Solution NMR characterizations of oligomerization and dynamics of equine infectious anemia virus matrix protein and its interaction with PIP2

Budding of retroviruses requires the structural precursor polyprotein, Gag, to target the plasma membrane through its N-terminal matrix (MA) domain. For HIV-1, the interaction between membrane signaling molecule phosphatidylinositol 4,5-diphosphate (PIP2) and MA induces the exposure of myristate and promotes membrane binding. Here we studied oligomerization of the naturally unmyristylated equine infectious anemia virus (EIAV) MA and its interaction with PIP2-C4 primarily using solution NMR spectroscopy. The measured H-1-N-15 residual dipolar coupling agrees with the atomic coordinates from the EIAV MA crystal structure. The analytical ultracentrifugation results show a dominant population of monomeric EIAV MA at a concentration of 63 mu M and 20 degrees C, along with a small trimer and a broad distribution of other oligomers. The monomer-trimer equilibrium model and the quaternary packing of the trimer were further established by the con centration-dependent N-15 spin relaxation rates and chemical shifts. Binding of MA to PIP2-C4 was detected by chemical shift mapping (CSM) with an apparent K-d of 182 +/- 56 mu M, a value similar to that reported for HIV-1 MA. The PIP2 binding site includes the Loop region between Helix2 and Helix3 in the EIAV MA. CSM and spin relaxation dispersion, reveal a coupling of conformational change and submillisecond dynamics, respectively, between the Loop and trimeric Interface Residues due to PIP2 binding. We infer that PIP2 participates in the initial trimer formation of EIAV MA, but more importantly, the concentration effect is dominant in shifting the equilibrium toward trimer, in line with the entropic switch mechanism proposed for myristylated HIV-1 MA.