Biology subjects

Georgieva, E. R.

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

8 recordsLinked to original sources

bioRxiv2026

Affinity and Conformations of Ca2+-Calmodulin Bound to Peptides Containing the Calmodulin-Binding Motif of HIV-1 Matrix Protein

Background: Protein-protein interactions enable viruses to adapt and replicate by modifying host factors. Thermodynamic characterization of viral-host complexes can reveal viral mechanisms, broader principles of protein interactions, and properties relevant to anti-HIV-1 inhibitor development. Here, we examine the binding of the HIV-1 matrix (MA) protein to host calmodulin (CaM). MA, the N-terminal domain of Gag (group-specific antigen), directs Gag to the plasma membrane for viral assembly and maturation and may recruit CaM to facilitate this trafficking. However, the MA-CaM interaction remains poorly characterized. MA contains a noncanonical N-terminal CaM-binding motif with hydrophobic regions that may mediate CaM binding and a central, positively charged KKK cluster. Objective: We aimed to determine the binding affinities of MA to calcium-bound CaM (Ca2+-CaM), elucidate protein regions that are critical to the heterocomplex formation, and monitor the conformational alterations in Ca2+-CaM upon binding to MA. Methods: We studied Ca2+-CaM binding to synthetic MA peptides representing full-length wild type (FL WT), a C-terminal truncation lacking the KKK cluster, and an FL K26N/K27H/K28Q triple mutant of the MA CaM-binding motif. The peptides were C-terminally labeled with Cy3, and FL CaM S39C with Cy5. Binding affinity was measured by ensemble Forster resonance energy transfer (eFRET) using 100 nM Cy3-MA and increasing Cy5-Ca2+-CaM concentrations in the range of 50 nM to 2.5 microM. Conformational changes in doubly spin-labeled Ca2+-CaM upon MA binding were probed by double electron-electron resonance (DEER) spectroscopy. Results: Cy3 quenching and FRET analyses of complexes with Cy5-Ca2+-CaM yielded dissociation constants of ~80 nM for FL WT MA, ~65 nM for the truncation, and ~250 nM for the triple mutant, with corresponding binding free energies of ~9.8 to ~9 kcal/mol. These values indicate stable, specific complexes and show that the KKK cluster contributes little to MA-Ca2+-CaM binding. DEER measurements of Ca2+-CaM labeled at S39C and A103C showed that FL WT MA binding shifted the ~5.3 nm interspin distance of extended Ca2+-CaM to a broader ~1.8-3 nm distribution, indicating a major CaM restructuring. Conclusion: These findings define key energetic and structural features of MA-Ca2+-CaM interactions and clarify how HIV-1 proteins exploit CaM.

biophysics↗

bioRxiv2026

Quantitative and mutational analysis of soluble HIV-1 Vpu and calmodulin interactions

HIV-1 Vpu supports viral adaptation through host-protein interactions. Although mainly membrane-associated, we recently identified a soluble Vpu form that forms a stable complex with Ca2+-bound calmodulin (Ca2+-CaM), potentially influencing Vpu trafficking. Here, to determine the binding affinity and identify regions of soluble Vpu involved in CaM binding, we used ensemble Forster Resonance Energy Transfer (eFRET). We tested Cy3-labeled full-length (FL) Vpu, a C-terminal fragment (helices 2 and 3), and a Cy3-labeled FL Vpu V22A/W23Y/I33N mutant with substitutions of key residues in Vpu helix 1 and helices 1-to-2 loop having a role in the interaction with Ca2+-CaM. All Vpu variants were labeled at residue L42C. Ca2+-CaM was tagged with Cy5 at residue S39C. eFRET analysis of 100 nM Cy3-Vpu variants mixed with Cy5-Ca2+-CaM (in the range 100 nM-2.5 microM) revealed heterocomplexes formation with dissociation constants (Kd) and binding free energies (deltaG). FL Vpu-Ca2+-CaM showed highest stability (Kd ~74 nM, deltaG ~-9.7 kcal/mol), while the truncated C-terminal region and V22A/W23Y/I33N mutant formed weaker complexes with Ca2+-CaM (Kd ~182 nM and 800 nM, deltaG ~-9.2 kcal/mol and ~-8.3 kcal/mol). The reduced Vpu-Ca2+-CaM stability after disruption of binding sites in and near Vpu helix 1 may explain how this interaction is regulated, including through lipid competition that promotes Vpu membrane insertion. We propose that, at the membrane, hydrophobic helix 1 dissociates from Ca2+-CaM and inserts into the lipid bilayer, weakening the complex and releasing CaM. These findings clarify HIV-1 Vpu interactions with cellular components and may inform antiviral development.

biophysics↗

bioRxiv2025

The soluble HIV-1 Vpu protein interacts with calmodulin in a Ca2+-dependent manner

The HIV-1 Vpu membrane protein is crucial to the virus lifecycle. Our recent studies revealed soluble Vpu oligomers, prompting further investigation into their interactions with cellular proteins. Notably, Vpu may form a complex with calmodulin (CaM) due to its putative CaM-binding motif; however, experimental proof of this association remained unavailable. Here, we present definitive experimental evidence that the soluble Vpu complex interacts in vitro with calcium-bound CaM (Ca2+-CaM) its active form. Using double electron electron-resonance (DEER) spectroscopy and protein spin labeling, we detected the formation of a soluble Vpu-Ca2+-CaM complex. Both the full-length (FL) and truncated C-terminal regions of Vpu bind Ca2+-CaM. DEER experiments on a spin-labeled CaM cysteine mutant S39C/A103C revealed that, upon association with Vpu, Ca2+-CaM undergoes a transition from an open to a more closed conformation, consistent with previous reports of Ca2+-CaM interactions with other proteins. Furthermore, we observed that the binding of Vpu to Ca2+-CaM leads to dissociation of soluble Vpu oligomers, as evidenced by a reduction in DEER modulation depth for FL Vpu spin-labeled at residue L42C. FRET analysis with a fluorescently labeled C-terminal cysteine mutant of Vpu confirmed this result. Like FL Vpu, the Vpu C-terminal region forms soluble homooligomers that dissociate upon binding to Ca2+-CaM. Collectively, our results suggest that soluble Vpu and Ca2+-CaM form an equimolar complex. DEER analysis of Vpu C-terminal region spin-labeled at residues Q36C/I61C demonstrated that Vpu undergoes significant conformational changes to facilitate Ca2+-CaM binding. These findings could be relevant to Vpu-CaM interactions under physiological conditions.

biophysics↗

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↗