Structural and dynamic studies of the 12 kDa form of protease from Mason-Pfizer monkey virus.

Václav Veverka1, Jan Lang1, Helena Bauerová2, Iva Pichová2 and Richard Hrabal1


1Laboratory of NMR Spectroscopy, Institute of Chemical Technology, Technická 5,

166 28  Prague 6, Czech Republic, e-mail:

2Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10  Prague 6, Czech Republic


Mason-Pfizer monkey virus encodes an aspartic protease (M-PMV PR), which is essential for the correct assembly and maturation of the virion particles. The protease processes viral protein precursors yielding fully functional structural proteins and enzymes. It was demonstrated that the enzyme exists in three active forms with molecular mass of 17, 13, and 12 kDa per monomer, which makes M-PMV PR quite unique among other retroviral proteases [1]. 

We will report a complete three-dimensional structure of the shortest form of the protease (12 kDa) where both cysteine residues (Cys7 and Cys106) were mutated for alanines to prevent their oxidation [2] and the activity of the protease was suppressed by an exchange of the catalytic aspartate for asparigine in the position 26. Doubly labeled (13C/15N) sample was prepared and the resonance assignment was based on triple resonance multidimensional NMR experiments [3].  Based on the calculation of chemical shift index (CSI) approximate positions of secondary structure elements were located. The refinement of the structure was carried out by ARIA software package [4] based on NOE contacts, dihedral angle restraints and hydrogen bonds. To support the structural results we also measured 15N relaxation properties of M-PMV PR to obtain a picture of dynamic behavior of the protein.

It turned out that the lower activity of the shortest form of the protease, as compared with the fully active 17 kDa form, is caused by the prevailing monomer in solution. This result was supported by ultracentrifugation experiments. We have proved that the monomeric form of the 12 kDa M-PMV PR is folded similarly as the other retroviral proteases with several distinct features, which will be discussed.



Acknowledgment: The work has been supported by the Grant Agency of the Czech Republic (Grant 203/00/1241)




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