Collagen's primary structure determines collagen:HSP47 complex stoichiometry

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.

Keywords: collagen; crystal structure; extracellular matrix proteins; protein–protein interaction; stoichiometry.

Figure 1

HSP47 binds only very few peptides in the N-terminal collagen II peptide library.A and B, binding of HSP47 was investigated using ELISA-style binding assays immobilizing different collagen model peptides and incubating with soluble HSP47. K_D values for all measured library peptides can be found in Table 1. C, the role of a glutamate residue at the X₀ position was investigated. K_D collagen I: 234 ± 34; GPRGPP: 36 ± 3; GER: 606 ± 322. D, leucine or phenylalanine at the X + 1 position does not prohibit HSP47 binding. The K_D determined b curve fittings are 21.7 ± 3, 12.0 ± 1.4, 24.1 ± 5.4 and 115.6 ± 7.6 nM for RGP, RGF, RGL, and collagen I, respectively. All measurements were performed in triplicates. In the data termed “Block,” no CMPs were immobilized, i.e., they indicate the interaction of HSP47 with just the blocking reagent.

Figure 2

Prediction RGF & RGL containing collagens bound to HSP47. Amino acids at X₊₁ position were replaced by a phenylalanine or leucine respectively in our previously solved crystal structure of HSP47 and the collagen model peptide. Close-ups of the binding interface of the A- and B-site indicate clashes, which are particularly clear for the RGF peptide. The leading, middle, and trailing strands of the collagen helix are depicted in yellow, green, and orange, respectively.

Figure 3

Stoichiometry in HSP47 crystal structures.A, mutant and wild-type HSP47 (here the H271N/H271N double mutant shown in pink) always crystallized as a 2:1 complex when combined with RGP peptides of various lengths. B, complexes with RGL or RGF containing collagen model peptides exclusively crystallized in a 1:1 complex stoichiometry. C, alignment of these structures using the collagen triple helix as a fixpoint showed an overall identical binding mode (RMSD over Cα < 0.7 Å), despite the difference in stoichiometry. In pink color is depicted the structure with the RGP collagen model peptide, in slate color the structures of the RGF and RGL complexes, with the hypothetical second HSP47 molecule depicted in gray color. The leading, middle, and trailing strands of the collagen helix are depicted in yellow, green, and orange, respectively.

Figure 4

Crystal structure of RGF and RGL containing collagen model peptides bound to HSP47. Crystal structures of HSP47 in complex with RGF and RGL containing collagen model peptides were solved. Inspection of the interface indicated a concerted movement of the His₂₇₄ containing loop as well as Met₂₂₅ and Arg₂₂₈ to create a minor groove, perfectly shielding the collagen’s phenylalanine from the solvent. The effect is less pronounced in RGL, where mostly His₂₇₄ moves. Left panels show in slate color the RGF/RGL structures with the molecular surface. The middle panels indicate in gray color the surface of the structures of the docked RGF and RGL peptides on the previously published structures (PDB entry 4AU2). The right panels show an overlay of HSP47 structure of the RGF and RGL structures with the modeled HSP47 structure with an RGP peptide (PDB 4AU2). The leading, middle, and trailing strands of the collagen helix are depicted in yellow, green, and orange, respectively.

Figure 5

Comparison of the 1:1 complex of the newly solved HSP47:RGF/RGL with the 2:1 complex of HSP47:RGP. The newly solved 1:1 complexes of RGF (A) and RGL (B) were aligned to the 2:1 HSP47:RPG complex with central collagen model peptide as fixpoint. The HSP47 molecules from the B-site are colored in gray. To visualize potential conformations, multiple rotamers are shown for the phenylalanine in the middle strand—the rotamer found in the crystal structure is highlighted in red (A). The leading, middle, and trailing strands of the collagen helix are depicted in yellow, green, and orange, respectively.

Figure 6

Stoichiometry of the HSP47 complexes in solution measured by AUC. Analytical ultracentrifugation was performed to determine the stoichiometry of the complex in solution. Collagen model peptides were fluorescently labeled and its sedimentation constant determined in dependency of the presence of different molar excess of HSP47 (A–C). For comparison, sedimentation coefficients were plotted against HSP47 concentrations (D). Ratios are given in CMP:HSP47; concentration of CMP was 1 μM. The g(s∗) analysis reflects the apparent sedimentation coefficient distribution of the particles in solution. For better comparability, the apparent sedimentation coefficient was normalized to water at 20 °C.

Figure 7

Stoichiometry of the HSP47 complexes in solution measured by negative staining EM. HSP47 was mixed with RGP, RGF, and RGL containing collagen model peptides and subjected to negative staining electron microscopy. For visualization HSP47 was directly coupled to 5 nm colloidal gold particles prior the experiment. For each visible collagen helix, the number of attached gold particles was counted. The EM images without markings are shown in Fig S2. Scale bars represent 50 nm.