The conserved helix C region in the superfamily of interferon-gamma /interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK.
HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the alpha-helical homodimeric secretory cytokine interferon-gamma (IFN-gamma). We screened solid-phase peptide libraries from human and mouse IFN-gamma to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN-gamma dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the alpha-helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-gamma, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.
Vandenbroeck
Koen
K
Biomolecular Sciences Research Group, McClay Research Centre for Pharmaceutical Sciences, Queen's University of Belfast, United Kingdom. k.vandenbroeck@qub.ac.uk
Alloza
Iraide
I
Brehmer
Dirk
D
Billiau
Alfons
A
Proost
Paul
P
McFerran
Neil
N
Rüdiger
Stefan
S
Walker
Brian
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000378:metabolism*; D016753:Interleukin-10 / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; C000717248:Hspa5 protein, mouse; D016753:Interleukin-10; D007371:Interferon-gamma; C063842:dnaK protein, E coli