CHAIN — A crystallographic modeling program

Sack, John S.

doi:10.1016/s0263-7855(98)80040-4

Cited by 452 publications

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“…The map was calculated using 15-1.6 Å data and contoured at 1.3 σ (0.28 e Å -3 ). This figure was drawn with CHAIN (Sack, 1988). (B) Ribbon diagram showing the secondary structure elements of T4 CH monomer.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

1999

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Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyldCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 Å resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (~20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built. Keywords: bacteriophage T4/deoxycytidylate hydroxymethylase/deoxyuridylate hydroxymethylase/ dNTP-synthesizing complex/thymidylate synthase

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Section: Resultsmentioning

confidence: 99%

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

1999

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“…During the course of refinement, residue conformations and solvent molecules were routinely examined using simulated annealed omit maps calculated with Fourier coefficients 2͉F o ͉ Ϫ ͉F c ͉ or ͉F o ͉ Ϫ ͉F c ͉ and phases derived from the in-progress model. Model building was performed with the graphics programs CHAIN (Sack, 1988) and O (Jones et al, 1991). The inhibitor molecule was modeled into density after the R factor dropped below 0.19; force field parameters for the inhibitor molecule were generated using the XPLO2D utility provided with the O package (Jones et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

Unexpected Binding Mode of the Sulfonamide Fluorophore 5-Dimethylamino-1-naphthalene Sulfonamide to Human Carbonic Anhydrase II

Nair¹,

Elbaum²,

Christianson³

1996

Journal of Biological Chemistry

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The three-dimensional structure of human carbonic anhydrase II (CAII) complexed with the sulfonamide fluorophore 5-dimethylamino-1-naphthalene sulfonamide (dansylamide) has been determined to 2.1-Å resolution by x-ray crystallographic methods. Unlike other arylsulfonamide inhibitors of CAII, the naphthyl ring of dansylamide binds in a hydrophobic pocket in the active site, making van der Waals contacts with Val-121, Phe-131, Val-143, Leu-198, and Trp-209. Interestingly, a conformational change of Leu-198 is required to accommodate dansylamide binding, which rationalizes the enhanced dansylamide affinity measured for certain Sensors based upon biological macromolecules are increasingly used for the selective detection of numerous chemical entities, including the detection of trace quantities of metal ions. The advantages of metalloprotein-based biosensors over classical fluorometric chemical indicators for such applications include greater recognition and affinity for the target metal ion, exquisite discrimination among transition metals, and kinetically rapid biosensor-analyte association and dissociation. Recently, Thompson and Jones (1993) have exploited the selective recognition of zinc by the metalloenzyme carbonic anhydrase II (CAII) 1 in the design of an enzyme-based zinc biosensor. The physical basis of this sensor is the binding of zinc to the CAII apoenzyme, followed by binding of the fluorophore inhibitor 5-dimethylamino-1-naphthalene sulfonamide (dansylamide, K d ϭ 0.93 M (Nair et al., 1995); see Fig. 1) to the zinccontaining CAII holoenzyme. This leads to a titratable change in the fluorescence emission wavelength and intensity (Chen and Kernohan, 1967); nanomolar concentrations of zinc can be detected and quantified by ratiometric methods by measuring the emission of free dansylamide at 580 nm and the emission of bound dansylamide at 470 nm when excited at 290 nm.The zinc ion of CAII resides at the base of a 15-Å deep cleft, where it is liganded by the imidazole side chains of His-94, His-96, and His-119 (Håkansson et al., 1992). Hydroxide ion, which is the catalytic nucleophile in the CO 2 hydration mechanism, completes a tetrahedral metal coordination polyhedron. Zinc-bound hydroxide donates a hydrogen bond to the hydroxyl group of Thr-199, which in turn donates a hydrogen bond to Glu-106 (Eriksson et al., 1986, 1988aHåkansson et al., 1992). The binding of sulfonamide inhibitors displaces zinc-bound hydroxide and maintains the tetrahedral metal coordination polyhedron by the coordination of an ionized sulfonamide NH group, which also maintains the hydrogen bond interaction with Thr-199 (e.g. see Eriksson et al. (1988b) and Vidgren et al.

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“…To observe the effect of crystal contacts on water sites, crystal contacts in the seven thrombin structures in space group C2 were calculated using Chain (Sack, 1988), where interactions were included for symmetry mate atoms within 4.0 A. Crystal contact residue and atom lists were generated for each of the crystallographic structures, with water sites represented by the microclusters observed in that protein.…”

Section: Crystal Contact Calculationsmentioning

confidence: 99%

Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity

Sanschagrin

Kuhn

1998

Protein Science

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Cluster analysis is presented as a technique for analyzing the conservation and chemistry of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster analysis provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water molecules, and also avoids reliance on any one structure as a reference. Water sites were clustered for ten superimposed thrombin structures. three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water molecules from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concentrated in buried regions including the solvent channel surrounding the Na' site in thrombin, which is associated with ligand selectivity. Cluster analysis also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addition to facilitating the analysis of water sites from multiple structures, cluster analysis provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.

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CHAIN — A crystallographic modeling program

Cited by 452 publications

References 0 publications

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

Unexpected Binding Mode of the Sulfonamide Fluorophore 5-Dimethylamino-1-naphthalene Sulfonamide to Human Carbonic Anhydrase II

Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity

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