Peptide and Protein Binding in the Axial Channel of Hsp104

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 ␣-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.Hsp104 is a AAAϩ protein disaggregase that functions in yeast in the resolubilization and reactivation of thermally denatured and aggregated proteins (1, 2). In unstressed cells, Hsp104 is critical to the mitotic stability of the yeast prions [, and [URE3] (3-5). Hsp104 and its bacterial orthologue ClpB are members of the Hsp100/Clp family of proteins (6). Other Hsp100s, such as ClpA, ClpX, and ClpY (HslU), unfold and unidirectionally translocate polypeptides through a central axial channel (7-11). Crystal structures of HslU (12, 13) and cryoelectron microscopic reconstructions of ClpB (14) reveal that the diameter of the axial channel is regulated by flexible loops whose conformation is regulated by the nucleotide status of the nucleotide binding domain of each AAAϩ module. Modification of these loops impairs protein translocation and/or degradation implying that these loops play critical roles in translocation (15-18). Likewise, mutation of the flexible loops of Hsp104 and ClpB results in refolding defects suggesting that all Hsp100s employ a similar unfolding/threading mechanism to process substrates whether they are ultimately degraded or refolded (16,19,20). Despite the growing body of knowledge regarding the unfolding and translocation mechanism of Hsp104, the determinants of the initial stage of the unfolding process, substrate recognition and binding, remain unclear.In other Hsp100s, recognition of specific peptide sequences initiates unfolding and translocation. Protein substrates of ClpXP generally contain recognition signals of roughly 10 -15 residues that can be located either at the N or C termini (21). The SsrA tag, an 11-amino acid peptide (AANDENYALAA) that is appended to the C terminus of polypeptides by the action of transfer-messenger RNA on stalled ribosomes (22), is a particularly well studied example of an Hsp10...

Insight into Molecular Basis of Curing of [PSI+] Prion by Overexpression of 104-kDa Heat Shock Protein (Hsp104)

“…Y662A lacking conserved tyrosine residues in the proposed axial channel loops in the first and second AAAϩ domains, respectively (35) (Fig. 2C).…”

“…Trap , a derivative of Hsp104 that can bind but not hydrolyze ATP (35). Md also inhibited fRCMLa binding (Fig.…”

“…It is known that specific short peptides (13-mers) (35), model unfolded proteins (36,43), and poly-L-lysine (44,45) can bind to Hsp104 and stimulate its ATPase activity. For comparison of the effect of Md on Hsp104, we used peptide p370 (KLFSDDVFEREYA) that has been previously characterized and found to stimulate the ATPase activity in the second AAAϩ domain of Hsp104 in a mutant of Hsp104 in which ATP hydrolysis of the first AAAϩ domain is inactivated by the substitution of Ala for the catalytic Glu residue in the Walker B motif (Hsp104 E285A ) (35). We found that Md stimulated the ATPase activity of Hsp104 E285A to about the same extent as p370 (Fig.…”

“…C, the number of red-pigmented colonies (cured) on all plates was scored as a percentage of the total number of colonies counted. D, the ATPase activity of Hsp104 E285A was measured in the presence of Md, the Hsp104-binding peptide p370, and the nonbinding peptide pSGG (35) and expressed as the fold-increase relative to unstimulated activity. E, fRCMLa binding to Hsp104…”

“…E285A were all performed as previously described (35). ] caused by the expression of Md was scored by the appearance of red-pigmented colonies on plates lacking copper indicating the loss of suppression of the ade1-14 allele present in OT55 (Fig.…”