Balancing ATP in the cell.
Explore the source record for details and available documents.
Adenosine Triphosphate↗
Biomedical subjects
R M Stroud
Publications and source records attributed to R M Stroud.
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. kcat/K(m) for dUMP measures the rate of dUMP binding to TS during the ordered bi-substrate reaction, and in the ternary complex dUMP provides a binding surface for the cofactor. kcat/K(m) reflects the ability of the enzyme to accept a properly oriented dUMP for catalysis and is less sensitive than is Kd to the changes in electrostatics at the phosphate binding site.
Arginine↗
Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site.
The crystal structure of human thymidylate synthase, a target for anti-cancer drugs, is determined to 3.0 A resolution and refined to a crystallographic residual of 17.8%. The structure implicates the enzyme in a mechanism for facilitating the docking of substrates into the active site. This mechanism involves a twist of approximately 180 degrees of the active site loop, pivoted around the neighboring residues 184 and 204, and implicates ordering of external, eukaryote specific loops along with the well-characterized closure of the active site upon substrate binding. The highly conserved, but eukaryote-specific insertion of twelve residues 90-101 (h117-128), and of eight residues between 156 and 157 (h146-h153) are known to be alpha-helical in other eukaryotes, and lie close together on the outside of the protein in regions of disordered electron density in this crystal form. Two cysteines [cys 202 (h199) and 213 (h210)] are close enough to form a disulfide bond within each subunit, and a third cysteine [cys 183 (h180)] is positioned to form a disulfide bond with the active site cysteine [cys 198 (h195)] in its unliganded conformation. The amino terminal 27 residues, unique to human TS, contains 8 proline residues, is also in a region of disordered electron density, and is likely to be flexible prior to substrate binding. The drug resistance mutation, Y6H, confers a 4-fold reduction in FdUMP affinity and 8-fold reduction in kcat for the dUMP reaction. Though indirectly connected to the active site, the structure suggests a mechanism of resistance that possibly involves a change in structure. This structure offers a unique opportunity for structure-based drug design aimed at the unliganded form of the human enzyme.
Amino Acid Sequence↗
Episelection: novel Ki approximately nanomolar inhibitors of serine proteases selected by binding or chemistry on an enzyme surface.
A novel class of mechanism-based inhibitors of the serine proteases is developed using epitaxial selection. Tripeptide boronates esterified by an alcohol or alcohols at the boron retain the tight binding to trypsin-like enzymes associated with transition-state analogs and incorporate additional groups that can be utilized for selectivity between proteases. Formed by reaction of a series of alcohols with the inhibitor boronate oxygen(s), the most structurally compatible alcohol-derivatized inhibitors are either selected by binding to the enzyme (epitaxial selection) or assembled by epitaxial reaction on the enzyme surface. Mass spectrometry of the derivatized boronates and X-ray crystallography of the complexes identify the chemical structures and the three-dimensional interactions of inhibitors generated. This scheme also engineers novel, potent (Ki approximately 7 nM), and more specific inhibitors of individual serine proteases, by derivitizations of compounds obtained by epitaxial selection.
Amino Acid Sequence↗
Significance of structural changes in proteins: expected errors in refined protein structures.
A quantitative expression key to evaluating significant structural differences or induced shifts between any two protein structures is derived. Because crystallography leads to reports of a single (or sometimes dual) position for each atom, the significance of any structural change based on comparison of two structures depends critically on knowing the expected precision of each median atomic position reported, and on extracting it for each atom, from the information provided in the Protein Data Bank and in the publication. The differences between structures of protein molecules that should be identical, and that are normally distributed, indicating that they are not affected by crystal contacts, were analyzed with respect to many potential indicators of structure precision, so as to extract, essentially by "machine learning" principles, a generally applicable expression involving the highest correlates. Eighteen refined crystal structures from the Protein Data Bank, in which there are multiple molecules in the crystallographic asymmetric unit, were selected and compared. The thermal B factor, the connectivity of the atom, and the ratio of the number of reflections to the number of atoms used in refinement correlate best with the magnitude of the positional differences between regions of the structures that otherwise would be expected to be the same. These results are embodied in a six-parameter equation that can be applied to any crystallographically refined structure to estimate the expected uncertainty in position of each atom. Structure change in a macromolecule can thus be referenced to the expected uncertainty in atomic position as reflected in the variance between otherwise identical structures with the observed values of correlated parameters.
Crystallization↗
Topochemistry for preparing ligands that dimerize receptors.
