Functional and structural analysis of the ABC transporter Rv2563/Rv2564 from Mycobacterium tuberculosis

Abstract

Membrane proteins correspond to 25% to 30% of all transcribed sequences in known genomes and perform a range of essential functions in cellular metabolism, representing 50% of the targets in the pharmaceutical industry. ATP-Binding Cassette (ABC) transporter family of membrane proteins has been largely involved in drug transport and resistance, in cancer cells and in microrganisms, allowing the development of the multi drug-resistant (MDR) and extensively drug-resistant (XDR) strains. For M. tuberculosis, the causative agent of the tuberculosis, the efflux mechanisms have become of increased importance, since the resistance is affecting the treatment, patient survival and statistics of the disease. In this sense, our project aims to study functional and structurally the M. tuberculosis ABC transporter Rv2563/Rv2564, which function is putatively related to the drug efflux. In a previous period at the Membrane Protein Laboratory, genes Rv2563 and Rv2564, which encode a permease and an ATPase, respectively, were expressed and purified from E. coli cells using DM as detergent for solubilization. We got crystals of the complex that when submitted to X-ray diffraction at I24 Beamline of the Diamond Light Source (UK) diffracted at 5 Å resolution with a diffraction pattern very anisotropic. The objective of this proposal is to obtain the complex in different ways to improve the quality of the crystals and diffraction experiments. Morever, using the expertise and facilities of MPL, we want to produce the complex in proteliposomes for functional characterization in presence of different drugs and to perform studies of interaction with MacA, a putative partner identified in M. tuberculosis. Using the MPL and Diamond Light Source infrastructure we also will try to solve the complex by cryo-electron microscopy (Cryo-EM). For this, we will use the Membrane Protein Laboratory facilities from the National Physical Laboratory (Research Complex Harwell Campus) in Oxfordshire, UK and the expertise of our collaborator, Dr. Isabel de Moraes, one of the biggest specialists in this field and that has been working with us for more than 3 years. (AU)