The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.

Tyrosine 146 of thymidylate synthase assists proton abstraction from the 5-position of 2'-deoxyuridine 5'-monophosphate.

Tyr 146 of TS has been proposed to assist in the removal of the proton from the 5-carbon of the pyrimidine in a steady-state intermediate [Hyatt, D. C., Maley, F.

D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine.

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except cysteine abolish activity. We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N.

Conformational substates in enzyme mechanism: the 120 K structure of alpha-lytic protease at 1.5 A resolution.

Insight into the dynamic properties of alpha-lytic protease (alpha LP) has been obtained through the use of low-temperature X-ray crystallography and multiple-conformation refinement. Previous studies of alpha LP have shown that the residues around the active site are able to move significantly to accommodate substrates of different sizes. Here we show a link between the ability to accommodate ligands and the dynamics of the binding pocket.

Signaling phosphate starvation.

Phosphate starvation induces the transcription of several genes involved in phosphate metabolism in the budding yeast Saccharomyces cerevisiae. The signal transduction pathway that mediates this response consists of components that resemble those used to regulate the eukaryotic cell cycle; these include a cyclin-dependent kinase or CDK (Pho85), a cyclin (Pho80) and a CDK inhibitor (Pho81). The possibility that this pathway mediates cell-cycle responses to phosphate starvation is discussed.

A purified Drosophila septin complex forms filaments and exhibits GTPase activity.

Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1.

The complex of the anti-cancer therapeutic, BW1843U89, with thymidylate synthase at 2.0 A resolution: implications for a new mode of inhibition.

Background: Thymidylate synthase (TS) is critical to DNA synthesis as it catalyzes the rate limiting step in the only biosynthetic pathway for deoxythymidine monophosphate (dTMP) production. TS is therefore an important target for anti-proliferative and anti-cancer drug design. The TS enzymatic mechanism involves the reductive methylation of the substrate, deoxyuridine monophosphate (dUMP), by transfer of a methylene group from the co-factor, methylenetetrahydrofolate (CH2H4folate), resulting in the production of deoxythymidine monophosphate (dTMP) and dihydrofolate (H2folate).

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase.

Invariant arginine 179, one of four arginines that are conserved in all thymidylate synthases (TS) and that bind the phosphate moiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP), can be altered even to a negatively charged glutamic acid with little effect on kcat. In the mutant structures, ordered water or the other phosphate-binding arginines compensate for the hydrogen bonds made by Arg179 in the wild-type enzyme and there is almost no change in the conformation or binding site of dUMP. Correlation of dUMP Kds for TS R179A and TS R179K with the structures of their binary complexes shows, that the positive charge on Arg179 contributes significantly to dUMP binding affinity.

Toward fully automated high-resolution electron tomography.

Electron tomography is a powerful tool in elucidating the three-dimensional architecture of large biological complexes and subcellular organelles. Its use can be expanded through the simplification of the tomographic procedure by automation of its tasks. In this paper, we describe our EMACT/EMCAT system, which automates both tomographic data collection and reconstruction.

IVE (Image Visualization Environment): a software platform for all three-dimensional microscopy applications.

IVE (Image Visualization Environment) is a software platform designed from the outset to handle all aspects of modern computerized multidimensional microscopy. This platform provides users with an execution environment in which 5D data (XYZ, wavelength, and time) can be easily manipulated for the purpose of data collection, processing, display, and analysis. During the entire process, powerful data display functions are readily available for extracting complicated three-dimensional information through data visualization.

A structural role for hormone in the thyroid hormone receptor.

The crystal structure of the rat alpha 1 thyroid hormone receptor ligand-binding domain bound with a thyroid hormone agonist reveals that ligand is completely buried within the domain as part of the hydrophobic core. In addition, the carboxy-terminal activation domain forms an amphipathic helix, with its hydrophobic face constituting part of the hormone binding cavity. These observations suggest a structural role for ligand, in establishing the active conformation of the receptor, that is likely to underlie hormonal regulation of gene expression for the nuclear receptors.

Microtubule nucleation by gamma-tubulin-containing rings in the centrosome.

The microtubule cytoskeleton of animal cells does not assemble spontaneously, but instead requires the centrosome. This organelle consists of a pair of centrioles surrounded by a complex collection of proteins known as the pericentriolar material (PCM). The PCM is required for microtubule nucleation.

Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex.

The highly conserved protein gamma-tubulin is required for microtubule nucleation in vivo. When viewed in the electron microscope, a highly purified gamma-tubulin complex from Xenopus consisting of at least seven different proteins is seen to have an open ring structure. This complex acts as an active microtubule-nucleating unit which can cap the minus ends of microtubules in vitro.

Identification of two novel arginine binding DNAs.

