title: Peptide and protein binding in the axial channel of Hsp104. Insights into the mechanism of protein unfolding.
abstract: The AAA+ molecular chaperone Hsp104 mediates the extraction of proteins from aggregates by unfolding and threading them through its axial channel in an ATP-driven process. An Hsp104-binding peptide selected from solid phase arrays enhanced the refolding of a firefly luciferase-peptide fusion protein. Analysis of peptide binding using tryptophan fluorescence revealed two distinct binding sites, one in each AAA+ module of Hsp104. As a further indication of the relevance of peptide binding to the Hsp104 mechanism, we found that it competes with the binding of a model unfolded protein, reduced carboxymethylated alpha-lactalbumin. Inactivation of the pore loops in either AAA+ module prevented stable peptide and protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of ATPase turnover in the second AAA+ module of this mutant was abolished. Drawing on these data, we propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module.
authors.affiliation: Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada.
authors.forename: Ronnie
authors.initials: R
authors.lastname: Lum
authors.forename: Monika
authors.initials: M
authors.lastname: Niggemann
authors.forename: John R
authors.initials: JR
authors.lastname: Glover