Associate Professor
Fax: (530) 752-8995
Email: fisher@chem.ucdavis.edu
X-ray crystallography is one of the most powerful tools to examine protein structure at atomic detail. My laboratory uses single crystal x-ray diffraction techniques to determine the three-dimensional structure of biologically important proteins. Knowledge of the atomic resolution structure provides tremendous insight into understanding how the protein functions. Hypotheses on structure - function relationships can be drawn from the crystal structure, which can be tested using crystallography and other biophysical and biochemical techniques. Currently, the laboratory is focusing on two areas of research; programmed cell death and sulfate activation.
APOPTOSIS: In the last decade, much of the research in cell proliferation has been concentrated in the signals that initiate cell growth. Very little is know about the opposite end of the cellular life cycle, particularly cell death. All multicelluar organisms contain genes that can trigger a specific cell's demise. These suicide genes along with the signals and factors that regulate their expression are just beginning to be discovered. Programmed cell death, also called apoptosis, is essential for normal development and tissue homeostasis. Unregulated apoptosis can contribute to several diseases that include; cancer, AIDS, Alzheimer's, Parkinson's, neurodegenerative disorders, and Huntington's disease. Apoptosis also provides a primitive form of defense against viral infections. Infected cells accept apoptosis to prevent the propagation of progeny virus thereby slowing down the spread of the infection. Some ingenious viruses have evolved proteins to circumvent this defense mechanism. The baculovirus carries a gene that encodes for a 35,000 Dalton protein. This protein, P35, blocks apoptosis in mammalian, murine, and insect cell lines as well as in developing embryos of the fruit fly Drosophila and the nematode C. elegans. The ability of P35 to block apoptosis in such a diverse range of organisms induced by different signals suggests that it acts at an evolutionary conserved step in the apoptotic pathway. P35 has been found to inhibit a new class of cellular proteases that are involved in the suicide pathway. These novel cysteine proteases bind to, and cleave P35 causing it to form a stronger complex with the protease. This sequesters the protease and prevents it from cleaving the cellular proteins that commence apoptosis.
The three dimensional crystal structure of P35 was recently solved from x-ray data collected in the UC-Davis x-ray facility and refined to higher resolution data collected at the Stanford synchrotron radiation laboratory (SSRL). This is the first known structure of a protein that inhibits the apoptotic caspases and reveals a novel protein fold. The structure has revealed many clues on its interaction and inhibition of the cellular proteins involved in carrying out the suicide signal. Site directed mutagenesis studies are planned to investigate the structural and functional importance of specific residues by using crystallography and other biochemical and biophysical techniques. Experiments have been initiated to investigate the crystal structure of a complex between P35 and one of the novel cysteine proteases. These structures will help elucidate the molecular mechanism of the two proteins and can lead to therapeutic agents that could combat the diseases linked with unregulated apoptosis.
SULFATE ACTIVATION: Another project of interest in the lab is understanding sulfur assimilation, or fixation. This project focuses on the structural features underlying the catalytic and regulatory properties of the sulfate activating enzymes, ATP sulfurylase and APS kinase, which catalyze in order, the reactions:
MgPPi + APS (ATP sulfurylase)
MgATP + APS PAPS + MgADP (APS Kinase)
ATP sulfurylase plays three different roles in nature: (a) In fungi, yeast, most heterotrophic bacteria, algae, and higher plants, this enzyme catalyzes the first intracellular reaction in the reductive assimilation of sulfate into organic molecules. Thus, in these organisms, APS is the "active sulfate" precursor of cysteine, methionine, etc. Plants and algae use APS as the substrate for reduction while fungi, yeast, and bacteria use PAPS. PAPS serves as the sulfuryl donor for sulfate ester biosynthesis e.g., chondroitin sulfate, cerebroside sulfate, protein tyrosyl sulfate, and heparin in animals (which do not reduce (P)APS); choline-O-sulfate in fungi; flavinol sulfates in plants. (b) In anaerobic sulfate reducing bacteria (e.g., Desulfovibrio), ATP sulfurylase forms APS solely to serve as the terminal electron acceptor of heterotrophic metabolism. (c) In certain chemo- and photolithotrophic bacteria (e.g., Thiobacillus and Chromatium), ATP sulfurylase catalyzes the last reaction in the oxidation of reduced inorganic sulfur compounds to sulfate i.e., the physiological reaction is in the opposite direction compared to that in sulfate assimilators and reducers. This "APS pyrophosphorylase" reaction may be the sole substrate level ATP - forming step in sulfur lithotrophs.
A long-term goal of this project is to identify the structural adaptations that optimize the catalytic and regulatory properties of each class of ATP sulfurylase and APS kinase for the physiologically relevant task. My laboratory recently determined the crystal structure of APS kinase and is in the process of determining the structure with substrates bound in the active site. Structure determination is also in progress for ATP sulfurylase from a number of organisms.
Fisher, A. J., MacRae, I. J., Beynon, J. D., Lansdon, E. B., and Segel, I. H. (2004). Optimizing an enzyme for its physiological role: Structural and functional comparisons of ATP sulfurylases from three different organisms. In: Conformational Proteomics of Macromolecular Architecture. Cheng, R. H., and Hammar, L. (Eds.) World Scientific Publishing Co., London. pp. 222-241.
