AR COBRE: PROTEIN INTERACTION WITH THE EXTRACELLULAR MATRIX

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。细菌胶原酶蛋白的晶体学和NMR研究的协调努力将在几个重要的生物医学问题中产生影响。我们克隆了超过10种不同的梭菌胶原酶，它们与哺乳动物胶原酶几乎没有相似性。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，要么可以提供理解重要生物化学问题所必需的结构细节。我们用采用两种不同构象的连接体以非常高的分辨率（1 º和1.65 º）确定了CBD的结构。 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，这些分子很容易被循环冲洗掉，因此表现出有限的靶向特异性和短的半衰期，使得它们的体内治疗效果不可预测。由于胶原蛋白是哺乳动物细胞外基质的主要组分，因此可以通过将信号分子连接到CBD来将信号分子锚到细胞外基质。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开发药物递送系统，必须了解CBD与胶原蛋白的相互作用。在不存在钙的情况下，CBD的接头是α螺旋的。然而，该接头区在钙的存在下采用平行的β-折叠。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，只有少数残基是关键参与的结构变化。涉及突变CBD的结构研究？s将阐明这一机制？转换和从短程相互作用到长程相互作用的变化。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，诱变已帮助我们确定锌配体。与哺乳动物胶原酶不同，其细菌对应物非特异性地切割胶原。因此，催化结构域的结构确定不仅将提供一个可能的解释其底物-蛋白质相互作用，但它也将支持基于结构的药物设计，以治疗气性坏疽的努力。此外，多囊肾病（PKD）结构域未知功能的结构的确定将可能有助于我们开发这些结构域的结构-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.