BACKGROUND: The cyclic, disulfide-containing peptide, cyclo-Ac-[Cys-His-Pro-Gln-Gly-Pro-Pro-Cys]-NH2, binds to streptavidin with high affinity. In streptavidin-peptide cocrystals of space group I222, cyclic peptide monomers are bound on adjacent streptavidin tetramers related by a crystallographic two-fold symmetry axis. We set out to determine whether the disulfide bonds of the peptide, presented close to one another in the crystal, could undergo disulfide interchange to form a dimer. RESULTS: When juxtaposed, the disulfides of neighboring peptides undergo disulfide interchange, breaking and forming covalent disulfide bonds, to produce a peptide dimer adopting the symmetry of the crystal. This is the first example of a chemical transformation mediated by a protein crystal lattice. The structure of the streptavidin-bound monomer, and that of the dimer that was eventually produced from it in the crystal, were both determined from the same single crystal studied at different times. The two-fold symmetric peptide dimer was independently synthesized and shown to form crystals of dimerized streptavidin. CONCLUSIONS: We have shown that formation of a covalently linked peptide dimer can be mediated by a protein crystal lattice. The dimer thus produced dimerizes its target, streptavidin, suggesting that solid-state (or topochemical) reactions of this kind may be broadly useful for the preparation of ligands that can dimerize other protein targets.
Chromatography, High Pressure Liquid↗
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. (1989) Science 246, 603-608]. To delineate the role of structure in the function of this complex, we have solved the structure of T4TS as a basis for mapping the complex by mutagenesis. The 3.1 A structure of the unliganded enzyme was determined by molecular replacement and refined to 19.9% for all data. Three inserts and one deletion in the coding region are unique to T4TS, and all sites lie on one side of the enzyme surface, possibly encoding unique T4 specific intermolecular interactions during the infective cycle. The crystal structure is generally in the open, unliganded conformation seen in unliganded E. coli TS, as opposed to the closed, ternary complex conformation, except that the critically important C-terminus is inserted into the active site hydrogen bonded to residue Asn85, as seen in functional ternary complex structures. Other differences between E. coli TS and T4TS appear to explain the enhanced binding of folyl polyglutamate to the latter.
Amino Acid Sequence↗
Water-mediated substrate/product discrimination: the product complex of thymidylate synthase at 1.83 A.
In an irreversible enzyme-catalyzed reaction, strong binding of the products would lead to substantial product inhibition. The X-ray crystal structure of the product complex of thymidylate synthase (1.83-A resolution, R factor = 0.183 for all data between 7.0 and 1.83 A) identifies a bound water molecule that serves to disfavor binding of the product nucleotide, dTMP. This water molecule is hydrogen bonded to absolutely conserved Tyr 146 (using the Lactobacillus casei numbering system) and is displaced by the C7 methyl group of the reaction product thymidylate. The relation between this observation and kinetic and thermodynamic values is discussed. The structure reveals a carbamate modified N-terminus that binds in a highly conserved site, replaced by side chains that can exploit the same site in other TS sequences. The enzyme-products complex is compared to the previously determined structure of enzyme-substrate-cofactor analog. This comparison reveals changes that occur between the first covalent complex formed between enzyme and substrate with an inhibitory cofactor analog and the completed reaction. The almost identical arrangement of ligands in these two structures contributes to our model for the TS reaction and verifies the physiological relevance of the mode in which potent inhibitors bind to this target for rational drug design.
Carbamates↗
The domain structure of the ion channel-forming protein colicin Ia.
Colicin Ia undergoes a transition from a soluble to a transmembrane state, forming an ion channel to effect its bactericidal activity. The X-ray crystal structure of soluble colicin Ia at an effective resolution of 4 A reveals that the molecule is highly alpha-helical and has an unusually elongated 'Y'-shape. The stalk and two arms of the 'Y' form three discrete structural domains which most likely correspond to the three functional regions identified for the channel-forming colicins. The channel-forming region of colicin Ia can be located to the larger of the two arms, the insertion domain, by its structural similarity to the ten alpha-helix motif found for the ion channel-forming fragments of colicins A and E1. The domain arrangement found in this structure provides novel insights into the mechanism of membrane insertion of colicin Ia.
Cell Membrane↗
Structure of the protease from simian immunodeficiency virus: complex with an irreversible nonpeptide inhibitor.
A variant of the simian immunodeficiency virus protease (SIV PR), covalently bound to the inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), was crystallized. The structure of the inhibited complex was determined by X-ray crystallography to a resolution of 2.4 A and refined to an R factor of 19%. The variant, SIV PR S4H, was shown to diminish the rate of autolysis by at least 4-fold without affecting enzymatic parameters. The overall root mean square (rms) deviation of the alpha-carbons from the structure of HIV-1PR complexed with a peptidomimetic inhibitor (7HVP) was 1.16 A. The major differences are concentrated in three surface loops with rms differences between 1.2 and 2.1 A. For 60% of the molecule the rms deviation was only 0.6 A. The structure reveals one molecule of EPNP bound per protease dimer, a stoichiometry confirmed by mass spectral analysis. The epoxide moiety forms a covalent bond with either of the active site aspartic acids of the dimer, and the phenyl moiety occupies the P1 binding site. The EPNP nitro group interacts with Arg 8. This structure suggests a starting template for the design of nonpeptide-based irreversible inhibitors of the SIV and related HIV-1 and HIV-2 PRs.
