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

组织毒性梭状芽孢杆菌产生胶原酶，导致气性坏疽的广泛组织破坏。这些酶的C端胶原结合结构域(CBD)是负责与胶原纤维结合的最小片段。我们已经证明，CBD可以用来锚定局部组织中的生长因子。原胶原蛋白的三个方向与CBD中的热点残基相互作用。CBD具有同位素富集性，在‘H-’5N HSQC谱中每个残基都有一个峰。交叉峰分散良好的光谱也表明CBD被正确折叠。使用原胶原类似物的核磁共振滴定研究缩小了裂隙的结合界面。自旋标记的胶原肽类似物的核磁共振滴定明确地确定了原胶原在CBD上的方向。马鞍状的结合裂隙会使原胶原蛋白弯曲。CBD可通过1)以一种取向与胶原肽结合以促进原纤维的解离，以及2)通过弯曲胶原三螺旋以帮助三螺旋解开，从而使可剪断的多肽键暴露，从而有助于水解性。甲状旁腺激素(PTH)用于治疗骨质疏松症，但它代谢很快，必须每天注射。为了延长其作用时间，我们合成了PTH和CBD的融合蛋白(PTH-CBD)。PTH-CBD在体外保留了其与胶原结合的能力，并在稳定转染PTH受体的LL-CPK细胞中刺激cAMP积聚，其效力和效力与人PTH相似。正常雌性小鼠每周注射PTH-CBD 8周后，脊柱骨密度显著增加。未观察到副作用。这种新的融合蛋白代表了一种新概念在药物设计中的应用，将单个蛋白质结构域结合起来创建具有独特性质的试剂

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