Probing the Structure of the Small Multidrug Resistance Efflux Pump EmrE by Solid-State Nuclear Magnetic Resonance Spectroscopy.

摘要

Multidrug resistance is a major medical problem in the treatment of infections. One of the ways in which bacteria gain resistance to antibiotics and antiseptics is with a class of membrane proteins known as efflux pumps. These pumps use energy to export drugs, against their concentration gradient, out of a cell, thereby conferring resistance to the cell. Among the efflux pumps, the small multidrug resistance (SMR) family has become the model system for studying ion-coupled drug efflux. Among the SMRs, EmrE is the model protein to study this problem. EmrE is a 4 transmembrane domain, 110 residue protein which forms a homodimer. EmrE couples the export of drugs with the import of two protons. It has been well characterized by biochemical methods, but structural details of the drug-free form and mechanistic details of the transport cycle have been lacking. In this dissertation, new methods we developed to study membrane proteins at atomic resolution with solid-state NMR are discussed. From these methods, we have obtained new insights into the structure and the dynamics of drug-free EmrE. We found that the drug-free dimer of EmrE is asymmetric and antiparallel in lipid bilayers. We also found that there is a structured loop linking the ligand binding domain and the dimerization domain and it is necessary for conferring resistance. Finally, we found that the drug-free form of EmrE is highly dynamic and undergoes rapid conformational exchange between an inward-open and outward-open state. The information learned here lays the building blocks towards obtaining a higher resolution structure of EmrE in order to fully understand the ion-coupled transport cycle.