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. There are three human trypsin isozymes: cationic trypsin, anionic trypsin, and mesotrypsin. All three are produced and secreted by the pancreas as digestive proenzymes, and are also produced to a lesser extent in other tissues. Trypsins are typically regulated through strong inhibition by endogenous small protein inhibitors, but the minor isozyme mesotrypsin is uniquely resistant to inhibition, binding with reduced affinity to small protein inhibitors and degrading them as substrates. The inhibitor-degrading activity of mesotrypsin has been postulated to play a role in the development of pancreatitis, through clearance of the protective pancreatic trypsin inhibitor SPINK1. Mesotrypsin is also highly expressed in the brain, and has been postulated to play a role in the processing of myelin basic protein in the pathological mechanism of multiple sclerosis. In addition, preliminary work in our own laboratory suggests that mesotrypsin plays a role in progression to malignancy in several cancers. A single amino acid change in the enzyme active site, substitution of arginine for a conserved glycine, appears to be wholly responsible for the resistance of mesotrypsin to protein inhibitors. Modeling studies based on existing X-ray structures for several trypsin/inhibitor complexes and the structure of uncomplexed mesotrypsin indicate that this arginine residue interferes with inhibitor docking in the active site, and that substantial conformational changes must occur at the enzyme/inhibitor interface before mesotrypsin can bind to a protein inhibitor. The relative binding affinities of mesotrypsin to a variety of trypsin inhibitors do not parallel the preferences of other trypsins, indicating that mesotrypsin/inhibitor binding energetics may be dominated by different interactions than those that determine the specificities of more well-studied trypsins. It is our aim to solve the structures of human mesotrypsin and human cationic trypsin in complex with several protein inhibitors, including bovine pancreatic trypsin inhibitor (BPTI), Alzheimer precursor protein inhibitor domain (APPI), and soybean trypsin inhibitor (SBTI). We expect that comparison of these structures (1) will reveal the conformational changes that allow mesotrypsin to bind inhibitors, (2) will give clues to explain why mesotrypsin, in contrast to cationic trypsin, is then able to cleave bound inhibitors, and (3) will give insight into the different specificities of mesotrypsin and cationic trypsin. The involvement of mesotrypsin in a number of human diseases makes it a potential drug target, and a structural understanding of the inhibitor resistance of this enzyme will facilitate efforts towards drug development.