In the present study, we provide direct experimental evidence suggesting why such non-traditional chaperone action may be required for procollagen triple helix folding. In particular, HSP47 appears to bind preferentially to the triple helix rather than unfolded chains –, opposite to most other ER chaperones. The best known triple helix chaperone is HSP47, but the molecular mechanism of its action remains controversial, –. However, folding of procollagen triple helix may not. Posttranslational modification of procollagen chains and folding of the globular C-propeptide may follow this pathway. Once the native state is achieved, the protein is believed to be released from its interactions with the chaperone(s). The traditional view is that chaperone molecules interact with unfolded and partially folded polypeptide chains, preventing their aggregation and other nonproductive interactions that may result in misfolding. Disruptions of this complex by recessive null mutations in CRTAP and P3H1 were recently discovered in several patients with delayed procollagen folding and severe/lethal skeletal deformities reminiscent of Osteogenesis Imperfecta –. P3H1 and CRTAP form a tight, ER-resident complex with cyclophilin B known for its peptidyl-prolyl-isomerase activity. The most recent additions to the latter family are prolyl-3-hydroxylase (P3H1) and cartilage-associated protein (CRTAP). Some are known mostly for their other functions, but may also act as collagen chaperones, e.g., SPARC –.
Some are collagen-specific, e.g., HSP47 and prolyl-4-hydroxylase. Some are general ER chaperones, e.g, calnexin, BiP, GRP94, and PDI. As with many other proteins, a variety of different chaperone molecules appear to be involved in procollagen folding. Procollagen is a collagen precursor, in which the triple helix is flanked by globular N- and C-terminal propeptides. The triple helix folding follows synthesis of procollagen chains within Endoplasmic Reticulum (ER). Folding defects result in severe/lethal bone fragility and deformities (Osteogenesis Imperfecta) –.
Proper folding of its triple helix is crucial for forming the matrix of bones and other tissues. Type I collagen is the most abundant protein in higher vertebrates.
#THE CHAPERONE REVIEW DESTINY FREE#
The required 50–200 µM concentration of free HSP47 is not unusual for heat-shock chaperones in ER, but it is 100 times higher than used in reported in vitro experiments, which did not reveal such stabilization. It takes over 20 HSP47 molecules to stabilize a single triple helix at body temperature. Based on the triple helix folding temperature measured here and published binding constants, we deduce that HSP47 is likely to do just that. We argue that folding of the triple helix requires stabilization by preferential binding of chaperones to its folded, native conformation. However, such binding only further destabilizes the triple helix. Common ER chaperones may prevent aggregation and misfolding of procollagen C-propeptide in their traditional role of binding unfolded polypeptide chains. ER-like molecular crowding by nonspecific proteins does not affect triple helix folding or aggregation of unfolded chains.
We find that human procollagen triple helix spontaneously folds into its native conformation at 30–34☌ but not at higher temperatures, even in an environment emulating Endoplasmic Reticulum (ER). In fibers, the triple helix is stabilized by neighbors, but how does it fold? The observations reported here reveal surprising features that may represent a new paradigm for folding of marginally stable proteins. Fibers composed of type I collagen triple helices form the organic scaffold of bone and many other tissues, yet the energetically preferred conformation of type I collagen at body temperature is a random coil.
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