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:
hrabalr@vscht.cz
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)
References:
1
Zábranský
A., Andreanský M., Hrušková-Heidingsfeldová O., Havlíček V., Hunter E., Ruml
T., Pichová I., Virology, 245(2), 250-256 (1998).
2.
Yamazaki,T.,
Nicholson L.K., Torchia D.A., Stahl S.J., Kaufman J.D., Wingfield P.T.,
Domaille P.J., Campbell-Burk S. Eur. J.Biochem., 219, 707-712 (1994).
3.
Veverka V., Bauerova H., Zabransky A., Pichova I., and Hrabal R., J.
Biomolecular NMR, 291-292
(2001).
4. Nilges M., Macias J., O’Donoghue S.I., Oschkinat H., J.Mol.Biol., 408-422 (1997).