Scientific Report 2005

Molecular Biology

High-Throughput Structure-Based Drug Discovery and Structural Neurobiology

R.C. Stevens, E.E. Abola, A. Alexandrov, J.W. Arndt, G. Asmar-Rovira, R. Benoit, F. Bi, M.H. Bracey, D. Carlton, Q. Chai, J.C. Chappie, E. Chien, T. Clayton, B. Collins, A. Gámez, M. Griffith, C. Grittini, M.A. Hanson, A. Houle, J. Joseph, K. Masuda, B. McManus, K. Moy, M. Nelson, R. Page, M.G. Patch, C. Roth, K. Saikatendu, V. Sridhar, M. Straub, V. Subramanian, J. Velasquez, L. Wang, M. Yadav

High-Throughput Structural Biology

For the past several years, we have focused on developing tools to change the field of structural biology by accelerating the rate of determination of protein structures, an endeavor that includes pioneering microliter expression/purification for structural studies, nanovolume crystallization, and automated image collection. Applications of these technologies were initially tested at the Joint Center for Structural Genomics (http://www.jcsg.org), where we showed the power of the new tools. In addition to the recent funding of the JCSG-2 as a second-phase production center of the National Institute of General Medical Sciences, 2 new centers funded by the National Institutes of Health have been spun off for technologic innovations in structural biology. The first center is called the Joint Center for Innovative Membrane Protein Technologies (http://jcimpt.scripps.edu). Here, in collaboration with G. Chang, S. Lesley, K. Wüthrich, and Q. Zhang, Department of Molecular Biology; P. Kuhn and M. Yeager, Department of Cell Biology; and M.G. Finn, Department of Chemistry, we do research exclusively on membrane proteins, including G protein–coupled receptors. The second center is the Accelerated Technologies Center for Gene to 3D Structure (http://www.atcg3d.org). Here we are doing collaborative studies with P. Kuhn, Department of Cell Biology, and researchers from deCODE biostructures, Bainbridge Island, Washington; Lyncean Technologies, Palo Alto, California; and the University of Chicago, Chicago, Illinois. In the near future, this center will build a synchrotron resource at Scripps Research.

Structural Neurobiology

Although we have developed high-throughput methods to accelerate the determination of protein structures, our primary interest is using these tools to study the chemistry and biology of neurotransmission and of diseases that affect neurons. Our goals are to understand how neuronal cells function on a molecular level and, on the basis of that understanding, create new molecules and materials that mimic neuronal signal transduction and recognition. We use high-throughput protein crystallography and biochemical methods to probe the structure and function of molecules involved in neurotransmission and neurochemistry.

Fatty Acid Amide Hydrolase

In collaboration with B.F. Cravatt, Department of Cell Biology, we solved the structure of fatty acid amide hydrolase (FAAH), a degradative integral membrane enzyme responsible for setting intracellular levels of endocannabinoids, to 2.8 Å. FAAH is intimately associated with CNS signaling processes such as retrograde synaptic transmission, a process that is also modulated by the illicit substance δ⁹-tetrahydrocannabinol. FAAH is a dimer capable of monotopic membrane insertion; it has an active-site structure consistent with the capacity for hydrolysis of hydrophobic fatty acid amides and structural features amenable to structure-based drug design. With our knowledge of the 3-dimensional structure, we are trying to understand how the enzyme works at a basic level and how it might be the basis for potential drug discovery.

Biosynthesis of Neurotransmitters

For neuronal signal transduction, the presynaptic cell synthesizes neurotransmitters that then traverse the synaptic cleft. We are using the high-throughput methods to determine the inclusive structures of complete biochemical pathways. Specifically, we are interested in determining the structures of all the enzymes in the biosynthesis pathways of neurotransmitters in order to understand the mechanistic details of each individual enzymatic reaction at the atomic level. This approach also allows us to determine the best path of drug discovery in the areas of neurotransmitter biosynthesis and catabolism.

Phenylalanine hydroxylase and tyrosine hydroxylase initiate the first committed step in the biosynthesis of the neurotransmitters dopamine, adrenaline, and noradrenaline, and tryptophan hydroxylase catalyzes the rate-determining step in the biosynthesis of serotonin. Because of the importance of these neurotransmitters in the proper functioning of the CNS, understanding the molecular details involved in the catalysis and regulation of these biosynthetic enzymes is crucial. We determined the 3-dimensional structures for tyrosine hydroxylase, tryptophan hydroxylase, and phenylalanine hydroxylase, and we are uncovering specific mechanistic details for these enzymes.

