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This study characterizes the binding interaction between HIV-1 matrix (MA) protein and host calmodulin (CaM), which may facilitate viral trafficking and assembly. Using fluorescence resonance energy transfer and electron spin resonance spectroscopy, researchers found that MA binds to calcium-bound CaM with nanomolar affinity (dissociation constants of 65-250 nM) and induces a major conformational change in CaM from an extended structure to a more compact form. The positively charged KKK cluster in MA contributes minimally to binding affinity, while hydrophobic regions appear more critical for the interaction.
Why it matters
Understanding how HIV-1 hijacks host proteins like calmodulin to facilitate viral replication could inform the development of antiviral therapies that disrupt these specific protein-protein interactions. The thermodynamic and structural data provided here establishes baseline characteristics that could guide rational drug design targeting the MA-CaM interface.
⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
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.