Liu, W., Peterson, P. E., Carter, R. J., Zhou, X., Langston, J., Fisher, A. J., and Toney, M. D. (2004). Crystal Structures of Unbound and Aminooxyacetate-bound Escherichia coli gamma-Aminobutyrate Aminotransferase. Biochemistry, 43:10896-10905. [PDF Reprint]
Lansdon, E. B., Fisher, A. J., and Segel, I. H. (2004). Human PAPS Synthetase (Isoform 1; Brain): Kinetic Properties of the ATP Sulfurylase and APS Kinase Domains. Biochemistry 43:4356-4365. [PDF Reprint]
Forsyth, C., M., Lemongello, D., LaCount, D. J., Friesen, P. D., and Fisher, A. J. (2004). Crystal Structure of an Invertebrate Caspase. J. Biol. Chem. 279:7001-7008. [PDF Reprint]
Hanna, E., Ng, K. F., MacRae, I. J., Bley, C. J., Fisher, A. J., and Segel, I. H. (2004). Kinetic and Stability Properties of P. Chrysogenum ATP Sulfurylase Missing the C-Terminal Regulatory Domain. J. Biol. Chem. 279:4415-4424. [PDF Reprint]
Corneillie, T. M., Fisher, A. J., and Meares, C. F. (2003). Crystal Structures of Two Complexes of the Rare Earth-DOTA-Binding Antibody 2D12.5: Ligand Generality from a Chiral System. J. Am. Chem. Soc. 125:15039-15048. [PDF Reprint]
Corneillie, T. M., Whetstone, P. A., Fisher, A. J., and Meares, C. F. (2003). A Rare Earth-DOTA-Binding Antibody: Probe Properties and Binding Affinity across the Lanthanide Series. J. Am. Chem. Soc. 125:3436-3437. [PDF Reprint]
MacRae, I. J., Segel, I. H., and Fisher, A. J. (2002). Allosteric inhibition via R-state destabilization in ATP sulfurylase from Penicillium chrysogenum. Nature Struc. Biol. 9:945-949. [PDF Reprint]
Lansdon, E. B., Segel, I. H., and Fisher, A. J. (2002). Ligand-induced structural changes in adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Biochemistry 41:13672-13680. [PDF Reprint]
Hanna, E., MacRae, I. J., Medina, D., Fisher, A. J., and Segel, I. H. (2002). ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus. Arch. Biochem. Biophys. 406:275 -288. [PDF Reprint]
Liu, W., Rogers, C. J., Fisher, A. J., and Toney, M. D. (2002). Aminophosphonate inhibitors of dialkylglycine decarboxylase: Structural basis for slow binding inhibition. Biochemistry 41:12320-12328 [PDF Reprint]
Eddins, M. J., Lemongello, D., Friesen, P. D., and Fisher, A. J. (2002). Crystallization and low-resolution structure of an effector-caspase/P35 complex: similarities and differences to an initiator-caspase/P35 complex . Acta Crystallographica D58:299-302. [PDF Reprint]
Beynon, J. D., MacRae, I. J., Huston, S. L., Nelson, D. C., Segel, I. H., and Fisher, A. J. (2001). Crystal structure of ATP Sulfurylase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila. Biochemistry 40:14509-14517. [PDF Reprint]
dela Cruz, W. P., Friesen, P. D., and Fisher, A. J. (2001). Crystal structure of baculovirus P35 reveals a large conformational change in the reactive site loop after caspase cleavage. J. Biol. Chem. 276:32933-32939. [PDF Reprint]
Medina, D., Hanna, E., MacRae, I. J., Fisher, A. J., and Segel, I. H. (2001). Temperature effects on the allosteric transition of ATP sulfurylase from Penicillium chrysogenum. Arch. Biochem. Biophys. 393:51-60. [PDF Reprint]
Mogilner, A., Fisher, A. J., and Baskin, R. J. (2001). Structural changes in the neck linker of kinesin explain the load dependence of the motor's mechanical cycle. J. Theor. Biol. 211:143-157. [PDF Reprint]
MacRae, I. J., Segel, I. H., and Fisher, A. J. (2001). Crystal structure of ATP sulfurylase from Penicillium chrysogenum: Insights into the allosteric regulation of sulfate assimilation. Biochemistry 40:6795-6804. [PDF Reprint]
Beynon, J., Rafanan Jr., E. R., Shen, B., and Fisher A. J. (2000). Crystallization and preliminary X-ray analysis of tetracenomycin A2 oxygenase: A flavoprotein hydroxylase involved in polyketide biosynthesis. Acta Crystallographica D56:1647-1651. [PDF Reprint]
MacRae, I. J., Hanna, E., Ho, J. D., Fisher, A. J., and Segel, I. H. (2000). Induction of positive cooperativity by amino acid replacements within the C-terminal domain of P. chrysogenum ATP sulfurylase. J. Biol. Chem. 275:36303-36310. [PDF Reprint]
MacRae, I. J., Segel, I. H., and Fisher, A. J. (2000). Crystal structure of adenosine 5'-phosphosulfate (APS) kinase from Penicillium chrysogenum. Biochemistry 39:1613-1621. [PDF Reprint]