Therapeutic Agents for Treatment of Phenylketonuria

In addition to the basic hydroxylase enzymology questions under investigation, recent clinical studies suggest that some patients with the metabolic disease phenylketonuria are responsive to (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin, the natural cofactor of phenylalanine hydroxylase. We are doing studies to correlate how structure can be used to predict which patients with phenylketonuria most likely will respond to treatment with this cofactor. Currently, the proprietary form of the cofactor, Phenoptin, is entering phase 3 clinical trials for the treatment of mild phenylketonuria. For classical phenylketonuria, we are developing an enzyme replacement therapeutic agent that is being tested in animal models. The therapy is based on administration of a modified form of phenylalanine ammonia lyase discovered in our structural studies (Fig. 1). Last, we are determining the structural basis of diseases caused by several other enzymes involved in the biosynthesis of neurotransmitters. Many of these disorders are rare or occur during childhood.

Fig. 1. A, Crystal structure of phenylalanine ammonia lyase (PAL) determined at 1.6-Å resolution. This protein structure was engineered and chemically modified as a potential once-a-week injectable therapeutic agent for treatment of phenylketonuria. B, ENU2 mice are used as a model for phenylketonuria in preclinical studies. C and D, Reduction in phenylalanine and immune response levels in ENU2 mice after the injection of PAL that has been chemically modified (pegylated). These PEG-PAL formulations show promise as therapeutic agents for treatment of phenylketonuria.

Neurotoxins

The clostridial neurotoxins include tetanus toxin and the 7 serotypes of botulinum toxin (Fig. 2). We are determining the molecular events involved in the binding, pore formation, translocation, and catalysis of botulinum neurotoxin. Although botulinum toxin is most known for its deadly effects, it is now being used therapeutically to treat involuntary muscle disorders.

Fig. 2. Serotype structures of botulinum neurotoxin (BoNT), its light chain (LC), and the closely related tetanus neurotoxin (TeNT).

Recently, we determined the structure of the 900-kD complex form of the toxin, the 150-kD holotoxin form, the catalytic domain, and the catalytic domain bound to substrates and inhibitors. These structures are being used to understand and redesign the toxin’s mechanism of action and to determine additional therapeutic applications of the toxin.

Publications

Arndt, J.W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, M.A., Lebeda, F.J., Stevens, R.C. The structure of the neurotoxin-associated protein HA33/A from Clostridium botulinum suggests a reoccurring β-trefoil fold in the progenitor toxin complex. J. Mol. Biol. 346:1083, 2005.

Arndt, J.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an α/β serine hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 Å resolution. Proteins 58:755, 2005.

Arndt, J.W., Yu, W., Bi, F., Stevens, R.C. Crystal structure of botulinum neurotoxin type G light chain: serotype divergence in substrate recognition. Biochemistry 44:9574, 2005.

Cànaves, J.M., Page, R., Stevens, R.C. Protein biophysical properties that correlate with crystallization success in Thermotoga maritima: maximum clustering strategy for structural genomics. J. Mol. Biol. 344:977, 2004.

Carter, D.C., Rhodes, P., McRee, D.E., Tari, L.W., Dougan, D.R., Snell, G., Abola, E., Stevens, R.C. Reduction in diffuso-convective disturbances in nanovolume protein crystallization experiments. J. Appl. Crystrallogr. 38:87, 2005.

Chappie, J.S., Cànaves, J.M., Han, G.W., Rife, C.L., Xu, Q., Stevens, R.C. The structure of a eukaryotic nicotinic acid phosphoribosyltransferase reveals structural heterogeneity among type II PRTases. Structure (Camb.) 13:1385, 2005.

Erlandsen, H., Pey, A.L., Gámez, A., Pérez, B., Desviat, L.R., Aguado, C., Koch, R., Surendran, S., Tyring, T., Matalon, R., Scriver, C.R., Ugarte, M., Martìnez, A., Stevens, R.C. Correction of kinetic and stability defects by the cofactor tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations. Proc. Natl. Acad. Sci. U. S. A. 101:16903, 2004.

Gámez, A., Sarkissian, C.N., Wang, L., Kim, W., Straub, M., Patch, M.G., Chen, L., Striepeke, S., Fitzpatrick, P., Lemontt, J.F., O’Neill, C., Scriver, C.R., Stevens, R.C. Development of pegylated forms of recombinant Rhodosporidium toruloides phenylalanine ammonia-lyase for the treatment of classical phenylketonuria. Mol. Ther. 11:986, 2005.

Han, G.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an alanine-glyoxylate aminotransferase from Anabena sp at 1.70 Å resolution reveals a noncovalently linked PLP cofactor. Proteins 58:971, 2005.

Levin, I., Miller, M.D., Schwarzenbacher, R., et al. Crystal structure of an indigoidine synthase A (IndA)-like protein (TM1464) from Thermotoga maritima at 1.90 Å resolution reveals a new fold. Proteins 59:864, 2005.

