Crystal Structure of β-Arrestin at 1.9 Å Possible Mechanism of Receptor Binding and Membrane Translocation

AbstractBackground: Arrestins are responsible for the desensitization of many sequence-divergent G protein-coupled receptors. They compete with G proteins for binding to activated phosphorylated receptors, initiate receptor internalization, and activate additional signaling pathways.Results: In order to understand the structural basis for receptor binding and arrestin's function as an adaptor molecule, we determined the X-ray crystal structure of two truncated forms of bovine β-arrestin in its cytosolic inactive state to 1.9 Å. Mutational analysis and chimera studies identify the regions in β-arrestin responsible for receptor binding specificity. β-arrestin demonstrates high structural homology with the previously solved visual arrestin. All key structural elements responsible for arrestin's mechanism of activation are conserved.Conclusions: Based on structural analysis and mutagenesis data, we propose a previously unappreciated part in β-arrestin's mode of action by which a cationic amphipathic helix may function as a reversible membrane anchor. This novel activation mechanism would facilitate the formation of a high-affinity complex between β-arrestin and an activated receptor regardless of its specific subtype. Like the interaction between β-arrestin's polar core and the phosphorylated receptor, such a general activation mechanism would contribute to β-arrestin's versatility as a regulator of many receptors

CpA containing oligoribonucleotides specifically inhibit protein synthesis in rabbit reticulocytes

AbstractThe diribonucleoside monophosphate CpA (and no others) inhibits polypeptide chain elongation in rabbit reticulocyte lysates at 10–50 μM. Furthermore, all the trinucleotides containing CpA, i.e., XpCpA and CpApX (X = U, C, A or G) block polypeptide chain elongation as well. At 10 μM the inhibition by XpCpA and not CpApX is transient because a 3'-exonucleolytic activity destroys the critical CpA moiety. The inhibitors do not appear to interfere with the aminoacylation of tRNAs or disrupt the interaction of aminoacyl-tRNAs with the protein synthetic machinery. High levels (200 μM) of CpA or the trinucleotides containing CpA have no effect on translation in a wheat germ cell-free system

Crystal Structure at 2.4 Å Resolution of the Complex of Transducin βγ and Its Regulator, Phosducin

AbstractThe crystal structure of transducin's βγ subunits complexed with phosducin, which regulates Gtβγ activity, has been solved to 2.4 Å resolution. Phosducin has two domains that wrap around Gtβγ to form an extensive interface. The N-terminal domain binds loops on the “top” Gtβ surface, overlapping the Gtα binding surface, explaining how phosducin blocks Gtβγ's interaction with Gtα. The C-terminal domain shows structural homology to thioredoxin and binds the outer strands of Gtβ's seventh and first blades in a manner likely to disrupt Gtβγ's normal orientation relative to the membrane and receptor. Phosducin's Ser-73, which when phosphorylated inhibits phosducin's function, points away from Gtβγ, toward a large flexible loop. Thus phosphorylation is not likely to affect the interface directly, but rather indirectly through an induced conformational change

Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains

The 2.8-Å crystal structure of the complex formed by estradiol and the human estrogen receptor-α ligand binding domain (hERαLBD) is described and compared with the recently reported structure of the progesterone complex of the human progesterone receptor ligand binding domain, as well as with similar structures of steroid/nuclear receptor LBDs solved elsewhere. The hormone-bound hERαLBD forms a distinctly different and probably more physiologically important dimer interface than its progesterone counterpart. A comparison of the specificity determinants of hormone binding reveals a common structural theme of mutually supported van der Waals and hydrogen-bonded interactions involving highly conserved residues. The previously suggested mechanism by which the estrogen receptor distinguishes estradiol’s unique 3-hydroxy group from the 3-keto function of most other steroids is now described in atomic detail. Mapping of mutagenesis results points to a coactivator-binding surface that includes the region around the “signature sequence” as well as helix 12, where the ligand-dependent conformation of the activation function 2 core is similar in all previously solved steroid/nuclear receptor LBDs. A peculiar crystal packing event displaces helix 12 in the hERαLBD reported here, suggesting a higher degree of dynamic variability than expected for this critical substructure

Structure of β2-bungarotoxin: potassium channel binding by Kunitz modules and targeted phospholipase action

AbstractBackground: β-bungarotoxin is a heterodimeric neurotoxin consisting of a phospholipase subunit linked by a disulfide bond to a K+ channel binding subunit which is a member of the Kunitz protease inhibitor superfamily. Toxicity, characterized by blockage of neural transmission, is achieved by the lipolytic action of the phospholipase targeted to the presynaptic membrane by the Kunitz module.Results The crystal structure at 2.45 å resolution suggests that the ion channel binding region of the Kunitz subunit is at the opposite end of the module from the loop typically involved in protease binding. Analysis of the phospholipase subunit reveals a partially occluded substrate-binding surface and reduced hydrophobicity.Conclusion Molecular recognition by this Kunitz module appears to diverge considerably from more conventional superfamily members. The ion channel binding region identified here may mimic the regulatory interaction of endogenous neuropeptides. Adaptations of the phospholipase subunit make it uniquely suited to targeting and explain the remarkable ability of the toxin to avoid binding to non-target membranes. Insight into the mechanism of β-bungarotoxin gained here may lead to the development of therapeutic strategies against not only pathological cells, but also enveloped viruses