Abstract
Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that the presence or absence of a single cysteine in type-I collagen’s C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca2+ in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.
Highlights
Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered
In each case of the heterotrimeric collagen types, at least one of the genetically encoded polypeptides is known to be capable of forming homotrimers, whereas the others are only capable of forming heterotrimers
We began by aligning collagen C-Pro domains from distantly related species across the animal kingdom to track the appearance of each factor that has been proposed to play a key role in collagen assembly through evolutionary history
Summary
Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Given the strong sequence similarity between the Colα1(I) and Colα2(I) C-Pro domains, an appealing hypothesis is that the absence of C2 is a fundamental factor abrogating the ability to form stable homotrimers[20]. Studies evaluating this hypothesis relied on the use of a collagen mini-gene system, comprised of the N-propeptide and C-propeptide of Colα2(I) or Colα1(III) C-Pro appended on short, ~200-amino acid triplehelical domains (note that Colα1(III) is a homotrimerizing collagen and that Colα1(I) was not studied)[21,22,23].
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