Structural and functional insights into calmodulin-mediated lipid binding and proteolytic cleavage of the M-PMV matrix protein

The matrix (MA) domain of the Mason-Pfizer monkey virus (M-PMV) Gag polyprotein plays a central role in retroviral assembly and trafficking, coordinating membrane association and proteolytic maturation. Unlike HIV-1, M-PMV assembles immature particles in the cytoplasm prior to plasma membrane targeting, but the molecular mechanisms governing this process remain poorly understood. Here, we identify calmodulin (CaM) as a calcium-dependent modulator of MA structural dynamics. Using a combination of biophysical and biochemical methods, we demonstrate that CaM directly interacts with myristoylated MA, promoting its oligomerization and enhancing its cleavage by the viral protease. In-depth characterization of MA-CaM complex by protein cross-linking mass spectrometry, hydrogen/deuterium exchange mass spectrometry and NMR spectroscopy reveals that the N-terminal parts of both proteins are in close proximity within the complex and that CaM binding induces increased conformational flexibility of key regions within MA, including the basic patch and C-terminal cleavage site. These dynamic changes suggest an allosteric mechanism by which CaM regulates MA function, potentially facilitating the temporal coordination of membrane targeting, the myristoyl switch and proteolytic processing. Our findings broaden the understanding of CaM as a regulatory factor in retroviral assembly and underscore the importance of conformational plasticity in viral maturation.