Abstract 1073 Production and reconstitution in lipid nanoparticles of an engineered chimera construct of the Mtb EfpA drug exporter with apolipoprotein
Olamide Ishola, Adeyemi Ogunbowale, Md Majharul Islam, Elaheh Hadadianpour, MAJEED SAMAN, Oluwatosin Adefunke Adetuyi, Elka R. Georgieva · Journal of Biological Chemistry · 2024
Mycobacterium tuberculosis (Mtb) drug exporters contribute an efficient mechanism for drug resistance. Therefore, understanding the structure–function relationship of 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 outsidethe 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) fusedto 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 used negative staining electron microscopy to visualize gold nanoparticle labeled EfpA in detergent and lipid. Through these experiments, we found that apoAI-EfpAform oligomers. Further analysis by using AlphaFold software confirmed the self-oligomerization tendency of this protein. 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 proposed14-transmembrane helices of the Mtb EfpA. We also found very high identity, >80%, among the EfpA exporters 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 the high specificity of EfpA mechanisms. Our developments provide the foundation for more comprehensive in vitro studieson the EfpA exporter. Furthermore, we engineered a chimera construct of ApoAI fused to a biomedically important transmembrane protein. This methodology facilitated both the expression of the target protein and its incorporation of lipid nano carriers.