Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.
Thursday, October 2, 2014
Mechanochemical basis of protein degradation by a double-ring AAA+ machine
Adrian O Olivares, Andrew R Nager, Ohad Iosefson, Robert T Sauer & Tania A Baker
Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.
Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.
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