News and Publications

Structure and Function of Enzymes and Fertilization Proteins

C.D. Stout, G.S. Prasad, J. Nowakowski, S.J. Lloyd, P.J. Shim, N. Kresge, A. Muhlberg, V. Sridhar

This laboratory focuses on experimental x-ray crystallography of macromolecules. Fundamental questions are addressed through structure determination of key proteins involved in biological processes. The research often involves collaboration with other scientists at TSRI. The experiments entail biochemical preparation, crystallization, and collection and analysis of x-ray diffraction data. Once a structure is solved, experiments are designed to study relationships between structure and function. These involve preparation of site-directed mutants and ligand complexes, structure analysis, and assays of biological function. Projects focused on iron-sulfur enzymes, fertilization proteins, protein enzymes that use nucleotides, and DNA enzymes are in progress.

Detailed study of the mechanism of the iron-sulfur enzyme aconitase is continuing with the structure determination of active-site mutants in complex with substrates and inhibitors; intermediates in the catalytic reaction can be trapped in the crystalline state. The aconitase system is also being used as a molecular laboratory for engineering iron-sulfur clusters. These experiments are designed to determine the steric and chemical requirements of the protein ligands for different classes of iron-sulfur clusters in proteins.

In collaboration with B.K. Burgess, University of California, Irvine, the properties of iron-sulfur clusters in proteins are being probed by using the 7-iron ferredoxin from Azotobacter vinelandii as a model system. Mutagenesis, chemical modification, reduction potential and spectroscopic measurements, and high-resolution structure refinement are being used. The structure of the electron-transfer partner of this protein, NAPH:ferredoxin oxidoreductase, has been solved (Fig. 1); both proteins function in an oxidative stress response pathway.

A long-term goal is the study of sperm-egg interaction at the molecular level. in collaboration with V.D. Vacquier, Scripps Institution of Oceanography, 4 structures of proteins from the acrosomal granule of abalone sperm are being analyzed at atomic resolution (1.3--2.0 Å): the green 16,000 molecular weight lysin dimer, the green 18,000 molecular weight lysin monomer, the red 16,000 molecular weight monomer, and the red 16,000 molecular weight dimer. These proteins dissolve the egg vitelline envelope and fuse the egg and sperm plasma membranes. The structures allow mapping of the molecular features of the protein surfaces responsible for species-specific interaction and egg receptor recognition.

Structural analyses of 4 enzymes that use nucleotide substrates are ongoing: dUTP pyrophosphatase from feline immunodeficiency virus, in collaboration with J. Elder, Department of Molecular Biology; ADP ribosyl cyclase, which synthesizes the secondary messenger cyclic ADP ribose from NAD; CD38, a cell-surface ectozyme and B-cell coreceptor homologous to cyclase; and dynamin, a multidomain, oligomeric GTPase involved in endocytosis, in collaboration with S. Schmid, Department of Molecular Biology.

A DNA enzyme with catalytic efficiency exceeding that of ribonuclease A has been evolved in the laboratory of G. Joyce, Department of Molecular Biology. A number of constructs in which the 5´ and 3´ ends of the DNA were varied were prepared and used for crystallization. Nine crystal forms were found, 1 of which diffracts to 3.0-Å resolution. The structure in this crystal form, solved by using 6 heavy atom derivatives, has been refined at 3.0 Å. The structure reveals that the DNA strands have rearranged to form a new Watson-Crick paired duplex on a 2-fold axis in the crystal while pairing with 2 RNA molecules. The overall dimeric, 4-stranded structure of 82 nucleotides is cross-shaped (Fig. 2) and consists of 1 DNA-DNA helix and 2 DNA-RNA helices. The structure has extensive base stacking, a novel loop, and 3 abrupt changes in chain direction, attesting to the flexibility of DNA. However, the structure is not consistent with solution studies for an active conformation of the enzyme. New experiments will be designed to trap the active conformation in a crystal lattice, in order to study the basis of catalysis by a DNA molecule.

PUBLICATIONS

Lloyd, S.J., Lauble, H., Prasad, G.S., Stout, C.D. The mechanism of aconitase: Trapping of citrate-mode in the S642A:fluorocitrate complex and an alternative citrate binding mode in the R644Q:fluorocitrate complex. Protein Sci., in press.

Lund, F.E., Muller-Steffner, H.M., Yu, N., Stout, C.D., Schuber, F., Howard, M. CD38 signaling is controlled by its ectodomain but occurs independently of enzymatically generated ADP-ribose or cyclic ADP-ribose. J. Biol. Chem., in press.

Prasad, G.S., Kresge, N., Muhlberg, A.B., Shaw, A., Jung, Y.S., Burgess, B.K., Stout, C.D. The crystal structure of NADPH:ferredoxin oxidoreductase from Azotobacter vinelandii. Protein Sci., in press.

Sridhar, V., Prasad, G.S., Burgess, B.K., Stout, C.D. Crystal structures of ferricyanide-oxidized [Fe-S] clusters in Azotobacter vinelandii ferredoxin I. J. Biol. Inorg. Chem. 3:140, 1998.

Stout, C.D., Stura, E.A., McRee, D.E. Structure of Azotobacter vinelandii 7Fe ferredoxin at 1.35-Å resolution and determination of the [Fe-S] bonds with 0.01-Å accuracy. J. Mol. Biol. 278:629, 1998.

Vacquier, V.D., Swanson, W.J., Metz, E.C., Stout, C.D. Acrosomal proteins of abalone spermatozoa. Adv. Dev. Biochem., in press.