Biology subjects

Georgieva, E. R.

Publications and source records attributed to Georgieva, E. R..

5 recordsLinked to original sources

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↗

bioRxiv2024

A comparative study of influenza A M2 protein conformations in DOPC/DOPS liposomes and in native E. coli membranes

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IAM2) protein reconstituted at pH 7.4 in DOPC/DOPS bilayers to those in isolated E. coli membranes, having preserved its native proteins and lipids. IAM2 is a single-pass transmembrane protein known to assemble into homo-tetrameric proton channel. To represent this channel, we made a construct containing the IAM2s TMD region flanked by the juxtamembrane residues. The single cysteine substitute, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the ESR (electron spin resonance) study. We compared the conformations of the spin-labeled IAM2 residing in DOPC/DOPS and native E. coli membranes using continuous-wave (CW) ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The CW ESR spectra corresponded to a nearly rigid limit spin label dynamics in both environments. In all cases, the DEER data were reconstructed into the distance distributions showing well-resolved peaks at 1.68 nm and 2.37 nm. The peak distance ratio was 1.41{+/-}0.2 and the amplitude ratio was 2:1. This is what one expects from four nitroxide spin-labels located at the corners of a square, indicative of an axially symmetric tetramer. Distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IAM2 tetramer in agreement with the NMR model. Thus, we can conclude that IAM2 TMD has similar conformations in model and native E. coli membranes of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

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↗

bioRxiv2023

HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self-association

We report our findings on the assembly of the HIV-1 protein Vpu into soluble oligomers. Vpu is a key to HIV-1 protein. It has been considered exclusively a single-pass membrane protein. However, we revealed that this protein forms stable oligomers in aqueous solution, which is an interesting and rather unique observation, as the number of proteins transitioning between soluble and membrane embedded states is limited. Therefore, we undertook a study to characterize these oligomers by utilizing protein engineering, size exclusion chromatography, cryoEM and electron paramagnetic resonance (EPR) spectroscopy. We found that Vpu oligomerizes via its N-terminal transmembrane domain (TM). CryoEM analyses suggest that the oligomeric state most likely is a hexamer or hexamer-to-heptamer equilibrium. Both cryoEM and EPR suggest that, within the oligomer, the distant C-terminal region of Vpu is highly flexible. To the best of our knowledge, this is the first comprehensive study on soluble Vpu. We propose that these oligomers are stabilized via possibly hydrophobic interactions between Vpu TMs. Our findings contribute valuable information about this protein properties and about protein supramolecular complexes formation. The acquired knowledge could be further used in protein engineering, and could also help to uncover possible physiological function of these Vpu oligomers.

biophysics↗

bioRxiv2022

Oligomeric polymorphism of HIV-1 Vpu protein in lipid environment and in solution

The HIV-1 encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with multiple host proteins to support virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. The structures of full-length Vpu in its monomeric and oligomeric forms are unknown, although both the monomer and oligomer are deemed important. Here, we report on the diversity of Vpu oligomeric structures and how the environment affects the Vpu oligomer formation. We produced a uniquely designed MBP-Vpu chimera protein in E. coli in soluble form. We subjected this protein to analytical size exclusion chromatography (SEC) and negative staining electron microscopy (nsEM). Strikingly, we found that MBP-Vpu forms stable oligomers in solution, presumably driven by Vpu transmembrane domain self-association. Our coarse modeling suggests that these oligomers are pentamers, in agreement with the pentameric membrane-bound Vpu. To the best of our knowledge, this is the first observation of Vpu self-association out of lipid membrane environment. We further found that MBP-Vpu oligomer stability decreases when the protein was reconstituted in lipid membrane mimetics, such as {beta}-DDM, and mixtures of lyso PC/PG or DHPC/DHPG--In these cases significant oligomer heterogeneity was observed with oligomeric order lesser than that of MBP-Vpu oligomer in solution, but larger oligomers were observed as well. Importantly, we found that in lyso PC/PG, above certain protein concentration, MBP-Vpu forms linear array-like structures, which is also novel. Thus, our studies provide unique information about Vpu protein quaternary organization by capturing multiple Vpu oligomeric structures, which we believe are physiologically relevant.

biochemistry↗