On mechanisms and conformational dynamics of Drug:H+ Antiporters

Abstract

Multidrug efflux is an active transport process involving energy consumption to drive antibacterial compounds out of cells. This is performed either through primary active transport, where chemical energy, such as hydrolysis of ATP, is directly coupled to the transport, or via secondary active transport, where substrate transport is coupled with another ion's transport down its concentration gradient. Drug: H+ Antiporters (DHAs) constitute two families within the Major Facilitator Superfamily (MFS), the largest group of secondary active transporters known to date. Members of the DHAs use the pH gradient across the bacterial membrane to drive the transport process. They are further divided into two families based on the number of transmembrane helices they harbor. These families are DHA1 and DHA2, where two domains harbor six helices each, and the DHA2 members possess an extra two transmembrane helices outside of these domains. Until recently, insights into how DHAs work was primarily derived from structurally and functionally characterized DHA1 transporters, and not much was known about the members of the DHA2 family. The Transporter Classification Database annotates nearly 80 curated proteins as members of DHA2 family, of which none are structurally characterized. This thesis describes our studies on two of them, called NorC and QacA, identified as multidrug efflux transporters from Staphylococcus aureus. Membrane proteins have always been known to be recalcitrant to crystallization because of the very low solvent-exposed surface they possess in detergent micelles and lipid bilayers. Also, members of the Major Facilitator Superfamily span between 400-550 amino acids, with inherent pseudo-two-fold symmetry that makes their structural characterization through CryoEM-based single particle analysis difficult. Single-domain camelid antibodies were employed to study both the transporters, and they served as vital tools as crystallization chaperones and fiducial markers for CryoEM. This thesis delves into the structural characterization of two DHA2 transporters with contrastingly different vestibular environments, traits that make them similar or different from other well-characterized DHA1 transporters, their conformational preferences, and the effect of VHH interactions on the conformational landscape of these transporters.