Abstract
Heat shock protein 47 (HSP47) is a single-substrate molecular chaperone crucial for collagen biosynthesis. Although its function is well established, the molecular mechanisms that govern binding to procollagen peptides and triple helices in the endoplasmic reticulum (followed by controlled release in the Golgi) are unclear. HSP47 binds procollagen at a neutral pH but releases at a pH similar to the pK(a) of the imidazole side chain of histidine residues. It thus seems likely that these residues are involved in this pH-dependent mechanism. Murine HSP47 has 14 histidine residues grouped into three clusters, known as the breach, gate, and shutter. Here, we report the use of histidine mutagenesis to demonstrate the relative contribution of these three clusters to HSP47 structure and the "pH switch." Many of the tested mutants are silent; however, breach mutants H197A and H198A show binding but no apparent pH switch and are unable to control release. Another breach mutant, H191A, shows perturbed collagen release characteristics, consistent with observed perturbations in pH-driven trans-conformational changes. Thus, His-198, His-197 and His-191 are important (if not central) to HSP47 mechanism of binding/release to collagen. This is consistent with the breach cluster residues being well conserved across the HSP47 family.
Highlights
Heat shock protein 47 (HSP47) is a single-substrate molecular chaperone essential for collagen biosynthesis
Because the pH dependence of collagen release is close to the pKa of histidine imidazole, one or more histidine residues have long been considered responsible for the pH sensitivity
We took a site-directed mutagenesis approach with mutant selection based on a homology model of HSP47 WT, constructed using the known crystal structure of ␣1-antitrypsin (a serpin present in mammalian blood plasma (Protein Data Bank code 1QLP) [29]
Summary
HSP47 is a single-substrate molecular chaperone essential for collagen biosynthesis. Results: H197A and H198A mutants show binding but no apparent pH switch, whereas H191A shows perturbed collagen release characteristics. His-198, His-197 and His-191 are important (if not central) to HSP47 mechanism of binding/release to collagen This is consistent with the breach cluster residues being well conserved across the HSP47 family. Breach Histidine Cluster Controls HSP47 pH Switch Mechanism substrate molecular chaperone essential for collagen biosynthesis. We have suggested that the pH profile of the switch mechanism would be consistent with an active role for histidine residues in procollagen polypeptide binding and release [22]. Ogy model of HSP47 WT that was developed in association with our previous work [14, 22] Using this model, we describe our efforts to devise a series of site-directed mutants of HSP47 that target evolutionarily conserved histidine clusters to evaluate their respective roles in HSP47 structure and in the pH switch mechanism. This histidine mutagenesis study is complemented by trptophan-tophenylalanine (WF) mutants designed to probe for those HSP47 subdomains affected by the pH-dependant conformational changes
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