Roles of the N-domains of the ClpA Unfoldase in Binding Substrate Proteins and in Stable Complex Formation with the ClpP Protease

Abstract

The hexameric cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding and translocation of recognized substrate proteins into the coaxially associated serine protease ClpP. Each subunit of ClpA is composed of an N-terminal domain of approximately 150 amino acids at the top of the cylinder followed by two AAA+ domains. In earlier studies, deletion of the N-domain was shown to have no effect on the rate of unfolding of substrate proteins bearing a C-terminal ssrA tag, but it did reduce the rate of degradation of these proteins (Lo, J. H., Baker, T. A., and Sauer, R. T. (2001) Protein Sci. 10, 551-559; Singh, S. K., Rozycki, J., Ortega, J., Ishikawa, T., Lo, J., Steven, A. C., and Maurizi, M. R. (2001) J. Biol. Chem. 276, 29420-29429). Here we demonstrate, using both fluorescence resonance energy transfer to measure the arrival of substrate at ClpP and competition between wild-type and an inactive mutant form of ClpP, that this effect on degradation is caused by diminished stability of the ClpA-ClpP complex during translocation and proteolysis, effectively disrupting the targeting of unfolded substrates to the protease. We have also examined two larger ssrA-tagged substrates, CFP-GFP-ssrA and luciferase-ssrA, and observed different behaviors. CFP-GFP-ssrA is not efficiently unfolded by the truncated chaperone whereas luciferase-ssrA is, suggesting that the former requires interaction with the N-domains, likely via the body of the protein, to stabilize its binding. Thus, the N-domains play a key allosteric role in complex formation with ClpP and may also have a critical role in recognizing certain tag elements and binding some substrate proteins.