Interaction of the M-PMV matrix protein with phosphatidylinositol bisphosphate

Jan Prchal, Jiri Vlach, Tomas Ruml, Richard Hrabal

Institute of Chemical Technology in Prague, Technická 5, Prague 6, Czech Republic

Polyprotein Gag as a precursor of structural proteins plays a key role in formation and budding of retroviral particles. The N-terminal domain of Gag, the matrix protein (MA) interacts with the cytoplasmic membrane of infected cell through the bipartite signal that involves a cluster of basic residues and myristic acid which is covalently attached to the amino-terminal glycine. In unbound state the myristate is seqestered in MA hydrophobic core. When Gag binds on cytoplasmic membrane, the myristate is released and anchors the MA on the membrane. This is called myristoyl switch. In HIV-1 MA the myristoyl switch is regulated by phosphatidylinositol-4,5-bisphosphate (PIP), which is a phospholipide and is a part of the cytoplasmic membrane. PIP binds on HIV-1 MA and triggers the release of myristoyl, one of its fatty acid chains interacts with MA, strengthening the binding on the membrane. Because PIP is also necessary for HIV-2 and MMLV MA`s binding, we believe that PIP plays an important role in the interaction of M-PMV MA with the membrane.

We have studied the interaction of PIP with MA using NMR spectroscopy and artificial liposomes. For NMR measurement we used soluble PIP whith C₈ fatty acid chains. Upon titration of MA with an increasing concentration of PIP we observed changes of MA chemical shifts. Therefore, STD-NMR (saturation transfer difference) experiment was measured at different MA/PIP ratios.demonstrating that the major binding epitope is located on the fatty acid chains.. These experiments allowed us to prove that the interaction is specific and to calculate an approximate structure of the PIP-MA complex. We also prepared artificial liposomes from phosphatidilcholine with 10% of PIP and mixed them with MA. Bound MA was separated from the ubound molecules by ultracentrifugation.We have proven, that M-PMV MA interacts with PIP specifically and that the binding mode is copletely different from HIV-1 and HIV-2.

This work was supported by the Czech Ministry of Education ME 904.