Eukaryotes and archaea use a protease called the proteasome that has an integral role in maintaining cellular function through the selective degradation of proteins1,2,3,4. Proteolysis occurs in a barrel-shaped 20S core particle, which in Thermoplasma acidophilum is built from four stacked homoheptameric rings of subunits, α and β, arranged α7β7β7α7 (ref. 5). These rings form three interconnected cavities, including a pair of antechambers (formed by α7β7) through which substrates are passed before degradation and a catalytic chamber (β7β7) where the peptide-bond hydrolysis reaction occurs4,5. Although it is clear that substrates must be unfolded to enter through narrow, gated passageways (13 Å in diameter) located on the α-rings1,6,7, the structural and dynamical properties of substrates inside the proteasome antechamber remain unclear. Confinement in the antechamber might be expected to promote folding and thus impede proteolysis. Here we investigate the folding, stability and dynamics of three small protein substrates in the antechamber by methyl transverse-relaxation-optimized NMR spectroscopy8. We show that these substrates interact actively with the antechamber walls and have drastically altered kinetic and equilibrium properties that maintain them in unstructured states so as to be accessible for hydrolysis.
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The authors would like to thank J. Forman-Kay for providing laboratory space and for discussions, R. Muhandiram for NMR support, H. Lin for help designing protein purification protocols, X. Li and A. Shimmer for assistance with the plate reader, and W. Baumeister for discussions. T.L.R. acknowledges The European Molecular Biology Organization (ALTF 827-2006) and The Canadian Institutes of Health Research (CIHR) for postdoctoral fellowships. L.E.K. holds a Canada Research Chair in Biochemistry. This work was supported by grants from the CIHR and the Natural Sciences and Engineering Research Council of Canada.
The authors declare no competing financial interests.
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Ruschak, A., Religa, T., Breuer, S. et al. The proteasome antechamber maintains substrates in an unfolded state. Nature 467, 868–871 (2010) doi:10.1038/nature09444
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