PubMed Health⌕ Search

Biomedical subjects

J E Ladner

Publications and source records attributed to J E Ladner.

At least 19 recordsLinked to original sources
PubMed2000

Stressing-out DNA? The contribution of serine-phosphodiester interactions in catalysis by uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates. From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5' pinching interaction. The S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fold damaging effects on k(cat)/K(m), respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect. A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5' and 3' phosphodiester groups flanking the bound uridine. Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation. However, in contrast to a recent proposal [Parikh, S. S., et al. (2000) Proc Natl. Acad. Sci. 94, 5083], there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10(12)-fold rate enhancement brought about by UDG.

Catalysis↗
PubMed2000

The 1.30 A resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimer.

The crystal structure of the Bacillus subtilis chorismate mutase, an enzyme of the aromatic amino acids biosynthetic pathway, was determined to 1.30 A resolution. The structure of the homotrimer was determined by molecular replacement using orthorhombic crystals of space group P2(1)2(1)2(1) with unit-cell parameters a = 52.2, b = 83. 8, c = 86.0 A. The ABC trimer of the monoclinic crystal structure [Chook et al. (1994), J. Mol. Biol. 240, 476-500] was used as the starting model. The final coordinates are composed of three complete polypeptide chains of 127 amino-acid residues. In addition, there are nine sulfate ions, five glycerol molecules and 424 water molecules clearly visible in the structure. This structure was refined with aniosotropic temperature factors, has excellent geometry and a crystallographic R factor of 0.169 with an R(free) of 0.236. The three active sites of the macromolecule are at the subunit interfaces, with residues from two subunits contributing to each site. This orthorhombic crystal form was grown using ammonium sulfate as the precipitant; glycerol was used as a cryoprotectant during data collection. A glycerol molecule and sulfate ion in each of the active sites was found mimicking a transition-state analog. In this structure, the C-terminal tails of the subunits of the trimer are hydrogen bonded to residues of the active site of neighboring trimers in the crystal and thus cross-link the molecules in the crystal lattice.

Bacillus subtilis↗
PubMed2000

Cryosalts: suppression of ice formation in macromolecular crystallography.

Quality data collection for macromolecular cryocrystallography requires suppressing the formation of crystalline or microcrystalline ice that may result from flash-freezing crystals. Described here is the use of lithium formate, lithium chloride and other highly soluble salts for forming ice-ring-free aqueous glasses upon cooling from ambient temperature to 100 K. These cryosalts are a new class of cryoprotectants that are shown to be effective with a variety of commonly used crystallization solutions and with proteins crystallized under different conditions. The influence of cryosalts on crystal mosaicity and diffraction resolution is comparable with or superior to traditional organic cryoprotectants.

Cryoprotective Agents↗
PubMed1999

Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction.

Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.

Animals↗
PubMed1999

The three-dimensional structures of two isoforms of nucleoside diphosphate kinase from bovine retina.

The crystal structures of two isoforms of nucleoside diphosphate kinase from bovine retina overexpressed in Escherischia coli have been determined to 2.4 A resolution. Both the isoforms, NBR-A and NBR-B, are hexameric and the fold of the monomer is in agreement with NDP-kinase structures from other biological sources. Although the polypeptide chains of the two isoforms differ by only two residues, they crystallize in different space groups. NBR-A crystallizes in space group P212121 with an entire hexamer in the asymmetric unit, while NBR-B crystallizes in space group P43212 with a trimer in the asymmetric unit. The highly conserved nucleotide-binding site observed in other nucleoside diphosphate kinase structures is also observed here. Both NBR-A and NBR-B were crystallized in the presence of cGMP. The nucleotide is bound with the base in the anti conformation. The NBR-A active site contained both cGMP and GDP each bound at half occupancy. Presumably, NBR-A had retained GDP (or GTP) from the purification process. The NBR-B active site contained only cGMP.

Animals↗
PubMed1998

Nucleoside diphosphate kinase from bovine retina: purification, subcellular localization, molecular cloning, and three-dimensional structure.

The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.

Amino Acid Sequence↗
PubMed1997

X-ray structure of a ribonuclease A-uridine vanadate complex at 1.3 A resolution.

