AR COBRE: PROTEIN INTERACTION WITH THE EXTRACELLULAR MATRIX

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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 CBDs 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.