Protein engineering, production, reconstitution in lipid nanoparticles, and initial characterization of theMycobacterium tuberculosisEfpA drug exporter

Abstract

Mycobacterium tuberculosis (Mtb) drug exporters contribute an efficient mechanism for drug resistance. Therefore, understanding the structure/function relationship in these proteins is important. We focused on the Mtb EfpA efflux pump, which belongs to the major facilitator superfamily (MSF) and transports anti-tuberculosis drugs outside the bacterial cell. Here, we report on our advancements in producing and in vitro characterization of this protein. We engineered a construct of apolipoprotein AI (apoAI) fused to the N-terminus of EfpA (apoAI-EfpA) and cloned it in an E. coli expression vector. This fusion construct was found in a membrane-bound form, unlike the deposited in inclusion bodies EfpA without apoAI. We purified the apoAI-EfpA in detergent to a sufficient degree and reconstituted it in DOPC/DOPS lipids. We found that upon reconstitution in lipid, the apoAI-EfpA forms discoidal protein-lipid nanostructures with a diameter of about 20 nm, resembling nanodiscs. We further detected apoAI-EfpA form oligomers in β-DDM and lipid. To the best of our knowledge, this is the first complete protocol on the expression, purification, and lipid reconstitution of the Mtb EfpA reported. Our bioinformatic analysis confirmed the earlier proposed 14-transmembrane helices of the Mtb EfpA. We also found very high identity, >80%, among the EfpAss of diverse mycobacterial species. Outside of mycobacteria, EfpA has no close homologues with only low identity with the QacA family of transporters. These findings possibly indicate high specificity of EfpA mechanisms. Our developments provide a foundation for more comprehensive in vitro studies on the EfpA exporter.

Engineered Chimera Protein Constructs to Facilitate the Production of Heterologous Transmembrane Proteins in E. coli

“…Therefore, using a mMBP solubilization tag fused to the N-termini of the TMPs of interest proved helpful in producing these proteins. It is tempting to mention that mMBP-tagged TMPs of Mtb were not found in the E. coli plasma membrane [19,57], which is the opposite to what we observed in the case of apoAI-EfpA [32]. These differences might be because of the TMPs size, i.e., single-pass small TMPs [65] vs. large multi-pass TMP [66], but future examinations might be needed to understand this better.…”

“…By doing so, we produced, for the first time to the best of our knowledge, highly pure FL Mtb-EfpA in quantities sufficient for downstream in vitro characterization. Remarkably, when reconstituted in lipid, because of the presence of apoAI in the apoAI-EfpA fusion construct, we observed by electron microscopy the formation of protein-lipid nanoparticles [32], which are similar to previously described nanodiscs [51][52][53]. This suggests that we can carry out future studies on EfpA's properties (e.g., drug binding, structure determination, assessing the conformational dynamics) using these two-component (apoAI-EfpA protein and lipid) nanoparticles.…”

“…Interestingly, apoAI is typically expressed as a soluble protein in E. coli [54]. We also found that the untagged EfpA is deposited in inclusion bodies upon expression [32]. Therefore, there is a question of how and why the apoAI-EfpA is directed to the membrane.…”

“…The yield of Mistic-aKv1.1 was approximately 2 mg/L culture [20] The Mistic-ARI yield amounted to roughly 0.12 mg/L culture [22] Apolipoprotein AI Mtb-EfpA [32], EmrE transporter [26], human cyt b5 [26], HSD17β3 [26], GluA2 [26], DsbB [26], CLDN1 [26], CLDN3 [26],…”

“…Recently, motivated by a study on soluble TMPs fused to the N-terminus of apoAI (discussed in more detail below) [26], our research group designed and expressed in E. coli a chimera construct of apoAI with the Mycobacterium tuberculosis EfpA (Mtb-EfpA) drug exporter [32]. By doing so, we produced, for the first time to the best of our knowledge, highly pure FL Mtb-EfpA in quantities sufficient for downstream in vitro characterization.…”