorrhoeae across the world. We identified that a C-terminal mutation H105Y, outside the DNA binding domain of MtrR, decreases the binding of MtrR with its promoter. We identified that the mutation alters the structure of the dimer, as well as enhances the antibiotic binding. We envisage that the altered structure of MtrR affects its DNA binding thereby increasing efflux of antibiotics and increased resistance. These findings could be used as a guide to design novel drugs that should either not bind to MtrR or could surpass the conformational change and decreased DNA binding.View Full Text