Jenny P. Glusker

Structural Aspects of Metal Liganding to Functional Groups in Proteins

Publisher Summary Metal ions serve a variety of functions in proteins. The most important function is to enhance the structural stability of the protein in the conformation required for biological function and to take part in the catalytic processes of enzymes. Metal ions can take part in trigger and control mechanisms by specifically altering or stabilizing a macromolecular conformation on binding. This chapter discusses the properties of metals that are useful in the structure and function of proteins, particularly enzymes, and the geometry of interaction of metals with the various chemical groups of proteins are emphasized. Ligands donate an electron pair to the bond and are generally negatively charged or neutral. Important in a study of metal-ligand interactions are the polarizability of both the metal ion and the ligand, the number of the ligands around each metal ion, and the stereochemistry of the resulting arrangement. The stereochemistry of liganding of metal ions in proteins is known for several proteins. Some selected examples follow with data derived from the Protein Data Bank are emphasized in the chapter. The metals in protein crystal structures discussed are— namely, (1) copper, (2) iron, (3) manganese, (4) zinc, (5) magnesium, and (6) calcium. In the cases in which two different metals are bound, information can be obtained on preferential sites for each metal in the presence of the other.

Metal Ion Roles and the Movement of Hydrogen during Reaction Catalyzed by D-Xylose Isomerase: A Joint X-Ray and Neutron Diffraction Study

Conversion of aldo to keto sugars by the metalloenzyme D-xylose isomerase (XI) is a multistep reaction that involves hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic D-glucose (before sugar ring opening has occurred), and the other containing nickel and linear D-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. The data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening and gains a proton after this; the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.

Structure of a dinucleoside phosphate--drug complex as model for nucleic acid--drug interaction.

The crystal structure of a 3 : 2 complex of the frameshift mutagen proflavine with the dinucleoside phosphate cytidylyl-3′5′-guanosine has been determined. The complex has one drug molecule intercalated between Watson–Crick base pairs of the nucleotide duplex. The other two proflavine molecules are bound to the exterior of the miniature double helix. The orientation of the base pairs in this miniature double helix has aspects similar to that found in RNA 11.

Directional Aspects of Intermolecular Interactions

Directional intermolecular interactions can be found by statistical analyses of the surroundings of functional groups in all crystal structures containing them. The most notable such interaction is the hydrogen bond, which, if strong as between OH or NH groups and oxygen or nitrogen acceptors, is approximately linear. This provides an important means of aligning molecules together. Analogous but weaker interactions described here are C-H...O, F...H and H... π, and also those between C-S-C groups and electrophiles or nucleophiles. Some directionality can also be identified in aromatic-aromatic interactions. Metal ion coordination can, in certain instances, also have a directional component, particularly if the coordination geometry is inflexible, as for the octahedral binding of divalent magnesium. The geometries of interactions of metal ions with various functional groups in proteins are described, and in many cases they are more rigid than the analogous interaction involving a hydrogen bond. The emerging use of crystal surfaces as probes of molecular recognition is then discussed. Finally some examples of molecular recognition in biological macromolecules are given; these stress the importance of pattern recognition in hydrogen bonding, together with the significance of weaker interactions.

Fluorocitrate Inhibition of Aconitase: Relative Configuration of Inhibitory Isomer by X-ray Crystallography

The fluorocitrate isomer that is a strong inhibitor and inactivator of aconitase has been shown by x-ray crystallographic studies on the rubidium ammonium salt to have the configurations (1R : 2R) or (1S : 2S) 1-fluoro-2-hydroxy-1,2,3-propanetricarboxylic acid. A possible mechanism for the action of fluorocitrate is proposed which involves the 1R : 2R isomer suggested from biochemical data.

Modes of binding substrates and their analogues to the enzyme D-xylose isomerase.

Studies of binding of substrates and inhibitors of the enzyme D-xylose isomerase show, from X-ray diffraction data at 1.6-1.9 A resolution, that there are a variety of binding modes. These vary in the manner in which the substrate or its analogue extend, on binding, across the carboxy end of the (betaalpha)(8)-barrel structure. These binding sites are His54 and the metal ion (magnesium or manganese) that is held in place by Glul81, Asp245, Glu217 and Asp287. Possible catalytic groups have been identified in proposed mechanisms and their role in the binding of ligands is illustrated.

Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of D-xylose isomerase with bound D-xylulose

The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme d-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, d-xylulose, shows, unexpectedly, that O5 of d-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in the proximity of C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.

Preclinical and clinical perspectives on the use of estramustine as an antimitotic drug

A variety of cell biological, pharmacological, crystallographic and clinical approaches have indicated that the antimitotic drug estramustine has interesting and unusual properties. Although designed as an alkylating agent, the marked stability of the carbamate linkage to the steroid carrier molecule prevents the formation of alkylating intermediates. The affinity of the parent molecule for microtubule associated proteins and the concomitant antimicrotubule activity have cytotoxic consequences in tumor cells. Both preclinical and clinical studies of estramustine in combination with other antimicrotubule agents have shown that this approach has great potential to achieve therapeutic advantage, especially in disease states such as hormone refractory prostate cancer.

The crystal structure of the antitumor agent 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazonato) copper(II)

Abstract The crystal structure of a triclinic form of the copper(II) complex of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone) (CuKTS) has been determined. The KTS coordinates to copper(II) as a tetradentate ligand via two nitrogen and two sulphur atoms. The unit cell is triclinic, space group P 1 , with dimensions a = 9.306(6), b = 10.443(7), c = 7.479(5) A, α = 90.63(5), β = 114.25(5), γ = 98.42(5)°, observed density 1.720 g.cm−1, calculated density 1.721 g.cm−1, Z = 2. The structure was refined by full-matrix least-squares methods with anisotropic temperature parameters for non-hydrogen atoms to R = 0.056 for 2454 reflections. The ethoxyethyl side chain is disordered. The molecules pack in sheets, but the hydrogen bonding is generally weak. In the crystal structure, sulphur atoms in adjacent molecules lie above and below the copper atom of a central molecule, completing coordination about the Cu(II) with two long CuS distances of 3.101(2) and 3.312(2) A. These values may be compared to the molecular plane CuS bond lengths of 2.263(1) and 2.267(1) A and CuN bond lengths of 1.980(2) and 1.959(2) A. Thus there are potential binding sites in the axial positions of the copper atom. Binding at at least one of these sites probably occurs when the complex is carrying out its biological function, during which it is believed to act on a thiol group.

Absolute configuration of the isomer of fluorocitrate that inhibits aconitase

Abstract The absolute configuration of the isomer of fluorocitrate that is a strong inhibitor and activator of aconitase has been determined to be 2 R , 3 R , as shown in the Fischer and perspective diagrams below. Fischer diagram Perspective diagram This is the isomer described by Dummel and Kun (1969, J. Biol. Chem. 244 , 2966) and defined as (−)- erythro -fluorocitrate by them. We report here structural studies of the mirror image (enantiomer) of this isomer. Crystals of the complex of the diethyl ester of (+)- erythro -fluorocitrate with (−)-methylbenzylamine were studied by X-ray diffraction techniques. Since the absolute configuration of (−)-methylbenzylamine has already been determined experimentally, the absolute configuration of the (+)-erythro isomer of fluorocitrate is thereby established. This isomer is shown to be noninhibitory with the enzyme aconitase, while its racemate is a powerful inhibitor. Thus it is proved that the absolute configuration of the isomer of fluorocitrate that is formed from fluoroacetyl-CoA by the enzyme citrate synthase, and that inhibits aconitase, is the 2 R , 3 R , isomer.