Is local flexibility of M-PMV matrix protein related to its binding affinity with a molecular motor?

Pavel Srb^1,2, Jan Lang ^1,2,Jiří Vlach², Marian Grocký ¹, Jan Prchal³, Jan Lipov³, Tomáš Ruml^3,4 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

⁴Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech republic, Flemingovo nám. 2, 166 10, 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 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 [1]. 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).

The NMR assignments and three-dimensional structures of WT and R55F have been solved recently by our group [2]. Both structures are composed of four a-helices, however the relative orientation of the N-terminal domain (helices 1 and 2) with respect to the C-terminal one (helices 3 and 4) is different. Such reorientation causes different accessibility of Cytoplasm Targetting/Retention Signal (CTRS) sequence for the interaction with a molecular motor dynein . It is expected that the degree of flexibility of the CTRS sequence will affect binding affinity with the Tctex-1 which is one of the light chains of dynein.

Therefore, we carried out a detailed motional analysis of the loop connecting helices 2 and 3, the least structured part of CTRS. Relaxation parameters of amide ¹⁵N and carbonyl ¹³C nuclei have been studied by NMR spectroscopy on uniformly labelled (¹⁵N and ¹⁵N/¹³C) samples. Additionally, MD trajectory analysis have provided a complementary view on the mobility of CTRS sequence which is crucial for understanding the interation of Gag with dynein.

1. S. S. Rhee and E. Hunter, Cell, 1990, 63, 77–86

2. J. Vlach, J. Lipov, V.Veverka, M.Rumlova, T. Ruml, R.Hrabal, J.Biomol. NMR, 2005, 4, 381-2