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Scientific Report 2006
Molecular Biology
Structural Neurobiology and
Development of Protein Therapeutic Agents
R.C. Stevens, E.E. Abola,
A.I. Alexandrov, H.M. Archer, J.W. Arndt, G.A. Asmar-Rovira, R.R. Benoit, M.H.
Bracey, A. Brooun, Q. Chai, V.G. Cherezov, E. Chien, A. Gámez, M.T.
Griffith, C. Grittini, M.A. Hanson, V.-P. Jaakola, J. Joseph, K. Masuda, M. Mileni,
K. Moy, M. Nelson, C. Roth, K. Saikatendu, V. Subramanian, J. Velasquez, L.
Wang, M.K. Yadav
High-Throughput Structural Biology
Out
of frustration with the rate at which information on structural biology became known
in the past, we focused on developing new tools to change the field of structural
biology by accelerating the rate of determination of protein structures. This endeavor
included pioneering microliter expression/purification for structural studies, nanovolume
crystallization, automated collection of images, and synchrotron beam line automation.
These technologies were initially tested by staff at the Joint Center for Structural
Genomics (http://www.jcsg.org), where the power of the new tools was demonstrated.
Although the Joint Center for Structural Genomics 2 has continued as a successful
second-phase structural genomics production center, in collaboration with P. Kuhn,
Department of Cell Biology, we have created 2 new technology-focused centers funded
by the National Institutes of Health.
The first center is the Joint Center
for Innovative Membrane Protein Technologies (http://jcimpt.scripps.edu). Here,
in collaboration with K. Wüthrich, Q. Zhang, and G. Chang, Department of Molecular
Biology; M.G. Finn, Department of Chemistry; and P. Kuhn and M. Yeager, Department
of Cell Biology, we do research exclusively on eukaryotic and prokaryotic membrane
proteins. The second center is the Accelerated Technologies Center for Gene to 3D
Structure (http://www.atcg3d.org). Here we are doing collaborative work with Dr.
Kuhn and with researchers from deCODE biostructures, Bainbridge Island, Washington;
Lyncean Technologies, Palo Alto, California; and the University of Chicago, Chicago,
Illinois. In 2005, scientists at the centers showed that high-resolution electron
density maps and refined models can be obtained from in situ diffraction of crystals
grown in microcapillaries. In 2007, the first laboratory-sized synchrotron will
be installed at Scripps Research. The synchrotron has performance characteristics
comparable to those of a synchrotron beam line in terms of intensity and tunability
and will enable us to use direct diffraction analysis of ongoing in situ crystallization
experiments to accelerate the determination of macromolecular structures.
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, particularly childhood neurologic disorders. 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.
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 for the biosynthesis of neurotransmitters.
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 in collaboration
with scientists at BioMarin Pharmaceutical Inc., Novato, California, to correlate
how structure can be used to predict which patients with phenylketonuria most likely
will respond to treatment with this cofactor. Phase 3 clinical trials for the treatment
of mild phenylketonuria with the proprietary form of the cofactor, Phenoptin, have
been completed.
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.
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| 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 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, A reduction in phenylalanine and immune response levels occurs
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. 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 such
as cerebral palsy and neuromuscular dystonias. Previously, we determined the structure
of the 150-kD holotoxin form of the toxin, the holotoxin bound to antibodies, the
catalytic domains of several serotypes (A, B, D, F, G), and the catalytic domain
bound to substrates and inhibitors (Fig. 2). These structures are being used to
understand and redesign the toxin’s mechanism of action and to determine additional
therapeutic applications of the toxin.
 |
| Fig. 2. Serotype structures of botulinum neurotoxin (BoNT), its light chain (LC), the closely
related tetanus neurotoxin (TeNT), and the crystal structure of 150-kD botulinum
neurotoxin A bound to a fragment of a neutralizing monoclonal antibody. |
Cannabinoid Signaling
In collaboration with B.F. Cravatt,
Department of Cell Biology, we solved the structure of fatty acid amide hydrolase,
a degradative integral membrane enzyme responsible for setting intracellular levels
of endocannabinoids, to 2.8 Å. Fatty acid amide hydrolase is intimately associated
with CNS signaling processes such as retrograde synaptic transmission, a process
that is also modulated by the illicit substance δ-9-tetrahydrocannabinol.
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.
Publications
Arndt, J.W., Chai, Q., Christian,
T., Stevens, R.C. Structure of botulinum neurotoxin
type D light chain at 1.65 Å resolution: repercussions for VAMP-2 substrate
specificity. Biochemistry 45:3255, 2006.
Arndt, J.W., Jacobson, M.J., Abola,
E.E., Tepp, W.H., Johnson, E.A., Stevens, R.C. A
structural perspective of the sequence variability within botulinum neurotoxin subtypes
A1-A4. J. Mol. Biol. 362:733, 2006.
