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  • 1
    ISSN: 1072-8368
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Sir—Matrix metalloproteinases (MMPs) are a family of enzymes that are responsible for specifically cleaving components of the extracellular matrix such as collagen and proteoglycans1. Normal physiological processes such as morphogenesis, tissue repair, angiogenesis, uterine involution and ...
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  • 2
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
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  • 3
    ISSN: 0887-3585
    Keywords: staphylococcal nuclease ; nonproductive substrate binding to ; subsites of ; active site mutants of ; oligonucleotide binding to ; Ca2+ binding to ; Mn2+ binding to ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: By a combination of NMR docking and model building, the substrate binding site on staphylococcal nuclease was found to accommodate a trinucleotide and to consist of three subsites, each interacting with a single nucleotidyl unit of DNA. Binding of the essential Ca2+ activator and substrate cleavage occur between subsites 1 and 2. Hence, catalytically productive binding would span subsites 1 and 2 while nonproductive binding would span subsites 2 and 3. Lys-49 is near subsite 1, and Lys-84 and Tyr-115 interact with substrates at sub site 3 [Weber, D. J., Gittis, A. G., Mullen, G. P., Abeygunawardana, C., Lattman, E. E., Mildvan, A. S. Proteins 13:275-287, 1992]. The proposed locations of these subsites were independently tested by the effects of the K49A, K84A, and Y115A mutations of staphylococcal nuclease on the binding of Mn2+, Ca2+, and the dinucleotide and trinucleotide substrates, 5′-pdTdA, dTdA, and dTdAdG. These three mutants have previously been shown to be fully active and to have CD and 2D NMR spectra very similar to those of the wild-type enzyme (Chuang, W.-J., Weber, D. J., Gittis, A. G., Mildvan, A. S. Proteins 17:36-48, 1993). All three mutant enzymes and their pdTdA and dTdA complexes (but not their dTdAdG complex) bind Mn2+ and Ca2+ more weakly than the wild-type enzyme by factors ranging from 2 to 11. The presence of a terminal phosphate as in 5′-pdTdA raises the affinity of the substrate for staphylococcal nuclease and its three mutants by two orders of magnitude and for the corresponding enzyme-metal complexes by three to four orders of magnitude, suggesting that the terminal phosphate is coordinated by the enzyme-bound divalent cation. Such complexation would result in the nonproductive binding of 5′-pdTdA at subsites 2 and 3. Accordingly, the K84A and Y115A mutations significantly weaken the binding of 5′-pdTdA and its metal to staphylococcal nuclease by factors of 2.2 to 37.8, while the K49A mutation has much smaller or no effect. Such nonproductive binding explains the low activity of staphylococcal nuclease with small substrates, especially those With a terminal phosphate. Similarly, the K84A and Y115A mutations weaken the binding of dTdA and its metal complexes to the enzyme by factors of 3.4 to 13.1 while the K49A mutation has smaller effects indicating significant nonproductive binding of dTdA. The trinucleotide dTdAdG binds more tightly to wild-type and mutant staphylococcal nuclease and to its metal complexes than does the dinucleotide dTdA by factors of 2.4 to 12.2, reflecting the occupancy of an additional subsite. Predominantly productive binding of dTdAdG is indicated by the 1.7- to 8.3-fold lower affinities of the K49A, K84A, and Y115A mutants for the trinucleotide and its metal complexes. The largest effects on dTdAdG binding are seen with the Y115A mutation presumably reflecting the dual role of Tyr-115 both in donating a hydrogen bond to a phosphodiester oxygen between subsites 2 and 3 and in stacking onto the guanine base at subsite 3. © 1994 John Wiley & Sons, Inc.
    Additional Material: 11 Ill.
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  • 4
    ISSN: 0887-3585
    Keywords: staphylococcal nuclease ; mechanism of ; ternary enzyme-La3+-dTdA complex ; active site ; trinucleotide complex of ; assignments of 1H aromatic resonances ; assignments of 15N resonances ; HMQC studies of ; NOESY-HMQC studies of ; energy minimization of ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The conformation of the staphylococcal nuclease-bound metal-dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425-7437] was docked into the X-ray structure of the enzyme-Ca2+-3′,5′-pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183-201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme-La3+-dTdA complex were sequence specifically assigned by 2D phase-sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronu-clear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY-HMQC spectros-copy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme-La3+-3′,5′-pdTp complex and the enzyme-Ca2+-3′,5′-pdTp complex, proton resonances of Tyr-85, Tyr-113, Tyr-115, and Phe-34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr-113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme-bound dTdA shows the 5′-dA leaving group to partially overlap the inhibitor, 3′,5′-pdTp (in the X-ray structure). Tne 3′-TMP moiety of dTdA points toward the solvent in a channel defined by Ile-18, Asp-19, Thr-22, Lys-45, and His-46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu-43 and to attack the phosphorus with inversion. Arg-35 and Arg-87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg-87 positioned to protonate the leaving 5′-oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5′-dGMP onto the 3′-oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu-80, Asp-83, Lys-84, and Tyr-115 with its 3′-OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide. © 1992 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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  • 5
    ISSN: 0887-3585
