Protein Structure and Enzymology Summary
We are interested in characterizing proteins as a means of understanding molecular mechanisms of action or for providing information useful in the design of therapeutically useful agents. We utilize x-ray crystallography as a tool for the study of macromolecular structure. Our earliest work in this area began as a collaboration with Stuart Schreiber on the binding of the natural product immunosuppressants FK506 and rapamycin to the peptidyl proline isomerase FKBP.(1),(2) Subsequent studies in the Schreiber group showed that the therapeutic activity of these immunosuppressants, rather than being associated with inhibition of FKBP activity as everyone had previously believed, was in fact caused by inhibition by the FKBP/immunosuppressant complex of a second enzyme. In the case of FK506 the target is the calcium-dependent phosphatase calcineurin,(4) The structure of the FKBP/FK506/calcineurin complex was first described by a group from Vertex Pharmaceuticals.(5) Our laboratory determined the structure of the FKBP/rapamycin/FRAP complex.(6) The ternary complex structures revealed structural aspects of the small molecules crucial for bringing the two proteins into association, an event not known to occur in the absence of the small molecule. Insights gained from the complex structures have helped guide further studies in other laboratories into using these small molecules as templates for protein dimerizers.
We have structural projects that aim to provide information useful in decreasing the incidence or severity of human disease. The pyrimidine biosynthesis pathway has been proposed as a desirable target of drugs for the treatment of malaria, caused by the parasitic microorganism Plasmodium falciparum.(7) We have determined the structure of the human(8) and P. falciparum(9) forms of dihydroorotate dehydrogenase (DHODH), an enzyme in the pyrimidine biosynthetic pathway whose activity is crucial for growth and survival. Comparison of the structures highlight differences in the size and shape of the active site and associated cofactor-binding sites that may be useful in the design of therapeutic agents that selectively inhibit P. falciparum DHODH in the presence of the human enzyme. We hope to use these results and techniques to guide and supplement our screening efforts aimed at identifying potent and selective inhibitors of P. falciparum DHODH.
In collaboration with Walter Leal we have studied structural aspects of the transport of hydrophobic insect pheremones across the aqueous medium that separates the external pores of antennae from the olefactory receptors. We studied the pheremone binding protein (PBP) from the model organism of insect olefaction, the silkworm moth Bombyx mori. We determined the structure of the small, hydrophillic PBP in complex with bombykol, a sex pheremone of B. mori produced by female moths to attract males.(10) To further probe the mechanism and specificity of PBP/pheremone interactions, we have recently determined the structure of PBP in the absence of ligand and in the presence of other potential ligands.(11) These studies also included variation in pH and have provided new insights and raised new questions about the means by which PBPs are capable of providing rapid transport of pheremones to the olefactory receptors.
One interesting recent development in our group has been the merging of the "protein-related" and "small molecule-related" viewpoints within the group. This convergence has resulted from our studies on environmental DNA and the tools it provides for studying biosynthesis. In particular, the fee biosynthetic cluster(12) has provided targets for both structural and enzymologic characterization. Comparison of the recently-determined structure(13) of the long-chain N-acyl tyrosine synthase FeeM with the structurally and functionally related GNAT (GCN5-related N-acetyl transferases) superfamily of proteins has provided a plausible mechanism of action for FeeM that is currently being evaluated by enzymological studies. We have additionally reconstituted the activity of FeeM/FeeL in vitro and have been using assays to explore substrate requirements and aspects of acyl transfer. We have also began studies into reconstituting the nitrogen-to-oxygen transposase activity of the enzyme FeeH as a prelude to a full mechanistic determination of this activity for which there is no precedence in the enzymological literature.
- (1) Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L., and Clardy, J. "Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant complex" Science 1991, 252, 839-842. (PubMed, PDB)
- (2) Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L., and Clardy, J. "Atomic structure of the rapamycin human immunophilin FKBP-12 complex" J. Am. Chem. Soc. 1991, 113, 7433-7434. (PDB)
- (3) Liu, J., Farmer, J.D., Jr., Lane, W.S., Friedman, J., Weissman, I., and Schreiber, S.L. "Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes" Cell 1991, 66, 807-815. (PubMed)
- (4) Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S., and Schreiber, S.L. "A mammalian protein targeted by G1-arresting rapamycin-receptor complex" Nature 1994, 369, 756-758. (PubMed)
- (5) Griffith, J.P., Kim, J.L., Kim, E.E., Sintchak, M.D., Thomson, J.A., Fitzgibbon, M.J., Fleming, M.A., Caron, P.R., Hsiao, K., and Navia, M.A. "X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex" Cell 1995, 82, 507-522. (PubMed, PDB)
- (6) Choi, J., Chen, J., Schreiber, S.L., and Clardy J. "Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP" Science 1996, 273, 239-242. (PubMed, PDB)
- (7) Gero, A.M., Brown, G.V., and O'Sullivan, W.J. "Pyrimidine denovo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum" J. Parisitol. 1984, 70, 536-541. (PubMed)
- (8) Liu, S., Neidhardt, E.A., Grossman, T.H., Ocain, T., and Clardy, J. "Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents" Structure Fold Des. 2000, 15, 25-33. (PubMed, PDB: brequinar complex, A771726 complex)
- (9) Manuscript in preparation.
- (10) Sandler, B.H., Nikonova, L., Leal, W.S., and Clardy, J. "Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein-bombykol complex" Chem. Biol. 2000, 7, 143-151. (PubMed, PDB)
- (11) Manuscript in preparation.
- (12) Brady, S.F., Chao, C.J., and Clardy J. "New natural product families from an environmental DNA (eDNA) gene cluster" J. Am. Chem. Soc. 2002, 124, 9968-9969. (PubMed)
- (13) Manuscript in preparation.
- Sinars, C.R., Cheung-Flynn, J., Rimerman, R.A., Scammell, J.G., Smith, D.F., and Clardy J. "Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes" Proc. Nat. Acad. Sci. U.S.A. 2003, 100, 868-873. (PubMed, PDB: human, squirrel monkey)
- Calero, G., Gupta, P., Nonato, M.C., Tandel, S., Biehl, E.R., Hofmann, S.L., and Clardy, J. "The crystal structure of palmitoyl protein thioesterase-2 (PPT2) reveals the basis for divergent substrate specificities of the two lysosomal thioesterases, PPT1 and PPT2" J. Biol. Chem. 2003, 278, 37957-37964. (PubMed, PDB)
- Calero, G., Wilson, K.F., Ly, T., Rios-Steiner, J.L., Clardy, J.C., and Cerione, R.A. "Structural basis of m7GpppG binding to the nuclear cap-binding protein complex" Nat. Struct. Biol. 2002, 9, 912-917. PubMed, PDB: with capped nucleotide, free)
- Nonato, M.C., Widom, J., and Clardy, J. "Crystal structure of the N-terminal segment of human eukaryotic translation initiation factor 2alpha" J. Biol. Chem. 2002, 277, 17057-17061. (PubMed, PDB)
- Bellizzi, J.J., 3rd, Widom, J., Kemp, C., Lu, J.Y., Das, A.K., Hofmann, S.L., and Clardy, J. "The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis" Proc. Nat. Acad. Sci. U.S.A. 2000, 97, 4573-4578. (PubMed, PDB: with palmitate, free)