12 research outputs found
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SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped β-Sandwich Fold Important for SOCE Modulation.
Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca2+ stores and generate intracellular Ca2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X-ray crystal structure of the SARAF luminal domain, SARAFL. This domain forms a novel 10-stranded β-sandwich fold that includes a set of three conserved disulfide bonds, denoted the SARAF-fold. The structure reveals a domain-swapped dimer in which the last two β-strands (β9 and β10) are exchanged forming a region denoted the SARAF luminal switch that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation
Recommended from our members
SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped β-Sandwich Fold Important for SOCE Modulation.
Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca2+ stores and generate intracellular Ca2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X-ray crystal structure of the SARAF luminal domain, SARAFL. This domain forms a novel 10-stranded β-sandwich fold that includes a set of three conserved disulfide bonds, denoted the "SARAF-fold." The structure reveals a domain-swapped dimer in which the last two β-strands (β9 and β10) are exchanged forming a region denoted the "SARAF luminal switch" that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation
Miniaturization of Scorpion β-Toxins Uncovers a Putative Ancestral Surface of Interaction with Voltage-gated Sodium Channels*♦
The bioactive surface of scorpion β-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved βαββ core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian β-toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated β-toxins (ΔΔBj-xtrIT and ΔΔCss4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, ΔΔBj-xtrIT and ΔΔCss4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion α-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion α-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of LqhαIT at insect sodium channels. Co-application of ΔΔBj-xtrIT or ΔΔCss4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at ΔΔBj-xtrIT and ΔΔCss4 capable of interaction with sodium channels at a site other than sites 3, 4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both β-toxins may be viewed as an exercise in “reverse evolution,” providing a clue as to their evolution from a smaller ancestor of similar scaffold
Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels
Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds