Mark A Saper
University Of Michigan At Ann Arbor
Project start date: 2012-02-01
Project end date: 2014-01-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Mark A Saper
PROTEIN TYROSINE PHOSPHATASE: DECIPHERING CATALYTIC MECH And SUBSTRATE SPECIFICITY
Mark A Saper, Associate Professor
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 3P41RR001646-16S10064 from National Center For Research Resources
Abstract: We collected an outstanding data set from a crystals of the dual specificity phosphatase VHR complexed with vanadate. The data extended to at least 1.6 , and had an Rsym of 3.5% to 1.8 . This structure recently has been solved and provides insights into the transition state of phosphate hydrolysis. Several data sets were also collected from the catalytic domain of the LAR receptor tyrosine phosphatase. Data collected from frozen crystals of an anti-DNA Fab fragment are now being used to refine the structure of this antibody.
Keywords: biological product, biomedical resource, protein, radiography, structural biology, bioimaging /biomedical imaging
Project start date: 1998-09-15
Project end date: 1999-08-14
STRUCTURE OF TYROSINE AND DUAL-SPECIFICITY PHOSPHATASES
Mark A Saper, Associate Professor
University Of Michigan At Ann Arbor 3003 South State Street, Room 1040 Ann Arbor, Mi 481091274
Grant 2R01AI034095-05 from National Institute Of Allergy And Infectious Diseases IRG: BBCB
Abstract: 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, 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 and 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. s have purified this amino-terminal domain for crystallization experiments (Aim 8).
Keywords: Yersinia pestis, bacterial protein, enzyme mechanism, protein tyrosine phosphatase, recombinant protein, chemical binding, cysteine, cytoskeletal protein, intermolecular interaction, molecular site, vanadium, X ray crystallography, crystallization, microorganism culture
Project start date: 1993-05-01
Project end date: 2002-04-30
2R01AI034095-05 (1997): $232453
5R01AI034095-09 (2001): $255188
5R01AI034095-08 (2000): $245813
5R01AI034095-07 (1999): $278780
5R01AI034095-06 (1998): $268984
CRYSTAL STRUCTURE OF A YERSINIA TYROSINE PHOSPHATASE
Mark A Saper, Associate Professor
University Of Michigan At Ann Arbor 3003 South State Street, Room 1040 Ann Arbor, Mi 481091274
Grant 5R01AI034095-03 from National Institute Of Allergy And Infectious Diseases IRG: BBCB
Abstract: Phosphorylation of protein tyrosine residues is a cell s primary mechanism for propagating membrane receptor signals and for regulating cell growth and oncogenic transformation. A recently identified and growing family of protein tyrosine phosphatases, enzymes that specifically remove the phosphate moieties from phosphotyrosine- containing proteins, are essential for priming these signalling pathways and for controlling the levels of cell growth. Despite the centrality of these regulatory processes, no tertiary structures are known of any of the key enzymes. This proposal describes preliminary results and strategies to determine the X-ray crystal structure of a bacterial protein tyrosine phosphatase, with and without bound inhibitors and substrates, which will serve as a paradigm for understanding the enzyme s function and specificity. The pathogenic bacterium Yersinia is responsible for a range of human and rodent diseases from diarrhea to the bubonic plague. An essential determinant of its virulence is a secreted protein tyrosine phosphatase termed Yop51. Yop51 is likely a toxin which may enter host cells, interfere with immune cell phosphorylation levels and activation pathways, and allow Yersinia to subvert the host s immune system surveillance. The catalytic portion of this enzyme is highly homologous to human tyrosine phosphatases, has similar substrate specificity, and has an identical catalytic mechanism centered around an essential cysteine. Additionally, it can be expressed in large amounts for biochemical and structural studies. Large, well-diffracting crystals have been obtained of the apoenzyme. Data have been collected and a search for heavy atom derivatives is in progress. Diffracting crystals of the phosphatase complexed with the potent oxyanion inhibitor tungstate were also obtained and differ from the native. This structure may mimick the unique phosphocysteine enzyme transition state. In addition, procedures are described for crystallizing a phosphotyrosine peptide substrate bound to an enzyme variant incapable of substrate turnover. This will reveal how the enzyme catalyzes phosphate removal, why it is specific for phosphotyrosine, and how substrate-protein interactions define the enzyme s specificity.