    Keywords: staphylococcal nuclease ; mutants of ; lattice artifactsd ; dissociation constants of 3′,5′-pdTp ; subdomains of ; Ca2+ binding to ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In the X-ray structure of the staphylococcal nuclease-Ca2+ -3′,5′-pdTp complex, the conformation of the inhibitor 3′,5′-pdTp is distroteed Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5 : 183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3′,5′-pdTp in the staphylococcal nuclease-metal-pdTp Complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D. J., Serpersu, E. H., Gittis, A. G., Lattman, E. E., Mildvan, A. S. (preceding paper)]. In the NMR-docked structure, the 5′-phophate of 3′,5′-pdTp overlaps with that in the X-ray Structure. However the 3′-phosphate accepts a hydrogen bond from Lys-49 (2.89Å) rather than from Lys-84 (8.63 Å), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 Å) which does not occur in the X-ray structure (5.28 Å). These interactions have been tested by binding studies of 3′,5′-pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca2+ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While thye K84A mutation did not significantly weaken 3′,5′-pdTp binding to the enzyme (1.5 ± 0.7 fold), the K49A mutation weakened 3′,5′-pdTp binding to the enzyme by the factor of 4.4 ± 1.8-fold. Similarly, the Y115A mutation weakened 3′,5′-pdTp binding to the enzyme 3.6 ± 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca2+-3′,5′-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3′,5′-pdTp than by the X-ray structure. © 1993 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 6
    ISSN: 0887-3585
    Keywords: staphylococcal nuclease active site ; conformation of 3′,5′-pdTp ; lattice contacts ; metal-nucleus distances ; nuclear Overhauser effect ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In the X-ray structure of the ternary staphylococcal nuclease-Ca2+ -3′,5′-pdTp complex, the conformation of the bound inhibitor 3′,5′-pdTp is distorted by Lys-70* Abbreviations used: 3′,5′-pdTp, thymidine 3′,5′-di-phosphate; Tris-HCl, tris-(hydroxymethyl)aminomethane hydrochloride; NOE, nuclear Overhauser effect; EDTA, ethylene-diaminetetraacetic acid; TES, N-[tris(hydroxymethyl)-methyl]-2-aminoethane sulfonic acid; Lys-70*, 71*, lysine residues from a neighboring molecule of staphylococcal nuclease in the crystal lattice. and Lys-71* from an adjacent molecule of the enzyme in the crystal lattice (Loll, P.J. and Lattman, E.E. Proteins 5:183-201, 1989; Serpersu, E.H., Hibler, D.W., Gerlt, J.A., and Mildvan, A.S. Biochemistry 28:1539-1548, 1989). Since this interaction does not occur in solution, the NMR docking procedure has been used to correct this problem. Based on 8 Co2+ -nucleus distances measured by paramagnetic effects on T1, and 9 measured and 45 lower limit interproton distances determined by 1D and 2D NOE studies of the ternary Ca2+ complex, the conformation of enzyme-bound 3′,5′-pdTp is high-anti (χ = 58 = 10°) with a C2′ endo/O1′ endo sugar pucker (δ = 143 ± 2°), (-) synclinal about the C3′-O3′ bond (ε = 273 ± 4°), trans, gauche about the C4′-C5′ bond (γ = 301 ± 29°) and either (-) or (+) clinal about the C5′-O5′ bond (β = 92 ± 8° or 274 ± 3°). The structure of 3′,5′-pdTp in the crystalline complex differs due to rotations about the C4′-C5′ bond (γ = 186 ± 12°, gauche, trans) and the C5′-O5′ bond [β = 136 + 10°, (+) anticlinal]. The undistorted conformation of enzyme-bound metal-3′,5′-pdTp determined by NMR was docked into the X-ray structure of the enzyme, using 19 intermolecular NOEs from ring proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the inhibitor. van der Waals overlaps were then removed by energy minimization. Subsequent molecular dynamics and energy minimization produced no significant changes, indicating the structure to be in a global rather than in a local minimum. While the metal-coordinated 5′-phosphate of the NMR-docked structure of 3′,5′-pdTp overlaps with that in the X-ray structure, and similarly receives bifunctional hydrogen bonds from both Arg-35 and Arg-87, the thymine, deoxyribose, and 3′-phosphate are significantly displaced from their positions in the X-ray structure, with the 3′-phosphate receiving hydrogen bonds from Lys-49 rather than from Lys-84 and Tyr-85. The repositioned thymine ring permits hydrogen bonding to the phenolic hydroxyl of Tyr-115. These new interactions, found in the NMR docked structure, are supported by reduced affinities for 3′,5′-pdTp by appropriate mutants of staphylococcal nuclease (Chuang, W.-J., Weber, D.J., Gittis, A.G., and Mildvan, A.S. (1993) accompanying paper, this issue). An inner sphere, rather than a second sphere water ligand of the metal, is optimally positioned to donate a hydrogen bond to Glu-43 and to attack the coordinated 5′-phosphate with inversion. It is concluded that the NMR docking procedure can be used to correct structural artifacts created by lattice contacts in crystals, when they occur at or near ligand binding sites, such as the active sites of enzymes. © 1993 Wiley-Liss, Inc.
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  • 7
    ISSN: 1072-8368
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Sir—A multitude of proteins regulate the assembly and crosslinking of the ubiquitous cytoskeletal protein actin1. The actin-depolymerizing factor/cofilin family is a phylogenetically wide-spread class of proteins that bind actin monomers and sever actin filaments2. These properties promote ...
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  • 8
    ISSN: 1072-8368
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Deletion of six amino acids in a surface loop transforms staphylococcal nuclease from a monomeric protein into a very stable dimer (Kd〈1×10−8M). A 2 Å X-ray crystal structure of the dimer (R=0.176) shows that the carboxy-terminal α-helix has been stripped from its normal ...
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  • 9
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
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  • 10
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
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