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).

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Michigan Ann Arbor
Organized Research Units
Ann Arbor
United States
Zip Code
Ivanov, Maya I; Stuckey, Jeanne A; Schubert, Heidi L et al. (2005) Two substrate-targeting sites in the Yersinia protein tyrosine phosphatase co-operate to promote bacterial virulence. Mol Microbiol 55:1346-56
Khandelwal, Purnima; Keliikuli, Kai; Smith, Craig L et al. (2002) Solution structure and phosphopeptide binding to the N-terminal domain of Yersinia YopH: comparison with a crystal structure. Biochemistry 41:11425-37
Khandelwal, P; Keliikuli, K; Smit, C L et al. (2001) 1H, 15N and 13C assignments of the N-terminal domain of Yersinia outer protein H in its apo form and in complex with a phosphotyrosine peptide. J Biomol NMR 21:69-70
Vijayalakshmi, J; Mukhergee, M K; Graumann, J et al. (2001) The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity. Structure 9:367-75
Fauman, E B; Cogswell, J P; Lovejoy, B et al. (1998) Crystal structure of the catalytic domain of the human cell cycle control phosphatase, Cdc25A. Cell 93:617-25
Fauman, E B; Yuvaniyama, C; Schubert, H L et al. (1996) The X-ray crystal structures of Yersinia tyrosine phosphatase with bound tungstate and nitrate. Mechanistic implications. J Biol Chem 271:18780-8
Fauman, E B; Saper, M A (1996) Structure and function of the protein tyrosine phosphatases. Trends Biochem Sci 21:413-7
Yuvaniyama, J; Denu, J M; Dixon, J E et al. (1996) Crystal structure of the dual specificity protein phosphatase VHR. Science 272:1328-31
Denu, J M; Lohse, D L; Vijayalakshmi, J et al. (1996) Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis. Proc Natl Acad Sci U S A 93:2493-8
Schubert, H L; Fauman, E B; Stuckey, J A et al. (1995) A ligand-induced conformational change in the Yersinia protein tyrosine phosphatase. Protein Sci 4:1904-13