Elucidation of Inositol Hexaphosphate and Heparin Interaction Sites and Conformational Changes in Arrestin-1 by Solution Nuclear Magnetic Resonance

Vanderbilt University, Нашвилл, Michigan, United States
Biochemistry (Impact Factor: 3.02). 11/2010; 49(49):10473-85. DOI: 10.1021/bi101596g
Source: PubMed


Arrestins specifically bind activated and phosphorylated G protein-coupled receptors and orchestrate both receptor trafficking and channel signaling through G protein-independent pathways via direct interactions with numerous nonreceptor partners. Here we report the first successful use of solution NMR in mapping the binding sites in arrestin-1 (visual arrestin) for two polyanionic compounds that mimic phosphorylated light-activated rhodopsin: inositol hexaphosphate (IP6) and heparin. This yielded an identification of residues involved in the binding with these ligands that was more complete than what has previously been feasible. IP6 and heparin appear to bind to the same site on arrestin-1, centered on a positively charged region in the N-domain. We present the first direct evidence that both IP6 and heparin induced a complete release of the arrestin C-tail. These observations provide novel insight into the nature of the transition of arrestin from the basal to active state and demonstrate the potential of NMR-based methods in the study of protein-protein interactions involving members of the arrestin family.

Download full-text


Available from: Tiandi Zhuang,
  • Source
    • "IP6 has been implicated as an important component in the regulation of different proteins. For instance, IP6 induces a conformational change of the normally disordered RNA editing enzyme hADAR2 (Macbeth et al., 2005), a vesicle membrane protein, synaptotagmin I (Joung et al., 2012), and a sensory perception-related protein, arrestin (Zhuang et al., 2010). IP6 is also known to activate bacterial toxins, including repeats in toxin (RTX) produced by Vibrio cholera (Lupardus et al., 2008) and toxin A produced by Clostridium difficile (Pruitt et al., 2009) after they enter animal cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Gram-negative bacteria inject type III secreted effectors (T3SEs) into host cells to manipulate the immune response. The YopJ family effector HopZ1a produced by the plant pathogen Pseudomonas syringae possesses acetyltransferase activity and acetylates plant proteins to facilitate infection. Using mass spectrometry, we identified a threonine residue, T346, as the main autoacetylation site of HopZ1a. Two neighboring serine residues, S349 and S351, are required for the acetyltransferase activity of HopZ1a in vitro and are indispensable for the virulence function of HopZ1a in Arabidopsis thaliana. Using proton nuclear magnetic resonance (NMR), we observed a conformational change of HopZ1a in the presence of inositol hexakisphosphate (IP6), which acts as a eukaryotic co-factor and significantly enhances the acetyltransferase activity of several YopJ family effectors. S349 and S351 are required for IP6-binding-mediated conformational change of HopZ1a. S349 and S351 are located in a conserved region in the C-terminal domain of YopJ family effectors. Mutations of the corresponding serine(s) in two other effectors, HopZ3 of P. syringae and PopP2 of Ralstonia solanacerum, also abolished their acetyltransferase activity. These results suggest that, in addition to the highly conserved catalytic residues, YopJ family effectors also require conserved serine(s) in the C-terminal domain for their enzymatic activity. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.
    New Phytologist 06/2015; DOI:10.1111/nph.13528 · 7.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.
    Journal of Biological Chemistry 04/2011; 286(27):24288-99. DOI:10.1074/jbc.M110.213835 · 4.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Visual arrestin-1 plays a key role in the rapid and reproducible shutoff of rhodopsin signaling. Its highly selective binding to light-activated phosphorylated rhodopsin is an integral part of the functional perfection of rod photoreceptors. Structure-function studies revealed key elements of the sophisticated molecular mechanism ensuring arrestin-1 selectivity and paved the way to the targeted manipulation of the arrestin-1 molecule to design mutants that can compensate for congenital defects in rhodopsin phosphorylation. Arrestin-1 self-association and light-dependent translocation in photoreceptor cells work together to keep a constant supply of active rhodopsin-binding arrestin-1 monomer in the outer segment. Recent discoveries of arrestin-1 interaction with other signaling proteins suggest that it is a much more versatile signaling regulator than previously thought, affecting the function of the synaptic terminals and rod survival. Elucidation of the fine molecular mechanisms of arrestin-1 interactions with rhodopsin and other binding partners is necessary for the comprehensive understanding of rod function and for devising novel molecular tools and therapeutic approaches to the treatment of visual disorders.
    Progress in Retinal and Eye Research 07/2011; 30(6):405-30. DOI:10.1016/j.preteyeres.2011.07.002 · 8.73 Impact Factor
Show more