Crystallographic studies of efflux transporters with transport substrates and inhibitors

Structures of protein-ligand complexes provide some of the most intuitive insights into substrate recognition mechanisms. In the case of multidrug efflux transporters, structural analysis of complexes between target proteins and candidate compounds, such as inhibitors, provides critical information for understanding the action of those compounds and advancing the development of new drugs. The detailed information helps guide Structure-Based Drug Design and provides data for potential machine learning approaches. The recognition of ligands by proteins is sometimes likened to a lock and key process, while other analogies implicate induced fit with mutual conformational adjustment. Still, the multi-substrate recognition mechanism of multidrug efflux transporters seems to be an exception to these paradigms. Allosteric communication within and between protomers in oligomeric assemblies of transporters is another key functional aspect of the molecular recognition. The mode of recognition underpins multidrug resistance, a significant clinical issue where many antibiotics are rendered ineffective. Understanding recognition can also help to distinguish how transport substrates and inhibitors differ in their interactions with drug transporters, despite binding to the same pockets. Analysis of complex structures between multidrug efflux transporters and their substrates remains a fascinating subject of study in protein science and is a highly important from an applied perspective. In this chapter, we will primarily discuss the methods for analyzing the detailed structures of multidrug efflux transporters in complexes with amphiphilic and hydrophobic substrates using X-ray crystallography. The techniques described here share many common elements with work on soluble proteins, and are therefore not limited to multidrug efflux transporters. We will address some specific issues and points to consider that are particularly relevant to multidrug efflux transporters.