Biology subjects

Ogunbowale, A.

Publications and source records attributed to Ogunbowale, A..

3 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↗

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↗