Aspartic Acid↗
Structure at 2.5 A of a designed peptide that maintains solubility of membrane proteins.
A 24-amino acid peptide designed to solubilize integral membrane proteins has been synthesized. The design was for an amphipathic alpha helix with a "flat" hydrophobic surface that would interact with a transmembrane protein as a detergent. When mixed with peptide, 85 percent of bacteriorhodopsin and 60 percent of rhodopsin remained in solution over a period of 2 days in their native forms. The crystal structure of peptide alone showed it to form an antiparallel four-helix bundle in which monomers interact, flat surface to flat surface, as predicted.
Amino Acid Sequence↗
Mapping a membrane-associated conformation of colicin Ia.
Channel-forming colicins exist in at least two different membrane-associated conformations: a voltage-independent closed-channel state and a voltage-dependent open-channel state. In a voltage-independent membrane-associated conformation, we find that two major regions of colicin Ia are protected from pepsin proteolysis after association with negatively charged membranes. In contrast, colicin Ia is rapidly and completely proteolyzed in the absence of membranes. The major protected region includes an electrophysiologically defined C-terminal channel-forming domain as well as 96 residues upstream of this region. Approximately 100 residues spanning Ala79- approximately Arg189 within the N-terminal domain are protected as well. The first N-terminal 76 residues of colicin Ia and a large region which includes much of the putative central receptor-binding domain are not protected from proteolysis. Both N- and C-termini of protected peptides have been identified using a combination of gel electrophoresis, N-terminal sequencing, and mass spectrometry, thereby defining specific residues that are located on the outside of the lipid bilayer. These data suggest a role for regions other than the electrophysiologically defined C-terminal channel-forming domain in membrane insertion and channel formation.
Amino Acid Sequence↗
Refined structures of substrate-bound and phosphate-bound thymidylate synthase from Lactobacillus casei.
Crystal structures of two crystal forms of the complex of Lactobacillus casei (TS) with its substrate dUMP have been solved and refined at 2.55 A resolution. The two crystal forms differ by approximately 5% in the c-axis length. The TS-dUMP complexes are symmetric dimers with dUMP bound equivalently in both active sites. dUMP is non-covalently bound in the same conformation as in ternary complexes of TS with dUMP and cofactor or cofactor analogs. The same hydrogen bonds are made between TS and substrate in the binary and ternary complexes. We have also determined the 2.36 A crystal structure of phosphate-bound L. casei TS. This structure has been refined to an R-factor of 19.3% with highly constrained geometry. Refinement has revealed the locations of all residues in the protein, including the disordered residues 90 to 119, which are part of an insert found only in the L. casei and Staphylococcus aureus transposon Tn4003 TS sequences. The 2.9 A multiple isomorphous replacement (MIR) structure of L. casei TS in a complex with its substrate dUMP has been refined to a crystallographic R-factor of 15.5%. Reducing agents were withheld from crystallization solutions during MIR structure determination to allow heavy-metal labeling of the cysteine residues. Therefore, the active-site cysteine residue in this structure is oxidized and the dUMP is found at half-occupancy in the active site. No significant conformational difference was found between the phosphate-bound and dUMP-bound structures. The TS-dUMP structures were better ordered than the phosphate-bound TS or the oxidized TS-dUMP, particularly Arg23, which is clearly hydrogen-bonded to the phosphate group of dUMP. A large and a small P6(1)22 crystal form are observed for both phosphate-bound and dUMP-bound L. casei TS. The small cell forms of the phosphate-bound and dUMP-bound enzyme are isomorphous, whereas the cell constants of the larger cell form change slightly when dUMP is bound (c = 240 A versus c = 243 A). For both liganded and unliganded enzyme, conversion from the small to the large crystal form sometimes occurs spontaneously, and the crystal packing changes at a single interface. Conversion may be the result of a small change in pH in the mother liquor surrounding the crystal. A single intermolecular contact between symmetry-related Asp287 residues is disrupted on going from the small to the large crystal form.
Amino Acid Sequence↗
Structures of thymidylate synthase with a C-terminal deletion: role of the C-terminus in alignment of 2'-deoxyuridine 5'-monophosphate and 5,10-methylenetetrahydrofolate.
