Nan Wu

Case Western Reserve University, Cleveland, OH, United States

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Publications (9)43.08 Total impact

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    ABSTRACT: Arrestins serve as multi-functional regulators of G-protein coupled receptors, interacting with hundreds of different receptor subtypes and a variety of other signaling proteins. Here we identify calmodulin as a novel arrestin interaction partner using three independent methods in vitro and in cells. Arrestin preferentially binds calcium-loaded calmodulin with a Kd value of approximately 7 microM, which is within range of endogenous calmodulin concentrations. The calmodulin binding site is localized on the concave side of the C-domain and a loop in the center of the arrestin molecule, significantly overlapping with receptor and microtubule-binding sites. Using purified proteins, we found that arrestins sequester calmodulin, preventing its binding to microtubules. Nanomolar affinity of arrestins for their cognate receptors makes calmodulin an ineffective competitor for arrestin binding at relatively high receptor concentrations. The arrestin-calmodulin interaction likely regulates the localization of both proteins and their availability for other interaction partners.
    Full-text · Article · Jan 2007 · Journal of Molecular Biology
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    ABSTRACT: Fusobacterium nucleatum is a gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: the intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 59.3, c = 125.7 A and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 A. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum.
    Full-text · Article · Jan 2007 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
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    ABSTRACT: Signal transduction by G protein-coupled receptors (GPCRs) is mediated by interactions between intracellular proteins and exposed motifs on the cytoplasmic face of these receptors. Arrestins bind to GPCRs and modulate receptor function either by interfering with heterotrimeric G protein signaling or by serving as signaling adaptors themselves. Calmodulin interacts with GPCRs triggering a calcium response. We have studied the interaction of arrestin2 and calmodulin with intracellular elements of the human V1-vascular vasopressin receptor (hV1R). For this purpose, we designed, expressed, and purified soluble fusion proteins with the maltose-binding protein (MBP) from Escherichia coli that mimic the intracellular surface of the hV1R. These MBP fusion proteins bind arrestin2 and calmodulin with affinities in the micromolar range. A different series of soluble receptor analogs, named vasopressin receptor 1 elements on a soluble scaffold (V1ROSS) proteins, consist of the third intracellular loop and/or the C-terminal segment of the hV1R receptor juxtaposed on the surface of the MBP. V1ROSS proteins bind calmodulin and a truncated, phosphorylation-independent form of arrestin2 more tightly than the corresponding linear fusion proteins. Thus, embedding receptor loops within the three-dimensional structure of the MBP yields a better representation of the active conformation of these receptor loops than linear receptor peptides fused onto the C terminus of the MBP. V1ROSS proteins provide a valuable tool to study receptor interactions because they are more amenable to structural analysis than the native membrane receptor. These findings set the stage for the detailed structural analysis of these protein-protein interactions that are important for understanding the mechanism of signaling.
    Full-text · Article · Aug 2006 · Molecular Pharmacology
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    ABSTRACT: The electron density and the electrostatic potential (ESP) distributions of estrone have been determined using X-ray diffraction analysis and compared with theoretical calculations in the solid and gas phases. X-ray diffraction measurements are performed with a Rigaku Rapid rotating anode diffractometer at 20 K. The electron density in the estrone crystal has been described with the multipole model, which allowed extensive topological analysis and calculation of the ESP. From DFT calculations in the solid state a theoretical X-ray diffraction data set has been produced and treated in the same way as the experimental data. Two sets of single molecule DFT calculations were performed: (a) An electron density distribution was obtained via a single-point calculation with a large basis set at the experimental geometry and subsequently analyzed according to the quantum theory of atoms in molecules (AIM) to obtain the bond and most atomic properties, and (b) another electron density distribution was obtained with a smaller basis set, but at a geometry optimized using the same basis set for the analysis of atomic energies. An interesting locally stabilizing hydrogen-hydrogen bond path linking H(1) and H(11B) is found which represents the first characterization of such bonding in a steroid molecule. AIM delocalization indices were shown to be well correlated to the experimental electron density at the bond critical points through an exponential relationship. The aromaticity of ring A, chemical bonding, the O(1)...O(2) distance necessary for estrogenic activity, and the electrostatic potential features are also discussed.
    Preview · Article · Aug 2006 · Journal of the American Chemical Society
