AR COBRE: PROTEIN INTERACTION WITH THE EXTRACELLULAR MATRIX

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。细菌胶原酶蛋白的晶体学和核磁共振研究的协同努力将对几个重要的生物医学问题产生影响。我们克隆了超过10种不同的梭菌胶原酶，它们与哺乳动物的胶原酶几乎没有相似之处。这些细菌胶原酶由催化结构域、多囊肾病I （PKD）结构域和一个或多个胶原结合结构域（CBD）组成。每个域要么在医学上有用，要么可以提供必要的结构细节来理解重要的生化问题。我们以非常高分辨率（1¿和1.65¿）确定了采用两种不同构象的连接剂的CBD结构。该CBD结构域已被证明是有效的位点特异性药物递送载体。自分泌/旁分泌肽信号分子如生长因子有多种临床应用。然而，这些分子很容易被血液循环冲走，因此表现出有限的靶向特异性和较短的半衰期，使其体内治疗效果难以预测。由于胶原蛋白是哺乳动物细胞外基质的主要成分，因此可以通过将信号分子连接到CBD来将其固定在细胞外基质上。携带碱性成纤维细胞生长因子的cbd融合蛋白在小鼠注射部位强烈刺激成纤维细胞生长长达10天。为了通过合理的药物设计来开发药物传递系统，了解CBD与胶原蛋白的相互作用是至关重要的。在缺乏钙的情况下，CBD的连接体呈α螺旋状。然而，在钙存在的情况下，该连接区域采用平行的β -片。这种蛋白质二级结构的剧烈变化被认为发生在阿尔茨海默病和朊病毒疾病中。我们的研究表明，只有少数残基在结构变化中起关键作用。涉及突变型CBD的结构研究？美国将阐明这一机制？？转换和从短程相互作用到远距离相互作用的变化。我们目前也有催化结构域的晶体，作为一种新的锌蛋白酶。到目前为止，诱变已经帮助我们鉴定了锌配体。与哺乳动物的胶原酶不同，细菌的对应物可以非特异性地切割胶原蛋白。因此，催化结构域的结构确定不仅将为其底物-蛋白质相互作用提供可能的解释，而且还将支持基于结构的药物设计以治疗气性坏疽。此外，确定功能未知的多囊肾病（PKD）结构域的结构可能有助于我们建立这些结构域的结构-活性关系。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Coordinated efforts of crystallographic and NMR studies of bacterial collagenase proteins will have an impact in several important biomedical questions. We have over 10 different Clostridial collagenases cloned that have little similarity to mamalian collagenases. These bacterial collagenases consist of a catalytic domain, polycystic kidney disease I (PKD) domain and one or more collagen binding domains (CBD). Each domain is either medically useful or can provide structural details necessary to understand important biochemical questions. We determined the structure of CBD with a linker adopting two different conformations at very high-resolution (1¿ and 1.65¿). This CBD domain has been shown to be effective as a site-specific drug delivery vehicle. There are diverse clinical applications for autocrine/paracrine peptide signaling molecules such as growth factors. However, these molecules are easily washed out by the circulation, and hence exhibit limited target specificity and short half-lives, making their in vivo therapeutic effects unpredictable. As collagen is the primary component of the mammalian extracellular matrix, it is possible to anchor signaling molecules to the extracellular matrix by linking them to a CBD. A CBD-fusion protein carrying the basic fibroblast growth factor strongly stimulated fibroblast growth at an injection site in mice for up to 10 days. To develop a drug delivery system by rational drug design, it is essential to understand how CBD interacts with collagen. In the absence of calcium, the linker of the CBD is alpha helical. However, this linker region adopts a parallel beta-sheet in the presence of calcium. Such drastic change in protein secondary structure has been proposed to take place in Alzheimers as well as in prion diseases. Our studies suggest that only a handful of residues are critically involved in the structure change. Structural studies involving mutant CBD?s will shed light on the mechanism of this ??? conversion and the changes from short-range interactions to long-range interactions. We also currently have crystals of a catalytic domain that acts as a novel zinc protease. Thus far, mutagenesis has aided us in identifying the zinc ligands. Unlike the mammalian collagenase, its bacterial counterpart cleaves collagen non-specifically. Thus, structure determination of the catalytic domain will not only provide a likely explanation for its substrate-protein interactions, but it will also support efforts in structure-based drug design to treat gas gangrene. In addition, the determination of the structure of the polycystic kidney disease (PKD) domain of unknown function will likely aid us in developing a structure-activity relationship for these domains.