Determination of structure of double mutant T41I/T78I of M-PMV matrix protein
Jan Prchal1, Jiri Vlach1, Jan Lipov2 , Richard Hrabal1, Tomas Ruml2
1Laboratory of NMR spectroscopy, Institute of Chemical Technology, 166 28 Prague
2 Department of Biochemistry and Microbiology, Institute of Chemical Technology, 166 28 Prague
The Mason-Pfizer`s Monkey Virus (M-PMV) is a prototype of D type retroviruses. In type B and D retroviruses inmature virus particles pre-assemble in cytoplasma, whereas in type C retroviruses (HIV) Gag is targeted to the plasma membrane, where the particle formation occurs. The N-terminal domain of Gag, the matrix protein (MA), plays a critical role in determining this morphogenic difference. Several single- or multi-point mutations of MA have been described that alter various stages of the M-PMV life cycle. Our goal is to characterise the influence of mutant proteins in M-PMV on the molecular level by studying their structures. The three- dimensional structures of the wild-type and R55F mutant, a mutant which changes capsid assembly from D type to C type, are known so far.
In this work we focus on the determination of the three-dimensional structure of the double-mutant T41I/T78I, which doesn`t affect assembly and transport of immature virus particles. However, these particles are unable to bud through the cytoplasmatic membrane and rather accumulate on it. We determined the three-dimensional structure of the MA mutant using heteronuclear NMR spectroscopy. For calculation of the structure we used distance restraints obtained from N and C edited NOESY spectra and residual dipolar couplings measured in polyakrylamide gel. Comparison of the calculated structure and the structure of the wild-type proteins shows that the mutation altered angles between protein helices. In contrast to wild-type proteins, mutated isoleucines 41 and 78 are oriented inside the protein core where they may interact with a myristic acid which is linked to the N-terminus. This finding supports a hypothesis that the phenotypic change of the mutant is caused by enhanced interaction of the myristic acid with the protein core, which prevents association of immature viral particles with the cytoplasmatic membrane.