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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

组织毒性梭菌产生胶原酶，导致气性坏疽中广泛的组织破坏。这些酶的C末端胶原结合结构域（CBD）是负责结合胶原原纤维的最小片段。我们已经证明CBD可以应用于局部组织中的锚生长因子。CBD的"热点"残基与原胶原的三种取向相互作用，CBD的"H-"~5N HSQC谱中每个残基都有一个峰。具有良好分散的交叉峰的光谱也表明CBD被适当折叠。用原胶原类似物的NMR滴定研究将结合界面缩小到裂缝。用胶原肽的自旋标记类似物的NMR滴定明确地确定了CBD上原胶原的取向。鞍状结合裂隙可使原胶原弯曲。CBD可以通过以下方式帮助水解：1）以一个方向结合胶原肽以促进原纤维的解散，和2）通过弯曲胶原三螺旋以帮助三螺旋解旋，使得易断裂的肽键暴露。甲状旁腺激素（PTH）用于治疗骨质疏松症，但它是如此之快代谢，它必须通过每天注射。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保留了其在体外结合胶原的能力，并在用PTH受体稳定转染的LL-CPK细胞中以与人PTH相似的效力和功效刺激cAMP积累。在正常年轻雌性小鼠中每周注射PTH-CBD 8周导致脊柱骨矿物质密度显著增加。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，创造出具有独特特性的药剂