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Chain alignment of collagen I deciphered using computationally designed heterotrimers

Abstract

The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.

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Fig. 1: Design and structure of register-specific heterotrimers.
Fig. 2: Binding affinity of registers for collagen-binding proteins.
Fig. 3: Peptide-induced DDR1 and DDR2 autophosphorylation.
Fig. 4: Nine possible modes for binding of COL1 to DDR2 and VWF A3.

Data availability

Atomic coordinates of AAB (6Q3P), ABA (6Q41) and BAA (6Q43) crystal structures have been deposited with the Protein Data Bank. Raw data associated with Figs. 1,2,3 can be provided by the corresponding author upon reasonable request.

Code availability

The code for computational design of heterotrimers may be requested from the corresponding author.

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Acknowledgements

A.A.J. was supported by Newton International Fellowship (NF140721) granted jointly by the Royal Society, the British Academy and the Academy of Medical Sciences. D.S. was supported by a PhD studentship from the Imperial College London–Royal Holloway BBSRC Doctoral Training Partnership. J.D.H. and D.R.W. were supported in part by the Welch Foundation (C1557) and the National Science Foundation (CHE1709631). R.W.F. was supported by a British Heart Foundation programme grant (RG/15/4/31268). The authors thank D. Chirgadze and M. Hyvonen in the Department of Biochemistry at the University of Cambridge for X-ray crystallography support and crystallographic data refinement, respectively; E. Hohenester in the Department of Life Sciences at Imperial College London for helpful discussion on solid-phase binding assays; and J.-D. Malcor and A. Bonna in the Department of Biochemistry at the University of Cambridge for support in peptide synthesis. The authors also thank Diamond Light Source for beamtime (proposal mx14043) and the staff of beamlines I03, I04 and I24 for assistance with crystal testing and data collection.

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A.A.J. and R.W.F. conceived the project. A.A.J. synthesized and characterized the peptides, obtained the heterotrimer crystals and solved their crystal structures, developed the methodology for covalent capture of heterotrimers and their subsequent purification, performed the solid-phase binding assays and analyzed the CD, NMR, MS and solid-phase binding assay data. J.D.H. wrote the code for computational design of heterotrimers. B.L. expressed DDR–Fc fusion constructs and analyzed cellular activation experiments performed by D.S. S.W.H. expressed the recombinant VWF A3 domain, and E.J.H. assisted in optimization of solid-phase assays. P.B. co-solved and refined the crystal structure of AAB. K.S. planned the NMR experiments and co-analyzed the NMR and CD data. D.R.W. wrote the script for the analysis of the helical twist of heterotrimers. A.A.J., R.W.F. and B.L. co-wrote the manuscript with input from other authors.

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Correspondence to Abhishek A. Jalan.

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Supplementary Tables 1–6, Supplementary Figs. 1–8

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Jalan, A.A., Sammon, D., Hartgerink, J.D. et al. Chain alignment of collagen I deciphered using computationally designed heterotrimers. Nat Chem Biol 16, 423–429 (2020). https://doi.org/10.1038/s41589-019-0435-y

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