Larry DeLucas

Calcium binding to D-glucuronate residues: crystal structure of a hydrated calcium bromide salt of D-glucuronic acid

Three-dimensional X-ray diffraction data were used to determine the crystal structure of alpha-D-glucuronate CaBr times 3H20, a model system for investigating the factors involved in the binding of calcium ions to D-glucuronate residues of oligo-and poly-saccharides. Crystals of the salt are monoclinic, space group P21, having a = 6.410 (1), b = 10.784 (2), c = 8.879 (1) A, betta = 92.07 (1)degrees, and Z = 2. Instensity data for 1082 reflections were measured with an automated diffractometer. A trial structure, obtained by the heavy-atom method, was refined by least squares to R = 0.025. The absolute configuration was confirmed by anomalous-dispersion effects. An outstanding feature of the crystal packing is the interaction of D-glucuronate anions with calcium ions. The calcium ion is coordinated to three symmetry-related D-glucuronate anions and to two water molecules. The D-glucuronate anion binds calcium cations through three chelation sites: one that involves a carboxyl-oxygen atom combined with O-5; one that includes the second carboxyl-oxygen atom acting in concert with O-4, and one composed of the O-1-O-2 pair of hydroxyl groups.

Real time evolution of concentration distribution around tetragonal lysozyme crystal: case study in gel and free solution

Two-beam Michaelson interferometry was used to study concentration gradient layers around gel-grown tetragonal lysozyme crystals. Crystals were grown in gel to depress convection and mimic microgravity. The evolution of the concentration profile near the growing surface, the width of the concentration layer, surface concentration, and concentration gradient were investigated and the correlation of these parameters with lysozyme crystal growth is discussed. The concentration gradient properties of gel-grown crystals were compared to those obtained for solution-grown crystals and were found to be different from their solution-grown counterparts. In particular, concentration gradients were wider and transport rate were slower for gel-grown crystals than for solution counterparts.

The three-dimensional structure of calcium-depleted human C-reactive protein from perfectly twinned crystals.

C-reactive protein is a member of the pentraxin family of oligomeric serum proteins which has been conserved through evolution, homologues having been found in every species in which they have been sought. Human C-reactive protein (hCRP) is the classical acute-phase reactant produced in large amounts in response to tissue damage and inflammation and is used almost universally as a clinical indicator of infection and inflammation. The role of hCRP in host defence and the calcium-dependent ligand-binding specificity of hCRP for phosphocholine moieties have long been recognized. In order to clarify the structural rearrangements associated with calcium binding, the reported affinity of calcium-depleted hCRP for polycations and other ligands, and the role of calcium in protection against denaturation and proteolysis, the structure of calcium-depleted hCRP has been determined by X-ray crystallography. Crystals of calcium-depleted hCRP are invariably twinned and those suitable for analysis are merohedral type II twins of point group 4 single crystals. The structure has been solved by molecular replacement using the calcium-bound hCRP structure [Shrive et al. (1996), Nature Struct. Biol. 3, 346-354]. It reveals two independent pentamers which form a face-to-face decamer across a dyad near-parallel to the twinning twofold axis. Cycles of intensity deconvolution, density modification (tenfold NCS) and model building, eventually including refinement, give a final R factor of 0.19 (R(free) = 0.20). Despite poor definition in some areas arising from the limited resolution of the data and from the twinning and disorder, the structure reveals the probable mode of twinning and the conformational changes, localized in one of the calcium-binding loops, which accompany calcium binding.

Polarized Raman Spectroscopic Studies of Tetragonal Lysozyme Single Crystals

Polarized Raman spectra have been obtained for tetragonal lysozyme single crystals of different relative quality. The Raman band at 507 cm-1, which corresponds to the totally symmetric stretch vibration of the gauche-gauche-gauche (ggg) disulfide bridges of the protein, has been shown to possess different polarization characteristics compared with the gauche-gauche-trans (ggt) disulfide bridge band at 528 cm-1. The relative intensities of the ggg and ggt bands in the polarized Raman spectra have been numerically estimated for a number of tetragonal lysozyme single crystals, the X-ray diffraction data of which are available from the Protein Data Bank. On the basis of comparison between the experimental and calculated polarization characteristics of the disulfide Raman lines, the following main conclusions have been drawn. The orientation of the protein molecules correlates with the average orientation of their ggg disulfide bridges. This in turn can be described by the rhoggg value which reflects the average orientation of the S-S bonds with respect to the Z crystallographic axis and can be determined from polarized Raman spectra. Crystals of better quality are characterized by a better alignment of the protein molecules with respect to the Z axis, a smaller perturbation of the protein molecules in the crystal lattice and a somewhat higher interlattice water content.

