19th-banner-rev.gif

Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Computational Modeling of Allostery-Driven Unfolding and Translocation of Substrate Proteins

Molecular chaperones employ diverse ATP-dependent mechanisms to effect protein folding and degradation. Chaperonin nanomachines assist protein folding through concerted allostery in bacteria1,2 and sequential allostery in eukaryotic and archaeal organisms3,4. Clp ATPases, which are hexameric ring-shaped AAA+ nanomachines that perform substrate protein (SP) unfolding and translocation for protein degradation, are suggested to undergo sequential allostery5. We use coarse-grained simulations to study the protein remodeling actions of ClpY, which contains a single ATP-binding domain per subunit, onto a tagged four-helix bundle SP (Fig. 1). Our results indicate that unfolding is initiated at the tagged C-terminus via an obligatory intermediate6. Translocation proceeds on a different timescale than unfolding and involves sharp stepped transitions. We find that an ordered sequential mechanism is more effective than random or concerted allostery. In the absence of allosteric motions, mechanical unfolding of the SP in atomic force microscopy experiments proceeds via multiple unfolding pathways. SP threading through a non-allosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.

The p97 nanomachine is a homologue of ClpA and ClpB, which contain two ATP-binding domains per subunit. We use coarse-grained and atomistic simulations to investigate the unfolding mechanism of the four-helix bundle protein coupled with ATP-driven conformational changes in the D2 domain of p97. Our simulations suggest that SP unfolding and translocation takes place as a result of the collaboration between strongly conserved sites, Arg586 and Arg599 residues and the D2 central pore loop. The mechanism of SP translocation involves a mechanical force exerted by the D2 central pore loop combined with substrate binding at the Arginine sites of adjacent subunits. Unlike ClpY-assisted action, SP unfolding and translocation actions effected by p97 are simultaneous. We find that accumulation of the SP chain within the central cavity of the D2 does not result in significant SP refolding.

Fig. 1 Unfolding and translocation of the fusion protein formed by a four-helix bundle protein (purple) and the SsrA peptide (yellow) by the ClpY ATPases (green). For clarity, two of the six ClpY subunits are not shown.

This research has been supported by a grant from the American Heart Association and by the National Science Foundation CAREER grant to G. S. and an University Research Council fellowship at the University of Cincinnati to M.J.

References

  1. D. Thirumalai and G. H. Lorimer Annu. Rev. Biophys. Biomol. Struct. 30, 245-269 (2001).
  2. G. Stan, G. H. Lorimer, D. Thirumalai, B. R. Brooks Proc. Natl. Acad. Sci. USA 104, 8803–8808 (2007).
  3. A. Horovitz and K. Willison Curr. Op. Struct. Biol. 15, 646-651 (2005).
  4. M. Jayasinghe, C. Tewmey, G. Stan Proteins: Structure, Function, and Bioinformatics 78, 1254-1265 (2010).
  5. A. Martin, T. A. Baker, R. T. Sauer Nature 437, 1115—1120 (2005).
  6. A. Kravats, M. Jayasinghe, G. Stan Proc. Natl. Acad. Sci. USA (in press).

George Stan* 1
Andrea Kravats1
Sam Tonddast-Navaei1
and Manori Jayasinghe 2

1Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221
2Department of Chemistry, Northern Kentucky University, Highland Heights, KY 41099

ph: 513-556-3049;
fx: 513-556-9239;

george.stan@uc.edu