Interdisciplinary study on the calcium-dependent conformational change of collagen-binding domain from clostridial collagenases

点击翻译按钮获取中文摘要

Histotoxic clostridia produce collagneases responsible for extensive tissue destruction in gas gangrene. C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment responsible to bind to collagen fibril. We have shown that CBD can be applied to anchor growth factors in local tissue. Three orientations of tropocollagen were proposed to interact with 'hot spot' residues in CBD.CBD was isotopically enriched and one peak is observed for each residue in the 'H-'5N HSQC spectrum. The spectrum with well dispersed cross peaks also indicated that CBD was properly folded. NMR titration study with a tropocollagen analog narrowed the binding interface to a cleft. NMR titrations with spin-labeled analog of collagenous peptide unambiguously identified the orientation of tropocollagen on CBD. The saddle like binding cleft could bend a tropocollagen. CBD may aid in hydrolysis by 1) binding to collagenous peptide in one orientation to facilitate disbanding of the fibril and 2) by bending the collagenous triple helix to assist unwinding of triple helix in such a way that the scissile peptide bond would be exposed. Pararthyroid hormone (PTH) is used for the treatment of osteoporosis, but it is so quickly metabolized that it must be given by daily injection. To prolong its duration of action, we have synthesized a fusion protein (PTH-CBD) of PTH and CBD. PTH-CBD retained its ability to bind collagen in vitro, and stimulated cAMP accumulation with similar potency and efficacy to human PTH in LL-CPK cells stably transfected with the PTH receptor. Weekly injections of PTH-CBD in normal young female mice for 8 weeks resulted in a significant increase in spinal bone mineral density. There were no side effects observed. This novel fusion protein represents an application of a new concept in drug design, combining individual protein domains to create an agent with unique properties