Matalon, R., Michals-Matalon, K., Koch, R., Grady, J., Tyring, S., Stevens, R.C. Response of patients with phenylketonuria in the US to tetrahydrobiopterin. Mol. Genet. Metab., in press.

Mathews, I., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of
S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) from Thermotoga maritima at 2.0 Å resolution reveals a new fold. Proteins 59:869, 2005.

Page, R., Deacon, A.M., Lesley, S., Stevens, R.C. Shotgun crystallization strategy for structural genomics, II: crystallization and conditions that produce high resolution structures for T maritima proteins. J. Funct. Struct. Genomics 6:209, 2005.

Page, R., Peti, W., Wilson, I.A., Stevens, R.C., Wüthrich, K. NMR screening and crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a structural genomics pipeline. Proc. Natl. Acad. Sci. U. S. A. 102:1901, 2005.

Pérez, B., Desviat, L.R., Gomez-Puertas, P., Martinez, A., Stevens, R.C., Ugarte, M. Kinetic and stability analysis of PKU mutations identified in BH4-responsive patients. Mol .Genet. Metab., in press.

Peti, W., Johnson, M.A., Hermann, T., Newman, B.W., Buchmeier, M.J., Nelson, M., Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural genomics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsP7. J. Virol . 79:12905, 2005.

Peti, W., Page, R., Wilson, I., Stevens, R., Wüthrich, K. Structural proteomics pipeline miniaturized using micro expression and microcoil NMR. J. Struct. Funct. Genomics, in press.

Pey, A.L., Pérez, B., Desviat, L.R., Martinez, M.A., Aguado, C., Erlandsen, H., Gámez, A., Stevens, R.C., Thorolfsson, M., Ugarte, M., Martinez, A. Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations. Hum. Mutat. 24:388, 2004.

Ricci, J.S., Stevens, R.C., McMullan, R.K., Klooster, W.T. The crystal structure of strontium hydroxide octahydrate, Sr(OH)2.8H₂O at 20, 100, and 200 K from neutron diffraction. Acta Crystrallogr. B 61:381. 2005.

Rife, C., Schwarzenbacher, R., McMullen, D., et al. Crystal structure of a putative modulator of DNA gyrase (pmbA) from Thermotoga maritima at 1.95 Å resolution reveals a new fold. Proteins 61:444, 2005.

Rife, C., Schwarzenbacher, R., McMullen, D., et al. Crystal structure of a global regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a new fold. Proteins 61:449, 2005.

Saikatendu, K.S., Joseph, J.S., Subramanian, V., Clayton, T., Griffith, M., Moy, K., Velasquez, J., Neuman, B.W., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Structural basis of severe acute respiratory syndrome coronavirus (SARS-CoV) ADP-ribose-1″-phosphate (Appr-1″-p) dephosphorylation by a conserved domain of nsP3. Structure, in press.

Scriver, C.R., Hurtubise, M., Prevost, L., Phommarinh, M., Konecki, D., Erlandsen, H., Stevens, R.C., Waters, P.J., Ryan, S., McDonald, D., Sarkissan C. A PAH gene knowledge base: content, informatics, utilization. In: PKU and BH₄: Advances in Phenylketonuria and Tetrahydrobiopterin Research. Blaue, N. (Ed.), SPS Publications, Heilbrun, Germany, in press.

Swaminathan, S., Stevens, R.C. Three-dimensional protein structures of botulinum neurotoxin light chains serotypes A, B, and E. In: Treatments from Toxins: The Therapeutic Potential of Clostridial Neurotoxins. Foster, K., Hambleton, P., Shone, C. (Eds.). CRC Press, Boca Raton, FL, in press.

Wang, L., Gámez, A., Sarkissian, C.N., Straub, M., Patch, M.G., Han, G.W., Striepeke, S., Fitzpatrick, P., Scriver, C.R., Stevens, R.C. Structure-based chemical modification strategy for enzyme replacement treatment of phenylketonuria. Mol. Genet. Metab. 86:134, 2005.

Xu, Q., Schwarzenbacher, R., McMullen, D., et al. Crystal structure of a formiminotetrahydrofolate cyclodeaminase (TM1560) from Thermotoga maritima at 2.80 Å resolution reveals a new fold. Proteins 58:976, 2005.

Yadav, M.K., Gerdts, C.J., Sanishvili, R., Smith, W., Roach, L.S., Ismagilov, R.F., Kuhn, P., Stevens, R.C. In situ data collection and structure refinement from microcapillary protein crystallization. J. Appl. Crystallogr., in press.