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    ABSTRACT: Whereas arginine vasopressin binds to its receptor subtypes V(1)R and V(2)R with equal affinity of approximately 2 nM, nonpeptide antagonists interact differently with vasopressin receptor subtypes. The V(2)R antagonist binding site was mapped by site-directed mutagenesis at six selected amino acid positions, K100D, A110W, M120V, L175Y, R202S, and F307I, predicted to be involved in antagonist binding differences between V(2) R and V(1)R. These mutations did not alter the affinity for arginine vasopressin. However, the affinity for six nonpeptide receptor antagonists SR121463B [1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4[(2 morpholinoethoxy)cy-clohexane]indoline-2-one, phosphate monohydrate cis-isomer], SR49059 [(2S)1-[(2R3S)-(5-chloro-3-(2 chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide], SSR149415 [(2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2pyrrolidine carboxamide, isomer(-)], OPC21268 [1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone], OPC41061 [(+/-)-4'-[(7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-toluidide], and OPC31260, [(+/-)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-1,2, 3,4,5-tetrahydro-1H-benzazepine monohydrochloride], was altered to varying degrees, resulting in differences up to 6000-fold. Replacement of the small alanine for the bulky tryptophan in position 110 resulted in a reduced affinity for all six antagonists. In contrast, replacement of the large methionine for the smaller valine in position 120 caused a dramatic increase in affinity, up to a K(i) of 7 fM for OPC31260. Molecular modeling revealed that the binding sites for arginine vasopressin and the nonpeptide antagonists are partially overlapping. Whereas arginine vasopressin binds on the extracellular surface of V(2) R, the nonpeptide antagonists penetrate deeper into the transmembrane region of the receptor, in particular OPC21268. The mutagenesis data point to significant differences in the shape of the V(1)R and V(2)R antagonist binding pockets. The most important factor determining the specificity of nonpeptide antagonists seems to be the shape of the binding pocket on the receptor.
    No preview · Article · Mar 2006 · Journal of Pharmacology and Experimental Therapeutics
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    ABSTRACT: The V1 vascular vasopressin receptor (V1R) is a G-protein-coupled receptor (GPCR) involved in the regulation of body-fluid osmolality, blood volume and blood pressure. Signal transduction is mediated by the third intracellular loop of this seven-transmembrane protein as well as by the C-terminal cytoplasmic segment. A chimera of the maltose-binding protein (MBP) and the C-terminal segment of V1R has been cloned, expressed, purified and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 51.10, b = 66.56, c = 115.72 A, beta = 95.99 degrees. The 1.8 A crystal structure reveals the conformation of MBP and part of the linker region of this chimera, with the C-terminal segment being unstructured. This may reflect a conformational plasticity in the C-terminal segment that may be necessary for proper function of V1R.
    Full-text · Article · May 2005 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
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    ABSTRACT: Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone
    Full-text · Article · May 2002 · Biochimie
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    ABSTRACT: Colicins kill E. coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it. The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site. The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 Å long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms. Active site residues are D510, H513, E517, and R545. IP is buried between T and C. Rather than blocking the active site, IP prevents access of the active site to the ribosome.
    Full-text · Article · Nov 2001 · Molecular Cell
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    ABSTRACT: 2-Phenylglycerol has been crystallized, structurally characterized, and its related conformations considered by ab initio calculations at the HF/6-31G* level. C9H12O3. $$ \frac{1}{2} $$ H2O, P21,a = 5.8786(1), b = 28.6605(7), c = 10.5553(3) Å, ß = 90.187 (1)° at 100(1) K, Z = 8, R = 0.0424 for 5913 unique observed reflections. The asymmetric unit contains four molecules of the title compound and two molecules of water. The structures are characterized by extensive intermolecular hydrogen bonds. The packing is dominated by a hydrophilic hydrogen bonded layer with the hydrophobic phenyl rings forming a herringbone packed intermediate layer. Computational studies suggest that an increase in energy of about 13 kcal/mol can provide for significant conformational flexibility, including the traversal of an orientation that is appropriately aligned with the reaction trajectory needed for closure to the oxetane. Progression along this reaction coordinate, however, will probably require at least another 15 kcal/mol in the gas phase in order to bring the two SN2 species into a proximity which approaches that of the oxetane's C-O bond distance.
    No preview · Article · Feb 2000 · Journal of Chemical Crystallography

Publication Stats

246 Citations
43.08 Total Impact Points


  • 2002-2007
    • Case Western Reserve University
      • • Department of Biochemistry
      • • School of Medicine
      Cleveland, OH, United States
  • 2000-2006
    • University of Toledo
      • Department of Chemistry
      Toledo, Ohio, United States
  • 2001
    • University of British Columbia - Vancouver
      Vancouver, British Columbia, Canada