Thymidylate synthase undergoes a major conformational change upon ligand binding, where the carboxyl terminus displays the largest movement (approximately 4 A). This movement from an "open" unliganded state to the "closed" complexed conformation plays a crucial role in the correct orientation of substrates and in product formation. The mutant lacking the C-terminal valine (V316Am) of the enzyme is inactive. X-ray crystal structures of V316Am and its complexes with dUMP, FdUMP, and both FdUMP and CH2H4folate are described. The structures show that ligands are bound within the active site, but in different modes than those in analogous, wild-type thymidylate synthase structures. The 2.7-A binary complex structures of V316Am with FdUMP and dUMP show that the pyrimidine and ribose moieties of the nucleotides are pivoted approximately 20 degrees around the 3'-hydroxyl compared to dUMP in the wild-type enzyme. The 2.7-A crystal structure of V316Am complexed with cofactor, CH2H4folate, and the substrate analog, FdUMP, shows these ligands bound in an open conformation similar to that of the unliganded enzyme. In this ternary complex, the imidazolidine ring of the cofactor is open and has reacted with water to form 5-HOCH2H4folate. 5-HOCH2H4folate is structural evidence for the 5-iminium ion intermediate, which is the proposed reactive form of CH2H4folate. The altered ligand binding modes observed in the three V316Am complex structures open new venues for the design of novel TS inhibitors.
Crystallization↗
Structure-based discovery of inhibitors of thymidylate synthase.
A molecular docking computer program (DOCK) was used to screen the Fine Chemical Directory, a database of commercially available compounds, for molecules that are complementary to thymidylate synthase (TS), a chemotherapeutic target. Besides retrieving the substrate and several known inhibitors, DOCK proposed putative inhibitors previously unknown to bind to the enzyme. Three of these compounds inhibited Lactobacillus casei TS at submillimolar concentrations. One of these inhibitors, sulisobenzone, crystallized with TS in two configurations that differed from the DOCK-favored geometry: a counterion was bound in the substrate site, which resulted in a 6 to 9 angstrom displacement of the inhibitor. The structure of the complexes suggested another binding region in the active site that could be exploited. This region was probed with molecules sterically similar to sulisobenzone, which led to the identification of a family of phenolphthalein analogs that inhibit TS in the 1 to 30 micromolar range. These inhibitors do not resemble the substrates of the enzyme. A crystal structure of phenolphthalein with TS shows that it binds in the target site in a configuration that resembles the one suggested by DOCK.
Amino Acid Sequence↗
Colicin Ia inserts into negatively charged membranes at low pH with a tertiary but little secondary structural change.
Colicin Ia, a member of the channel-forming family of colicins, inserts into model membranes in a pH- and lipid-dependent fashion. This insertion occurs with single-hit kinetics, requires negatively charged lipids in the target membrane, and increases in rate as the pH is reduced below 5.2. The low-pH requirement does not act by inducing a secondary structural change in colicin Ia, which remains 66% +/- 4% alpha-helical between pHs 7.3 and 3.1 as determined by circular dichroism. The secondary structure also remains unchanged between pHs 7.3 and 4.2 in the hydrophobic environment provided by the detergent octyl beta-D-glucopyranoside (beta-OG). However, at pH 3.1 in the presence of beta-OG, an 11% +/- 3% decrease in the alpha-helical content is observed. Further, beta-OG induces a change in tryptophan fluorescence and an altered pattern of proteolytic digestion, indicative of a tertiary structural changes. This suggests that colicin Ia undergoes a tertiary but little or no secondary structural change in its transition from a soluble to a transmembrane protein.
Chromatography, Gel↗
Bacteriorhodopsin D85N: three spectroscopic species in equilibrium.
Ground-state absorbance measurements show that BR from Halobacterium halobium containing asparagine at residue 85 (D85N) exists as three distinct chromophoric states in equilibrium. In the pH range 6-12 the absorbance spectra of the three states are demonstrated to be similar to flash-induced spectral intermediates which comprise the latter portion of the wild-type BR photocycle. One of the states absorbs maximally at 405 nm, has a deprotonated Schiff base, and contains predominantly the 13-cis form of retinal, identifying it as a close homologue of the M intermediate in the BR photocycle. The other species possess absorbance maxima with correspondence to those of the wild-type N (570 nm) and O (615 nm) photointermediates. The retinal composition of the O-like form was found to be dominated by all-trans isomer. The pH dependence of the concentrations of the equilibrium species corresponds closely with the pH dependence of the M, N, and O photointermediates. These data support kinetic models which emphasize the role of back-reactions during the photocycle of bacteriorhodopsin. Energetic and spectral characterization of the D85N ground-state equilibrium supports its use as a model for elucidating molecular transitions comprising the latter portion of the BR photocycle.
Bacteriorhodopsins↗