NH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution.

The final step of NAD+ biosynthesis includes an amide transfer to nicotinic acid adenine dinucleotide (NaAD) catalyzed by NAD+ synthetase. This enzyme was co-crystallized in microgravity with natural substrates NaAD and ATP at pH 8.5. The crystal was exposed to ammonium ions, synchrotron diffraction data were collected and the atomic model was refined anisotropically at 1 A resolution to R = 11.63%. Both binding sites are occupied by the NAD-adenylate intermediate, pyrophosphate and two magnesium ions. The atomic resolution of the structure allows better definition of non-planar peptide groups, reveals a low mean anisotropy of protein and substrate atoms and indicates the H-atom positions of the phosphoester group of the reaction intermediate. The phosphoester group is protonated at the carbonyl O atom O7N, suggesting a carbenium-ion structure stabilized by interactions with two solvent sites presumably occupied by ammonia and a water molecule. A mechanism is proposed for the second catalytic step, which includes a nucleophilic attack by the ammonia molecule on the intermediate.

Stabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis.

The NH(3)-dependent NAD(+) synthetase (NADS) participates in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) by transforming nicotinic acid adenine dinucleotide (NaAD) to NAD(+). The structural behavior of the active site, including stabilization of flexible loops 82-87 and 204-225, has been studied by determination of the crystal structures of complexes of NADS with natural substrates and a substrate analog. Both loops are stabilized independently of NaAD and solely from the ATP-binding site. Analysis of the binding contacts suggests that the minor loop 82-87 is stabilized primarily by a hydrogen bond with the adenine base of ATP. Formation of a coordination complex with Mg(2+) in the ATP-binding site may contribute to the stabilization of the major loop 204-225. The major loop has a role in substrate recognition and stabilization, in addition to the protection of the reaction intermediate described previously. A second and novel Mg(2+) position has been observed closer to the NaAD-binding site in the structure crystallized at pH 7.5, where the enzyme is active. This could therefore be the catalytically active Mg(2+).

Large scale preparation of homogeneous bacteriorhodopsin

Homogeneous bacteriorhodopsin was obtained preparatively (100 mg batches) from purple membrane of Halobacterium halobium cells. The homogeneity of the protein was considerably affected by variations in the growth conditions of the bacteria. Fully maturated bacteriorhodopsin having a blocked N‐terminus and a homogeneous C‐terminus, was reproducibly obtained when cells were grown in a sufficiently aerated medium.

Structural adaptation of an interacting non-native C-terminal helical extension revealed in the crystal structure of NAD + synthetase from Bacillus anthracis

The crystal structures of NH(3)-dependent NAD+ synthetase from Bacillus anthracis as the apoenzyme (1.9 A), in complex with the natural catalytic products AMP and pyrophosphate (2.4 A) and in complex with the substrate analog adenosine 5-(alpha,beta-methylene)triphosphate (2.0 A) have been determined. NAD+ synthetase catalyzes the last step in the biosynthesis of the vitally important cofactor NAD+. In comparison to other NAD+ synthetase crystal structures, the C-terminal His-tagged end of the apoenzyme adopts a novel helical conformation, causing significant compensatory changes in the region. The structural accommodations observed in B. anthracis NAD+ synthetase are remarkable in the absence of adverse affects on enzyme activity. They also illustrate a rare example of the influence of a non-native C-terminal His-tag extension on the structure of a native protein. In contrast to the apoenzyme, when AMP and pyrophosphate or adenosine 5-(alpha,beta-methylene)triphosphate are bound, the C-terminus adopts a conformation that allows ATP binding and overall the structure then resembles other NAD+ synthetase structures. The structures of NAD+ synthetase complexes from B. anthracis are compared with published X-ray crystal structures of the enzyme from B. subtilis, Escherichia coli and Helicobacter pylori. These comparisons support the novel observation that P1 and P2 loop ordering is not a consequence of crystal contacts but rather a consequence of intrinsic intramolecular interactions within the ordered subunit.

Preliminary X-ray study of crystals of human transferrin

Abstract Large crystals of human transferrin have been grown from polyethylene glycol solutions. The crystals are orthorhombic, space group P212121, with a = 78 A , b = 94 A and c = 112 A . Density measurements indicate that there are four transferrin molecules in the unit cell.

Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 μm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.