Abstract
Collagens play important roles in development and homeostasis in most higher organisms. In order to function, collagens require the specific chaperone HSP47 for proper folding and secretion. HSP47 is known to bind to the collagen triple helix, but the exact positions and numbers of binding sites are not clear. Here, we employed a collagen II peptide library to characterize high-affinity binding sites for HSP47. We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. In contrast, large hydrophobic amino acids such as phenylalanine at certain positions in the collagen sequence increase binding strength. For further characterization, we determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large hydrophobic side chain from the middle strand of the collagen triple helix and, most surprisingly, possess an altered binding stoichiometry in the form of a 1:1 complex. This altered stoichiometry is explained by steric collisions with the second HSP47 molecule present in all structures determined thus far caused by the newly introduced large hydrophobic residue placed on the trailing strand. This exemplifies the importance of considering all three sites of homotrimeric collagen as independent interaction surfaces and may provide insight into the formation of higher oligomeric complexes at promiscuous collagen-binding sites.
Highlights
Some studies have investigated the influence of adjacent residues on the HSP47 interaction (17, 18), none has checked the actual binding site of HSP47 on a native collagen triple helix sequence
In a first in-silico approach to explain the discrepancy between the published prediction and our observation on the number of sites, we used the MutaBind[2] algorithm to predict the ΔΔG values for all binding sites individually based on our Journal previously solved crystal structure of HSP47 with a GPR containing collagen model peptide (21)
The collagen sequence of peptide 5 for example is (GPP)6GARGFPGTPGLPGVKGHRGYPGLDG AK(GPP)[6], which contains two potential binding sites marked by underlines
Summary
We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. Large hydrophobic amino acids like phenylalanine at certain positions in the collagen sequence increase binding strength. We determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large
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