Keywords: bacterial protein, enzyme complex, enzyme mechanism, enzyme structure, protein tyrosine phosphatase, enzyme substrate, enzyme substrate analog, enzyme substrate complex, mutant, phosphatase inhibitor, structural model, synthetic peptide, tyrosine, X ray crystallography, Yersinia pestis, computer program /software, computer simulation, crystallization, freezing, high performance liquid chromatography, isomorphous substitution, protein purification
Project start date: 1993-05-01
Project end date: 1997-04-30
5R01AI034095-03 (1995): $129934
5R01AI034095-02 (1994): $120130
1R01AI034095-01 (1993): $134876
Structures Of A Conserved Type III Effector Domain
Mark A Saper, Associate Professor
University Of Michigan At Ann Arbor 3003 South State Street, Room 1040 Ann Arbor, Mi 481091274
Grant 5R21AI055955-02 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Many animal and plant pathogenic bacteria, including at least seven on the NIAID priority pathogen list, deliver virulence proteins (effectors) directly into host cells through type III secretion (TTS) systems. There they disrupt cell signaling to manipulate the cell for the bacteria s advantage. This proposal investigates the structural determinants of how specific effector proteins are recognized by the TTS apparatus for subsequent secretion and translocation. Salmonella typhimurium is a class B pathogen that causes severe diarrhea in people and livestock. For some effectors, a small chaperone protein binds to a non-conserved region near the amino-terminus of the effector to ensure efficient translocation. Interestingly, in S. typhimurium, a set of nine effectors have homologous amino-terminal domains (about 145 residues, termed WEKIF domains) but are unrelated elsewhere. These domains are required and sufficient for translocation; no secretion chaperones have been identified and they may not be required. The conserved nature of the domain suggests that it also may localize the effector to specific compartments or proteins in the host cell. The long-term goal of the research is to define the structural basis for the proteinprotein interactions involving this domain. This is the first step for discovering potential targets for antimicrobial development. Comparative structural studies of the individual WEKIF domains are proposed here. Aim 1 of the proposed research will improve existing crystals of the WEKIF domain of the SspH1 effector, and begin crystallographic structure determination. Aim 2 proposes to express and purify four other WEKIF domains, SIrP, SifA, Ssel, and SseJ, and screen for crystallization conditions. Following up on a related, but positive result, Aim 3 will screen the full-length SspH1, containing the leucine-rich repeat effector domains, for crystals.
Keywords: Salmonella typhimurium, bacteria infection mechanism, host organism interaction, protein protein interaction, protein structure, protein transport, secretion, intracellular transport, X ray crystallography, protein purification
Project start date: 2003-07-01
Project end date: 2006-06-30
5R21AI055955-02 (2004): $153000
Sponsored Links Excellgen http://Excellgen.com
1R21AI055955-01 (2003): $153000
STRUCTURE OF TYROSINE AND DUAL SPECIFICITY PHOSPHATASES
Mark A Saper, Associate Professor
Biophysics Research Divisionuniversity Of Michigan At Ann Arbor
3003 South State Street, Room 1040
ann Arbor, Mi 481091274
Grant 3R01AI034095-05S1 from National Institute Of Allergy And Infectious Diseases IRG: BBCB
Project start date: 1993-05-01
Project end date: 2002-04-30
3R01AI034095-05S1 (1997): $36463
CRYSTAL STRUCTURE OF CATALYTIC DOMAIN OF RAT LAR, RECEPTOR TYROSINE PHOSPHATASE
Mark A Saper, Associate Professor
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 5P41RR001646-150062 from National Center For Research Resources
Keywords: Mammalia, bacteria, biological product, biomedical resource, enzyme, structural biology
Project start date: 1997-09-01
Project end date: 1998-08-31
PROTEIN TYROSINE PHOSPHATASE STRUCT: CATALYTIC MECHANISM And SUBSTRATE SPECIFICITY
Mark A Saper, Associate Professor
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 5P41RR001646-150063 from National Center For Research Resources
Keywords: biological product, biomedical resource, enzyme, structural biology
Project start date: 1997-09-01
Project end date: 1998-08-31
CRYSTAL STRUCTURE OF A YERSINIA TYROSINE PHOSPHATASE
Mark A Saper, Associate Professor
University Of Michigan At Ann Arbor 3003 South State Street, Room 1040 Ann Arbor, Mi 481091274
Grant 5R01AI034095-04 from National Institute Of Allergy And Infectious Diseases IRG: BBCB
Project start date: 1993-05-01
Project end date: 1997-04-30
5R01AI034095-04 (1996): $132793