Abstract
Precise mapping and unraveling the mechanism of interaction or degradation of a certain type of collagen triple helix requires the generation of short and stable collagenous fragments. This is a great challenge especially for hetero-trimeric collagens, where chain composition and register (stagger) are important factors. No system has been reported that can be efficiently used to generate a natural collagenous fragment with exact chain composition and desired chain register. The NC2 domain (only 35-50 residues) of FACIT collagens is a potent trimerization domain. In the case of type IX collagen it provides the efficient selection and hetero-trimerization of three distinct chains. The ability of the NC2 domain to determine the chain register of the triple helix is studied. We generated three possible sequence combinations (α1α1α2, α1α2α1, α2α1α1) of a type I collagen fragment (the binding region for the von Willebrand factor A3 domain) attached to the NC2 domain. In addition, two control combinations were produced that constitute homo-trimers of (α1)(3) or (α2)(3). For the hetero-trimeric constructs, α1α1α2 demonstrated a higher melting temperature than the other two. Binding experiments with the von Willebrand factor A3 domain revealed the homo-trimer of (α1)(3) as the strongest binding construct, whereas the homo-trimer of (α2)(3) showed no binding. For hetero-trimers, α1α1α2 was found to be the strongest binding construct. Differences in thermal stability and binding to the A3 domain unambiguously demonstrate that the NC2 domain of type IX collagen determines not only the chain composition but also the chain register of the adjacent triple helix.
Highlights
The chains of the collagen triple helix are staggered by one residue
We have recently identified small (35–55 residues) and efficient trimerization domains in collagen types XVIII and XV [19, 20] and XIX and IX [21, 22], which are useful tools for folding and stabilization of collagenous peptides because they represent a natural way to initiate folding and stabilize the triple helix
C-terminally added sequence of type III collagen cysteine knot GPCCGGV (Fig. 1) was crystallized, and its structure was solved by molecular replacement and refined to 1.5 Å resolution
Summary
The chains of the collagen triple helix are staggered by one residue. Results: The NC2 domain of type IX collagen establishes a stagger for the adjacent triple helix. Precise mapping and unraveling the mechanism of interaction or degradation of a certain type of collagen triple helix requires the generation of short and stable collagenous fragments This is a great challenge especially for hetero-trimeric collagens, where chain composition and register (stagger) are important factors. The recent crystal structure of type III collagen fragment with the natural cysteine knot [15] and a new crystal structure reported here raised a question of whether it can determine (lock) a specific stagger Another approach in achieving the determined stagger is a system of three artificial collagen-like chains bearing compensating charges that allow a specific assembly of hetero-trimeric molecules [16]. The type IX collagen hetero-trimerization domain is a light-weight non-triplehelical fragment that provides a technically simple and natural way for the generation of triple helical collagenous fragments of any type and length with predicted chain composition and stagger
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