title: The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair.
abstract: Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident collagen-specific chaperone and essential for proper formation of the characteristic collagen triple helix. It preferentially binds to the folded conformation of its clients and accompanies them from the ER to the Golgi compartment, where it releases them and is recycled back to the ER. Unlike other chaperones, the binding and release cycles are not governed by nucleotide exchange and hydrolysis, but presumably the dissociation of the HSP47-procollagen complex is triggered by the lower pH in the Golgi (pH 6.3) compared with the ER (pH 7.4). Histidine residues have been suggested as triggers due to their approximate textbook pKa value of 6.1 for their side chains. We present here an extensive theoretical and experimental study of the 14 histidine residues present in canine HSP47, where we have mutated all histidine residues in the collagen binding interface and additionally all of those that were predicted to undergo a significant change in protonation state between pH 7 and 6. These mutants were characterized by biolayer interferometry for their pH-dependent binding to a collagen model. One mutant (H238N) loses binding, which can be explained by a rearrangement of the Arg(222) and Asp(385) residues, which are crucial for specific collagen recognition. Most of the other mutants were remarkably silent, but a double mutant with His(273) and His(274) exchanged for asparagines exhibits a much less pronounced pH dependence of collagen binding. This effect is mainly caused by a lower koff at the low pH values.
authors.affiliation: Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse 12-14, D-50674 Cologne, Germany.
authors.forename: Sinan
authors.initials: S
authors.lastname: Oecal
authors.affiliation: Division of Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Fahrstrasse 17, D-91054 Erlangen, Germany.
authors.forename: Eileen
authors.initials: E
authors.lastname: Socher
authors.affiliation: Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse 12-14, D-50674 Cologne, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Uthoff
authors.affiliation: Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse 12-14, D-50674 Cologne, Germany.
authors.forename: Corvin
authors.initials: C
authors.lastname: Ernst
authors.affiliation: Center for Biochemistry, Medical Faculty, University of Cologne, Joseph Stelzmann Strasse 52, D-50931 Cologne, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Zaucke
authors.affiliation: Division of Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Fahrstrasse 17, D-91054 Erlangen, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sticht
authors.affiliation: Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse 12-14, D-50674 Cologne, Germany. Electronic address: ubaumann@uni-koeln.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Baumann
authors.affiliation: Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse 12-14, D-50674 Cologne, Germany. Electronic address: jan.gebauer@uni-koeln.de.
authors.forename: Jan M
authors.initials: JM
authors.lastname: Gebauer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D050958:HSP47 Heat-Shock Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010802:Phylogeny; D011485:Protein Binding; D011489:Protein Denaturation; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D050958:HSP47 Heat-Shock Proteins; D018832:Molecular Chaperones; D006639:Histidine; D003094:Collagen
keywords: collagen; heat shock protein (HSP); histidine; molecular chaperone; protein folding