Blau, N., Koch, R., Matalon,
R., Stevens, R.C. Five years of synergistic scientific
effort on phenylketonuria therapeutic development and molecular understanding. Mol.
Genet. Metab. 86(Suppl. 1):S1, 2005.
Collins, B., Stevens, R.C.,
Page, R. Crystallization optimum solubility screening:
using crystallization results to identify the optimal buffer for protein crystal
formation. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61(Pt. 12):1035,
2005.
DiDonato, M., Krishna, S.S.,
Schwarzenbacher, R., et al. Crystal structure of
a single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at
2.60 Å resolution. Proteins 63:256, 2006.
Han, G.W., Schwarzenbacher,
R., McMullan, D., et al. Crystal structure of an
apo mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins
60:797, 2005.
Han, G.W., Krishna, S.S., Schwarzenbacher,
R., et al. Crystal structure of the ApbE protein
(TM1553) from Thermotoga maritima at 1.58 Å resolution. Proteins 64:1083,
2006.
Jaroszewski, L., Schwarzenbacher,
R., McMullan, D., et al. Crystal structure of Hsp33
chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution.
Proteins
61:669, 2005.
Jin, K.K., Krishna, S.S., Schwarzenbacher,
R., et al. Crystal structure of TM1367 from Thermotoga
maritima at 1.90 Å resolution reveals an atypical member of the cyclophilin
(peptidylprolyl isomerase) fold. Proteins 63:1112, 2006.
Joseph, J.S., Saikatendu, K.S.,
Subramanian, V., Neuman, B.W., Brooun, A., Griffith, M., Moy, K., Yadav, M.K., Velazquez,
J., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Crystal
structure of non-structural protein-10 (nsp10) from the SARS coronavirus reveals
a novel fold with two zinc-binding motifs. J. Virol. 80:7894, 2006.
Klock, H.E., Schwarzenbacher,
R., Xu, Q., et al. Crystal structure of a conserved
hypothetical protein (gi: 13879369) from mouse at 1.90 Å resolution reveals
a new fold. Proteins 61:1132, 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.
86(Suppl. 1):S17, 2005.
Mathews, I.I., Krishna, S.S.,
Schwarzenbacher, R., et al. Crystal structure of
phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima
at 2.15 Å resolution. Proteins 63:1106, 2006.
Mathews, I.I., Krishna, S.S.,
Schwarzenbacher, R., et al. Crystal structure of
phosphoribosylformyl-glycinamidine synthase II, PurS subunit (TM1244) from Thermotoga
maritima at 1.90 Å resolution. Proteins 65:249, 2006.
Pérez, B., Desviat, L.R.,
Gómez-Puertas, P., Martìnez, A., Stevens, R.C., Ugarte, M. Kinetic
and stability analysis of PKU mutations identified in BH4-responsive
patients. Mol. Genet. Metab. 86(Suppl. 1):S11, 2005.
Peti, W., Page, R., Moy, K.,
O’Neil-Johnson, M., Wilson, I.A., Stevens, R.C., Wüthrich, K.
Towards miniaturization of a structural genomics pipeline using micro-expression
and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005.
Ratia, K., Saikatendu, K.S.,
Santarsiero, B.D., Barretto, N., Baker, S.C., Stevens, R.C., Mesecar, A.D.
Severe acute respiratory syndrome coronavirus papain-like protease: structure of
a viral deubiquitinating enzyme. Proc. Natl. Acad. Sci. U. S. A. 103:5717, 2006.
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 ADP-ribose-1′′-phosphate
dephosphorylation by a conserved domain of nsP3. Structure 13:1665, 2005.
Schwarzenbacher, R., McMullan,
D., Krishna, S.S., et al. Crystal structure of a
glycerate kinase (TM1585) from Thermotoga maritima at 2.70 Å resolution
reveals a new fold. Proteins 65:243, 2006.
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. Blau, N. (Ed.). SPS
Publications, Heilbrun, Germany, 2006, p. 434.
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.A., Hambleton, P.,
Shone, C.C. (Eds.). CRC Press: Boca Raton, FL, in press.
Xu, Q., Schwarzenbacher, R.,
Krishna, S.S., et al. Crystal structure of acireductone
dioxygenase (ARD) from Mus musculus at 2.06 Å resolution. Proteins 64:808,
2006.
Xu, Q., Schwarzenbacher, R.,
McMullan, D., et al. Crystal structure of virulence
factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals
a new fold. Proteins 62:292, 2006.
Yadav, M.K., Gerdts, C.J.,
Sanishvili, R., Smith, W.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. 38:900, 2005.
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