The X-ray crystal structure of a uridine vanadate-ribonuclease A complex has been determined at 1.3 A resolution. The resulting structure includes all 124 amino-acid residues, a uridine vanadate, 131 water molecules, and a single bound 2-methyl-2-propanol. Side chains of 11 surface residues showing discrete disorder were modeled with multiple conformations. The final crystallographic R factor is 0.197. Structures obtained from high-level ab initio quantum calculations of model anionic oxyvanadate compounds were used to probe the effects of starting structure on the refinement process and final structure of the penta-coordinate phosphorane analog, uridine vanadate. The least-squares refinement procedure gave rise to the same final structure of the inhibitor despite significantly different starting models. Comparison with the previously determined complex of ribonuclease A with uridine vanadate obtained from a joint X-ray/neutron analysis (6RSA) [Wlodawer, Miller & Sjölin (1983). Proc. Natl Acad. Sci. USA, 80, 3628-3631] reveals similarities in the overall enzyme structure and the relative position of the key active-site residues, Hisl2, His119 and Lys41, but significant differences in the V-O bond distances and angles. The influence of ligand binding on the enzyme structure is assessed by a comparison of the current X-ray structure with the phosphate-free ribonuclease A structure (7RSA) [Wlodawer, Svensson, Sjölin & Gilliland (1988). Biochemistry, 27, 2705-2717]. Ligand binding alters the solvent structure, distribution and number of residues with multiple conformations, and temperature factors of the protein atoms. In fact, the temperature factors of atoms of several residues that interact with the ligand are reduced, but those of the atoms of several residues remote from the active site exhibit marked increases.

Journal Article↗
PubMed1996

Crystallization of biological macromolecules for X-ray diffraction studies.

Advances in the crystallization of biological macromolecules have come not only from the application of biochemical, molecular biological and immunological principles and techniques, but also from continued efforts to understand the crystallization process. Developments in crystallization methodologies, protocols, and reagents are also facilitating crystallization efforts.

Animals↗
PubMed1994

Biological Macromolecule Crystallization Database, Version 3.0: new features, data and the NASA archive for protein crystal growth data.

Version 3.0 of the NIST/NASA/CARB Biological Macromolecule Crystallization Database (BMCD) includes crystal and crystallization data on all forms of biological macromolecules which have produced crystals suitable for X-ray diffraction studies. The data include summary information on each of the macromolecules, crystal data, crystallization conditions and comments about the crystallization procedure if it varies from the traditional methods employed for crystal growth. The database-management software maintains continuity with previous versions providing similar search procedures and displays. Version 3.0 of the BMCD includes protocols and results of crystallization experiments undertaken in space. These new data are comprised of both the NASA Protein Crystal Growth Archive, which includes information on all NASA-sponsored protein crystal growth experiments, and data describing other internationally sponsored microgravity macromolecule crystallization studies. The entries for the space growth crystallization experiments contain the crystallization protocols, apparatus descriptions, flight summary data, indication of success or failure of the experiments, references, etc. Other new features of the BMCD include the addition of crystallization procedures for small peptides and cross references to other structural biology databases.

Journal Article↗
PubMed1982

Gross quaternary changes in aspartate carbamoyltransferase are induced by the binding of N-(phosphonacetyl)-L-aspartate: A 3.5-A resolution study.

The three-dimensional structure of the complex of N-(phosphonacetyl)-L-aspartate with aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) has been determined to a nominal resolution of 3.5 A by single-crystal x-ray diffraction methods. Initial phases were obtained by the method of "molecular tectonics": beginning with the structure of the CTP-protein complex, the domains of the catalytic and regulatory chains were manipulated as separate rigid bodies. The resulting coordinates were used to calculate an electron density map, which was then back transformed to give a set of calculated amplitudes and phases. Using all observed data, we obtained a crystallographic R factor between observed and calculated amplitudes Fo and Fc of 0.46. An envelope was then applied to a 2Fo - Fc map and the density was averaged across the molecular twofold axis. Two cycles of averaging yielded an R factor of 0.25. In this complex, we find that the two catalytic trimers have separated from each other along the threefold axis by 11-12 A and have rotated in opposing directions around the threefold axis such that the total relative reorientation is 8-9 degrees. This rotation places the trimers in a more nearly eclipsed configuration. In addition, two domains in a single catalytic chain have changed slightly their spatial relationship to each other. Finally, the two chains of one regulatory dimer have rotated 14-15 degrees around the twofold axis, and the Zn domains have separated from each other by 4 A along the threefold axis. These movements enlarge the central cavity of the molecule and allow increased accessibility to this cavity through the six channels from the exterior surface of the enzyme.

Allosteric Regulation↗
PubMed1975

Structure of yeast phenylalanine transfer RNA at 2.5 A resolution.

The x-ray analysis of the monoclinic form of yeast tRNAPhe has been taken to a resolution of 2.5 A by the method of isomorphous replacement. The model proposed at 3 A has been confirmed and extended to reveal additional features of the tertiary structure and of the stereochemistry. An extensive hydrogen bonding network is described involving specific interactions between bases and the ribose-phosphate backbone. The structure of a G-U base pair has been solved.

Base Sequence↗