Abstract
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.
Highlights
Interferon-␥ (IFN-␥)1 is a small protein secreted by activated T lymphocytes and natural killer cells
Support for a role of the endoplasmic reticulum DnaK homolog BiP/GRP78 in the de novo folding of IFN-␥ comes from experiments in which IFN-␥-producing Chinese hamster ovary cells were transfected with a BiP ATPase mutant (T37G) that was previously shown to be able to bind peptide, but to be unable to release it upon binding of ATP [17]
The DnaK chaperone and its HSP70 homologs, including the endoplasmic reticulum variant BiP/GRP78, bind preferentially to stretches of hydrophobic amino acids that are usually buried in the core of completely folded proteins, but exposed on unfolded or partially folded proteins
Summary
Screening of DnaK Binding to IFN-␥-derived Cellulose-bound Peptides by Fractionated Electroblotting—Peptide libraries consisting of 44 and 41 13-mer peptides covering the entire mature protein portions of human and mouse IFN-␥, respectively, were prepared by automated spot synthesis [24, 25]. Peptide membranes were allowed to react with 100 nM DnaK (Stressgen Biotech Corp.) in buffer containing 31 mM Tris-HCl, pH 7.6, 170 mM NaCl, 6.4 mM KCl, 0.05% (v/v) Tween 20, and 5.0% (w/v) sucrose for 1 h at 25 °C with gentle shaking. Electrotransfer of peptide-bound DnaK onto polyvinylidene difluoride (PVDF) membranes was performed using a semidry blotter. Cellulose and PVDF membranes were sandwiched between blotting paper soaked in anode buffer (anode buffer I: 30 mM Tris base and 20% MeOH; anode buffer II: 300 mM Tris base and 20% MeOH) and cathode buffer (25 mM Tris base, 40 mM 6-aminohexane acid, and 20% MeOH supplemented with increasing concentrations of SDS). 70 nM DnaK was incubated with 40 mM RCMLA for 2 h at 37 °C in buffer containing 25 mM Tris-HCl, 20 mM HEPES, pH 7.15, 47.5 mM KCl, and 2.25 mM Mg(OAc) in the presence of competing peptide (0 –500 M).
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