Protein tyrosine phosphatases (PTPs), and the structurally-similar dual-specificity protein phosphatases (dsPTP), play critical roles in the regulation of cellular functions such as cell growth, cell division, and immune cell activation. Several crystal structures have revealed details of the catalytic mechanism and mode of substrate binding and inhibition, but little is known about how PTPs target specific substrates, or how they are regulated. This question is addressed by determining structures by X-ray crystallography of PTPs bound to functionally-relevant ligands. During the next 5-year period, the applicant proposes to continue studies of the dsPTP catalytic mechanism by solving a transition state mimic of VHR complexed with vanadate (Aim 1) and visualizing the novel phosphocysteine intermediate in a catalytically-deficient mutant (Aim 2). They will also co-crystallize an inactive VHR mutant with a phosphopeptide and small molecule substrate (Aims 3 & 4). Crystals have been obtained of the catalytic domain of LAR, a receptor PTP involved in cell adhesion and cytoskeletal reorganization. Diffraction data will be collected and the structure solved by molecular replacement (Aim 5). Recent structures suggest that receptor PTPs in vivo may dimerize to regulate PTP activity. Secondly, a peptide substrate will be co-crystallized with the LAR domain to compare its specificity with the recently-solved PTPa (Aim 6). The second, inactive PTP domain from LAR which binds cytoskeletal-associated proteins, will be expressed and purified for crystallization (Aim 7). Pathogenic Yersinia bacteria secrete a highly-active PTP into host macrophages which inhibits phagocytosis and suppresses an effective immune response. Like LAR, these is evidence that a non-catalytic domain in the Yersinia PTP targets the enzyme to cytoskeletal proteins. The applicants have purified this amino-terminal domain for crystallization experiments (Aim 8).
