Hadadianpour, E. | Search bioRxiv

bioRxiv2024

Highly versatile small virus-encoded proteins in cellular membranes: A structural perspective on how proteins' inherent conformational plasticity couples with host membranes properties to control cellular processes

We investigated several small viral proteins that reside and function in cellular membranes, which belong to the viroporin family because they assemble into ion-conducting oligomers. However, despite forming similar oligomeric structures with analogous functions, these proteins have diverse amino acid sequences. In particular, the amino acid compositions of the proposed channel-forming transmembrane (TM) helices are vastly different--some contain residues (e.g., His, Trp, Asp, Ser) that could facilitate cation transport. Still, other voroporins TM helices encompass exclusively hydrophobic residues; therefore, it is difficult to explain their channels activity, unless other mechanisms (e.g., involving a negative lipid headgroup) take place. For this study, we selected the M2, Vpu, E, p13II, p7, and 2B proteins from the influenza A, HIV-1, human T-cell leukemia, hepatitis C, and picorna viruses, respectively. We discuss the current knowledge of these proteins structures as well as remaining questions about a more comprehensive understanding of their structures, conformational dynamics, and function. Finally, we outline strategies to utilize a multi-prong structural approach to overcome current deficiencies in the knowledge about these proteins. HighlightsO_LISmall viral proteins encoded homo-oligomerize and function in cellular membranes as ion channels C_LIO_LIThese proteins were combined in the family of viroporins C_LIO_LIDespite the similarity in their oligomeric structures and functions, these proteins have vastly different primary structures C_LIO_LIIt is imperative to understand how proteins with no homology in their primary structures fulfill similar functions for diverse viruses C_LIO_LIThere is a need for a multi-prong structural approach to explain the structure, conformational dynamics, and function of these proteins C_LI

biophysics↗

bioRxiv2023

Protein engineering, production, reconstitution in lipid nanoparticles, and initial characterization of the Mycobacterium tuberculosis EfpA drug exporter

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 characterization of this protein. We engineered a construct of apolipoprotein A-I (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 oligomers in {beta}-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 transported. AlphaFold2 also predicted EfpA oligomers and further bioinformatic analysis confirmed the earlier proposed 14-transmembrane helices of the Mtb EfpA. We also found very high identity, >80%, among the EfpA-s 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.

biochemistry↗