DYNAMICAL ANALYSIS OF WT M-PMV MATRIX PROTEIN AND ITS SINGLE POINT MUTANT R55F WITH RESPECT TO THEIR DIFFERENT OLIGOMERIZATION PROPERTIES

 

Pavel Srb1,2, Jan Prchal2,3, Jiří Vlach2 Jan Lang 1,2, Marián Grocký1, Jan, Tomáš Ruml3 and Richard Hrabal2

 

1Department of Low Temperature Physics, Faculty of Mathematics and Physics, Charles University, V Holešovičkách 2, 180 00 Prague 8, Czech Republic

2Laboratory of NMR Spectroscopy and

3Department of Biochemistry and Microbiology

Institute of Chemical Technology, Technická 5, 166 28 Prague 6, Czech Republic

richard.hrabal@vscht.cz

 

Mason-Pfizer monkey virus (M-PMV) belongs to the genus of betaretroviruses, in which the matrix protein (MA) plays the essential role in certain stages of their life cycle (e.g. in the assembly, transport and budding of new viral particles). Several single or double point mutants are known to cause dramatic changes in the virus life cycle. In particular, the single point mutation R55F in MA redirects the assembly of the viral capsid to the plasma membrane instead of to cytoplasm, which is the place of assembly of the wild type form (WT).

In our recent work [1] we found that M-PMV WT MA exists in a monomer–dimer–trimer equilibrium in solution with the corresponding dissociation constants of 2.3 mM and 0.24 mM, respectively. Conversely, almost negligible and nonspecific oligomerization was observed for the R55F mutant. Structural comparison of matrix proteins explained their different behavior in solution, concluding that the key residues involved in the intermonomeric interaction are exposed in the WT MA while being buried in the mutant, which prevents the oligomerization of R55F MA.

Here, we present the motional analyses of the experimental 15N NMR relaxation and “in silico” molecular dynamic simulations (MD) for WT and R55F MAs. Both approaches enlighten molecular motions from the microscopic point of view. Analysis of experimental data provides information about global reorientation of molecule, flexibility of each residue (through order parameters, S2) and for most sites also the timescale of local motion. Although MD simulations provide a wealth of information giving almost complete picture of protein dynamics, careful analysis of results is necessary to avoid possible artifacts coming from poor sampling of rare dynamical processes during simulated trajectory. Two computational approaches were used here; the first one was the isotropical reorientational eigenmode dynamics (iRED) [2] which provided especially information about correlated motions. The second one was the calculation of the timescale window-dependent order parameters, S2(w), which provided information inaccessible by experiment. Computational routine is implemented in program PAIN [3]. Important changes in local dynamics caused by the single point mutation will be demonstrated and discussed.

References

            [1] J. Vlach et al. J. Mol. Biol., 390, 5, 967-980, (2009)

[2] J. Prompers, R. Brüschweiler, J.Am.Chem.Soc., 124, 4522-4534, (2002)

            [3] P. Macek, et al., J.Phys.Chem.B, 111, 5731-5739, (2007)