title: Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine.
abstract: In the axial channels of ClpX and related hexameric AAA+ protein-remodeling rings, the pore-1 loops are thought to play important roles in engaging, mechanically unfolding, and translocating protein substrates. How these loops perform these functions and whether they also prevent substrate dissociation to ensure processive degradation by AAA+ proteases are open questions. Using ClpX pore-1-loop variants, single-molecule force spectroscopy, and ensemble assays, we find that the six pore-1 loops function synchronously to grip and unfold protein substrates during a power stroke but are not important in preventing substrate slipping between power strokes. The importance of grip strength is task dependent. ClpX variants with multiple mutant pore-1 loops translocate substrates as well as the wild-type enzyme against a resisting force but show unfolding defects and a higher frequency of substrate release. These problems are magnified for more mechanically stable target proteins, supporting a threshold model of substrate gripping.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Ohad
authors.initials: O
authors.lastname: Iosefson
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Adrian O
authors.initials: AO
authors.lastname: Olivares
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: bobsauer@mit.edu.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D017510:Protein Folding; D012240:Rhodococcus / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins