Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen.
As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.
Thomson
Christy A
CA
Department of Biochemistry, McMaster University, 1200 Main Street, Hamilton, Ontario, Canada L8N 3Z5.
Tenni
Ruggero
R
Ananthanarayanan
Vettai S
VS
D001667:Binding, Competitive; D002942:Circular Dichroism; D024042:Collagen Type I / Q000737:chemistry / Q000378:metabolism*; D024043:Collagen Type II / Q000737:chemistry / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism; D003488:Cyanogen Bromide / Q000737:chemistry*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D011869:Radioligand Assay; D013379:Substrate Specificity; D013460:Sulfur Isotopes
D024042:Collagen Type I; D024043:Collagen Type II; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D013460:Sulfur Isotopes; D017364:Collagenases; D003488:Cyanogen Bromide