Affinity and Conformations of Ca 2+ -Calmodulin Bound to Peptides Containing the Calmodulin-Binding Motif of HIV-1 Matrix Protein
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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 (Ca 2+ -CaM), elucidate protein regions that are critical to the heterocomplex formation, and monitor the conformational alterations in Ca 2+ -CaM upon binding to MA.
Methods
We studied Ca 2+ -CaM binding to synthetic MA peptides representing full-length wild type (FL WT), a C-terminal truncation lacking the KKK cluster, and an FL K→N/K→H/K→Q 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 Förster resonance energy transfer (eFRET) using 100 nM Cy3-MA and increasing Cy5-Ca 2+ -CaM concentrations in the range of 50 nM to 2.5 µM. Conformational changes in doubly spin-labeled Ca 2+ -CaM upon MA binding were probed by double electron-electron resonance (DEER) spectroscopy.
Results
Cy3 quenching and FRET analyses of complexes with Cy5-Ca 2+ -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–Ca 2+ -CaM binding. DEER measurements of Ca 2+ -CaM labeled at S39C and A103C showed that FL WT MA binding shifted the ∼5.3 nm interspin distance of extended Ca 2+ -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-Ca 2+ -CaM interactions and clarify how HIV-1 proteins exploit CaM.
