AR COBRE: PROTEIN INTERACTION WITH THE EXTRACELLULAR MATRIX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细菌胶原酶蛋白质的结晶学和核磁共振研究的协调努力将对几个重要的生物医学问题产生影响。我们克隆了10多种不同的梭状芽胞杆菌胶原酶，它们与哺乳动物胶原酶几乎没有相似之处。这些细菌胶原酶由一个催化结构域、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。每个领域要么在医学上有用，要么可以提供理解重要生化问题所需的结构细节。我们用具有两种不同构象的连接子在非常高的分辨率(1？和1.65？)下确定了CBD的结构。这种CBD结构域已被证明是一种有效的特定部位药物输送工具。自分泌/旁分泌多肽信号分子有多种临床应用，如生长因子等。然而，这些分子很容易被循环冲走，因此表现出有限的靶向特异性和较短的半衰期，使得它们在体内的治疗效果不可预测。由于胶原蛋白是哺乳动物细胞外基质的主要成分，因此有可能通过将信号分子连接到CBD来将它们锚定到细胞外基质上。携带碱性成纤维细胞生长因子的CBD融合蛋白强烈刺激小鼠注射部位的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释放系统，了解CBD与胶原的相互作用是至关重要的。在没有钙的情况下，CBD的连接子是α螺旋的。然而，在有钙存在的情况下，这个连接区采用平行的β-折叠。蛋白质二级结构的这种剧烈变化被认为发生在阿尔茨海默病和普恩病毒疾病中。我们的研究表明，只有少数残基与结构变化密切相关。涉及突变CBD的结构研究？S将阐明这一机制？转换以及从短程相互作用到远程相互作用的变化。我们目前也有一个催化结构域的晶体，作为一种新的锌蛋白酶。到目前为止，诱变已经帮助我们确定了锌配体。与哺乳动物胶原酶不同，它的细菌对应物非特异性地裂解胶原蛋白。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，而且将为基于结构的药物设计治疗气性坏疽提供支持。此外，功能未知的多囊肾病(PKD)结构域的确定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.