The first crystal structure of monomeric retroviral protease solved by online game players

Miroslaw Gilski 1,2Maciej Kazmierczyk 1Szymon Krzywda 1Helena Zabranska 3Iva Pichova 3Firas Khatib 4Frank DiMaio 4David Baker 4Seth Cooper 5Zoran Popovic 5Mariusz Jaskólski 1,2

1. Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, Poznań 60-780, Poland
2. Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, PAS, Noskowskiego 12/14, Poznań 61-704, Poland
3. Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry, Flemingovo n. 2, Prague 16610, Czech Republic
4. University of Washington, Deptment of Biochemistry, Box 357350, Seattle, WA 98195, United States
5. University of Washington, Deptment of Computer Science and Engineering, Box 352350, Seattle, WA 98195, United States

Abstract

Mason-Pfizer Monkey Virus (M-PMV) causes acquired immunodeficiency syndrome (AIDS) in rhesus monkeys. As in all retroviruses, such as HIV-1, dimerization of its protease (PR) is a obligatory for processing of retroviral polyproteins and virion maturation. All previously determined crystal structures of retroviral proteases show interlaced dimers, representing the proteolytically active enzyme. Retroviral PR (retropepsin) is an unusual homodimeric form of aspartic protease, composed of two protein chains, each contributing a DTG triad to the catalytic center. The active site is covered by two symmetric loops called flaps. Structure-based design of protease inhibitors has led to a number of drugs used in the clinical treatment of AIDS. However their effectiveness is limited because the HIV virus is able to mutate quickly into a drug-resistant forms thus it is necessary to search for new methods of antiretroviral therapy.

An interesting approach would be to block the mandatory dimerization of the protease. Biophysical and NMR studies have indicated that in the absence of a substrate/inhibitor, M-PMV PR should fold into a stable monomer. The retroviral protease of M-PMV indeed crystallizes as a monomer, but despite the availability of several crystal forms, the crystal structure of this protein could not be solved and over a decade has resisted all molecular replacement efforts. Finally, the protein folding puzzle was presented to players of the computer game named Foldit who were challenged with the task to fold the polypeptide chain of the protein.

Foldit is a multiplayer online computer game in which players try to most accurately predict protein structures using human three-dimensional problem-solving skills. They collaborate with teammates while competing with other players to obtain the highest scoring (lowest energy) model. During the three weeks in which the puzzle was active, Foldit players generated over one million of structure model predictions. Finally the monomeric structure of M-PMV was solved by molecular replacement software, using one of the models constructed by Foldit players team Contenders.

The new structure indeed shows a monomeric protein with the termini completely disordered but the flap loop can be clearly identified in the electron density map. The flap has a completely new conformation with an unusual shape and orientation, different from both the open and closed states known from other retropepsins. Thanks to the original idea of the authors of Foldit, and to surprising intuition and three-dimensional skills of anonymous gamers, an important scientific problem could be finally solved. The structure of monomeric retropepsin determined at high resolution provides valuable extra information for the design of dimerization inhibitors that might help in development of new drugs for the treatment of retroviral infections, including AIDS.

 

Related papers
  1. Processing and analysis of synchrotron diffraction images of protein crystals
  2. Mutagenesis and expression of an active-site mutant of yellow lupine L-asparaginase

Presentation: Invited oral at IX Krajowe Sympozjum Użytkowników Promieniowania Synchrotronowego, by Miroslaw Gilski
See On-line Journal of IX Krajowe Sympozjum Użytkowników Promieniowania Synchrotronowego

Submitted: 2011-07-18 18:45
Revised:   2011-09-05 16:17