Affinity and Conformations of Ca 2+ -Calmodulin Bound to Peptides Containing the Calmodulin-Binding Motif of HIV-1 Matrix Protein.

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 (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.

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