We investigate the molecular mechanisms of the Mycobacterium tuberculosis (Mtb) EfpA membrane exporter. EfpA is a ca. 56 kDa monomeric integral membrane protein, which belongs to the Major Facilitator Superfamily transporters, and the QacA subfamily. It has fourteen predicted transmembrane segments/helices (TMs) with both N‐and C‐termini located in the intracellular space. EfpA exports anti‐Mtb drugs (e.g., isoniazid, fluoroquinolones, rifampicin, tetracycline, clofazimine) out of the cell coupled to the antiport of H+. Thus, EfpA supports a major mechanism of Mtb drug resistance. However, its structure and structure‐function relationship are currently poorly understood. What is more, to the best of our knowledge, the protein was not produced recombinantly in quantities needed for large‐scale in vitro investigations. To overcome this deficiency, we cloned the gene encoding EfpA and expressed the protein in E. coli host. Two fusion constructs of EfpA with respectively His8‐FLAG tag and His8‐maltose binding protein (MBP) tags were produced. These tags were designed to facilitate protein purification utilizing double affinity chromatography. We used SDS‐PAGE and western blotting to identify the protein localization in E. coli cellular fractions. We found that although these fusion constructs were expressed to a minor extent in the E. coli plasma membrane and can be partially purified, the proteins were mostly deposited in inclusion bodies, similarly to what was observed previously for other Mtb membrane proteins. We purified to a high degree and refolded these proteins from inclusion bodies. Currently, we are working on producing large quantities of highly‐pure EfpA protein for functional and structural studies. We further used Swiss Model program and the structures of the distant homologues NorC transporter and peptide transporter DtpA solved in outward‐ and inward‐facing states, respectively, to predict the structure of EfpA in the two major functionally‐relevant conformations. After that, we designed two double cysteine EfpA mutants E141C/F398C and F170C/Q401C, which report on large conformational rearrangements upon transition from outward‐ to inward‐facing conformations, for spin labeling and electron paramagnetic resonance (EPR) study. These mutants were generated via site‐directed mutagenesis on the background of cysteine‐free EfpA construct and are being produced in E. coli. We will further discuss results from EfpA‐drug binding and EPR experiments. Our studies have the potential to significantly advance the detailed characterization of EfpA functional mechanism.
Read full abstract