RNA tertiary structure is known to play critical roles in RNA-protein recognition and RNA function. To examine how DNA tertiary structure might relate to RNA structure, we performed in vitro selection experiments to identify single-stranded DNAs that specifically bind arginine, and compared the results with analogous experiments performed with RNA. In the case of RNA, a motif related to the arginine binding site in human immunodeficiency virus TAR RNA was commonly found, whereas in the case of DNA, two novel motifs and no TAR-like structures were found.

Three-dimensional structural characterization of centrosomes from early Drosophila embryos.

An understanding of the mechanism and structure of microtubule (MT)-nucleating sites within the pericentriolar material (PCM) of the centrosome has been elusive. This is partly due to the difficulty in obtaining large quantities of centrosomes for analysis, as well as to the problem of attaining interpretable structural data with conventional EM techniques. We describe a protocol for isolating a large quantity of functional centrosomes from early Drosophila embryos.

Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy-loss spectroscopic imaging.

With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0.3-1.0 microns).

Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos.

Heterochromatin protein 1 is associated with centromeric heterochromatin in Drosophila, mice, and humans. Loss of function mutations in the gene encoding heterochromatin protein 1 in Drosophila, Suppressor of variegation2-5, decrease the mosaic repression observed for euchromatic genes that have been juxtaposed to centromeric heterochromatin. These heterochromatin protein 1 mutations not only suppress this position-effect variegation, but also cause recessive embryonic lethality.

Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos.

Heterochromatin protein 1 (HP1) was initially discovered as a protein that is associated with the heterochromatin at the chromocenter of polytene chromosomes in Drosophila larval salivary glands. In this paper we investigate the localization of heterochromatin protein 1 in the diploid nuclei of Drosophila embryos. We focus on its association with the interphase heterochromatin in fixed embryos before and during cycle 14, the developmental time at which heterochromatin becomes most conspicuous, and also follow its localization during mitosis.

Neural transmission. Synaptotagmin is just a calcium sensor.

Ligand-binding studies have suggested many functions for the synaptic vesicle protein, synaptotagmin. But genetic elimination of the protein supports only one role: translating arriving Ca2+ signals into vesicle release.

Crystallization and preliminary structural studies of the ncd motor domain.

The motor domain of the kinesin homolog ncd has been crystallized in the presence of MgATP by the vapor diffusion method using polyethylene glycol as the precipitant. The crystals belong to the orthorhombic space group I222 with unit cell dimensions a = 127.1 A, b = 122.

Crystal structure of thymidylate synthase from T4 phage: component of a deoxynucleoside triphosphate-synthesizing complex.

Thymidylate synthase from phage T4 (T4TS) is part of a complex of several enzymes required for coordinate DNA synthesis in infected Escherichia coli cells. It has been proposed that similar complexes of enzymes related to DNA synthesis are also functional in eukaryotes [Pardee, A. B.

Subunits of the Saccharomyces cerevisiae signal recognition particle required for its functional expression.

The signal recognition particle (SRP) is an evolutionarily conserved ribonucleoprotein (RNP) complex that functions in protein targeting to the endoplasmic reticulum (ER) membrane. Only two protein subunits of the SRP, Srp54p and Sec65p, and the RNA subunit, scR1, were previously known in the yeast Saccharomyces cerevisiae. Purification of yeast SRP by immunoaffinity chromatography revealed five additional proteins.

Direct inhibition of the yeast cyclin-dependent kinase Cdc28-Cln by Far1.

Cell cycle arrest of Saccharomyces cerevisiae in G1 by the antimitogen alpha-factor is mediated by activation of a signal transduction pathway that results in inhibition of the cyclin-dependent kinase Cdc28-Cln. The Far1 protein is required for cell cycle arrest and associates with the Cdc28-Cln complex. The kinase activity of Cdc28-Cln was directly inhibited by Far1 both in vivo and in vitro, thus demonstrating that Far1 acts at the final step in the alpha-factor response pathway by inhibiting a G1 cyclin-dependent kinase.

Protein translocation across the endoplasmic reticulum.

In the past year, dramatic progress has been made in our understanding of protein biogenesis at the initial steps of the eukaryotic secretory pathway. New insights have refined our view of protein targeting to the endoplasmic reticulum membrane and provided the best glimpse so far of the subsequent translocation step. The interactions of three GTP-binding proteins have been found to result in a novel cycle of GTP binding and hydrolysis to regulate protein targeting.

Cryo automated electron tomography: towards high-resolution reconstructions of plastic-embedded structures.

The use of fully automated data collection methods for electron tomography allows a substantial reduction in beam dose. The goal has been to develop new protocols for data collection defining optimal approaches for maintaining data self-consistency and maximizing the useful resolution of the reconstruction. The effects of irradiation and post-cure microwaving were examined for a variety of embedding media (Epon, Epox, Lowicryl) in order to quantify beam damage with the goal of identifying the most beam stable embedding medium.