Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.10.19.760223

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

Saffarian Delkhosh, A.·Ogunbowale, A.·Hadadianpour, E.·Mahmud, K. M.·Borbat, P. P.·Georgieva, E. R.
View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Saffarian Delkhosh, A., Ogunbowale, A., Hadadianpour, E., Mahmud, K. M., Borbat, P. P., Georgieva, E. R.. 2026-10-24. Affinity and Conformations of Ca2+-Calmodulin Bound to Peptides Containing the Calmodulin-Binding Motif of HIV-1 Matrix Protein. https://doi.org/10.64898/2026.10.19.760223

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Cancers relax selection on charge at the mitochondrial complex I quinone cavity

Mitochondrial complex I translocates protons through hydrated channels whose connectivity and electrostatics are both defined by structure, but whether they are distinctly constrained is unknown. From a 2.39 cryo-EM structure modelling 2,945 ordered waters, we built the proton wire of the seven mtDNA-encoded ND subunits and scored all 40,166 missense substitutions on two near-orthogonal axes (r = 0.17): electrostatic shift at proton-loading sites, and topological disruption of the wire. Across 858 germline variant sites, loading sites (0.51) and cut vertices (0.67) are both depleted. In tumours only loading sites are enriched (1.42 TCGA, 1.67 PCAWG, 1.29 MSK-IMPACT), a 3.06-fold difference tested as a wire class tissue-type interaction (P = 3.6 10-). It survives adjustment for conservation, burial and coverage, and is concentrated in ND1 within 15 of the quinone cavity, not spread along the proton path. Selection separates charge from connectivity, without establishing a fitness benefit.

biophysics↗
bioRxiv2026

Rugnux: from diffraction images to structure at synchrotrons and XFELs in one program

Rising data rates in macromolecular crystallography outpace multi-program pipelines on computing clusters. Rugnux, a single GPU-accelerated program, turns rotation or serial still diffraction images into merged data and an initial structure. At synchrotrons, results arrive seconds after the last image; on one workstation, 207 public datasets from 36 sources took a median of 15 s each, with deposition-quality. Serial XFEL data were processed faster than the collection rate, enabling real-time structure determination.

biophysics↗
bioRxiv2026

Phase Separation of Intracellular Proteins Influences Synaptic Bistability

Synaptic bistability at the post synaptic density has been proposed to be the result of the nucleation and evaporation of receptor clusters. The exact biophysical mechanism underlying these inter-receptor attractions that can trigger nucleation remains to be elucidated. Emerging evidence demonstrates that numerous postsynaptic density submembrane proteins, that interact with various membrane receptors, can undergo phase separation, necessitating a revised conceptual model. By employing Langevin dynamics simulations of coarse-grained scaffold proteins and interacting membrane receptors, we study how phase transitions of scaffold proteins alter receptor clustering at the membrane. In our model, scaffold macromolecules can undergo phase separation and interact with receptor molecules at the model membranes. We observed that the presence of scaffold polymers across the membrane creates an attractive interaction between otherwise purely repulsive receptors. By systematically varying the number of receptors at the membrane, we dissected the coupling between receptor clustering and the phase behavior of scaffold macromolecules. We show that the propensity for receptor clustering is enhanced by the condensation of scaffold proteins. We then used two different scaffolds and systematically varied the interaction between them, to change the spatial organization of two condensed phases w.r.t. one another. Interestingly, we observed that receptor clustering can be reduced by co-phase separation of scaffold macromolecules, indicating the role of competing interactions in receptor clustering. Our work advances understanding of receptor-mediated signaling and phase separation, providing insights into cellular compartmentalization and biochemical regulation at the synapse.

biophysics↗