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. The problem to be studied is the relationship between the two phosphatase domains of CD45 under oxidizing and reducing conditions. CD45 belongs to a family of transmembrane phosphatases that contain two tandem phosphatase domains. In all of these proteins, the N-terminal domain is catalytically active, while the C-terminal domain has little to no activity despite being conserved at sequence and structural levels. All phosphatase domains contain a conserved cysteine in their catalytic or pseudocatalytic site. This cysteine is absolutely essential for catalytic activity, and its oxidation has been shown to induce a significant change in the x-ray crystal structure of a single domain phosphatase, PTP1B. We believe the catalytically inactive C-terminal domain of CD45 may regulate the activity of the N-terminal domain, perhaps as a redox sensor via its cysteine, which is more sensitive to alkylation. This regulation may occur through a conformational change in the C-terminal domain upon oxidation of its cysteine, that then disrupts the interaction of the two domains. We have reason to believe that such an interaction is important for activity. Domain swaps with other related phosphatases do not reconstitute activity, suggesting a specific functional interaction. X-ray crystallography has also suggested that at least one conformation has these two domains interacting tightly as a kinked rod. However, we suspect there may be conformational flexibility between these domains that may be regulated by the redox state of the C-terminal domain. Therefore we would like to study the dynamic nature of the interaction between these two domains under reducing and oxidizing conditions. We anticipate conversion from a kinked rod under reducing conditions, to a more floppy conformation under oxidizing conditions.
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