Molecular Rationale Behind the Differential Substrate Specificity of Homologous RND Transporters in E. coli and P. aeruginosa

The discovery of medicinal antibiotics was a crucial breakthrough in the treatment of infectious diseases. Unfortunately, bacteria proved to be highly competitive by expeditiously developing various survival mechanisms to deal with most (if not all) antibiotics available today. One such highly efficient mechanism is the over expression of specific and general transporters that recognize a wide spectrum of substrates (including many chemically different antibiotics) and actively expel them out of the cells, thereby contributing to multidrug resistance (MDR). Resistance-Nodulation-Division (RND) transporters like AcrB and AcrD in Escherichia coli, and MexB and MexY in Pseudomonas aeruginosa are the most prominent multi-component drug efflux pumps exporting a wide range of substrates ranging from lipophilic to amphiphilic molecules. Despite a comparable overall sequence homology among these RND transporters of E. coli and P. aeruginosa, they exhibit varied substrate specificity, the underlying basis of which still remains elusive. In an attempt to provide better insights into the substrate-transporter complementarity underlying the recognition and transport events in Acr pumps of E. coli and Mex pumps of P. aeruginosa, we performed a comparative analysis of multi-copy microsecond-long MD simulations of the apo-forms of AcrB, AcrD, MexB and MexY transporters. To this effect, we chose a set of important descriptors like pocket volume, molecular lipophilic potential, electrostatic potential and hydration to characterize the putative binding pockets (Access and Deep Pockets) in these transporters. Owing to the absence of crystal structure for AcrD and MexY, we also modeled their 3D-structures based on the high-resolution crystal structure of their closest homologues for this study. Our results suggest that the interactions of ligands with and their affinity to these transporters arise from an interplay between physicochemical properties, such as volume, lipophilicity, electrostatic potential, and certain specific features like changes in the loop conformations, altogether tuned by the dynamics of the systems. The thesis discusses in detail the important findings from our microsecond-long MD simulations of AcrB, AcrD, MexB and MexY proteins in the absence of a bound substrate, emphasizing the molecular determinants governing the partially different substrate specificity of the two couples of proteins in E. coli and P. aeruginosa. In addition, certain key interaction types needed for a substrate to bind to its transporter and/or for a transporter to recognize its substrate are also discussed for Acr transporters in E. coli.