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

梭菌胶原酶胶原结合域钙依赖性构象变化的跨学科研究

基本信息

批准号：
18590429
负责人：
MATSUSHITA Osamu
金额：
$ 2.52万
依托单位：
Kitasato University (2007)Kagawa University (2006)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

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

组织毒性梭状芽胞杆菌产生的胶原蛋白酶导致气体坏疽的大规模组织破坏。这些酶的C末端胶原结合结构域（CBD）是负责与胶原原纤维结合的最小段。我们已经表明，CBD可以应用于局部组织中的锚定生长因子。提出了三个方向的Tropocollagen与CBD.CBD中的“热点”残基相互作用，并且在同位素上富集，并且在“ H-'5N HSQC频谱中的每个残基”中观察到一个峰。具有良好分散横峰的光谱也表明CBD正确折叠。 Tropocollagen类似物的NMR滴定研究将结合界面缩小到裂缝。 NMR滴定胶原肽的自旋标记类似物明确鉴定了Tropocollagen在CBD上的方向。像绑定的裂缝一样，马鞍可以弯曲tropocollagen。 CBD可以通过一个方向与胶原肽结合1）与胶原肽结合，以促进原纤维解散，并通过弯曲胶原三重螺旋弯曲胶原蛋白三重螺旋，以这种方式有助于将s骨肽键的方式漏出。甲状旁腺激素（PTH）用于治疗骨质疏松症，但它很快代谢，必须通过每日注射给出。为了延长其作用持续时间，我们合成了PTH和CBD的融合蛋白（PTH-CBD）。 PTH-CBD保留了其在体外结合胶原蛋白的能力，并刺激了cAMP的积累，其效力和功效与人类PTH的LL-CPK细胞中稳定转染的LL-CPK细胞中的效力和功效相似。每周对正常雌性小鼠PTH-CBD注射8周，导致脊柱骨矿物质密度显着增加。没有观察到副作用。这种新颖的融合蛋白代表了在药物设计中的新概念的应用，结合了单个蛋白质结构域以创建具有独特特性的代理