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In a calcium-dependent interaction critical for blood coagulation, vitamin K-dependent blood coagulation proteins bind cell membranes containing phosphatidylserine via ?-carboxyglutamic acid-rich (Gla) domains. Gla domain-mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologic time scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovine prothrombin Gla domain. The serine head group binds Gla domain-bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gla domains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are being identified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in other physiologic processes. [ABSTRACT FROM AUTHOR]

A procedure for the preparation of specific molecular species of d- erythro-lactosylceramide involving deacylation and reacylation of lactosylceramide prepared from bovine brain gangliosides is described. Lactosylceramide was N-deacylated by alkaline hydrolysis and the resulting four lysolactosylceramides, which contained d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases, were simultaneously re- N-acylated with the N-succinimidyl ester of either 16∶0, 18∶0, 20∶0, 24∶0, 20∶1, 22∶1 or 24∶1 fatty acid. The resulting lactosylceramide contained four molecular species of lactosylceramides, i.e., d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases coupled with the fatty acid that was introduced. Lactosylceramides prepared in this manner were separated into four individual molecular species by high-performance liquid chromatography (HPLC). Each of the purified molecular species of lactosylceramide was quantitated by HPLC after derivatization with benzoylchloride and was characterized by mass spectrometry. The yields of reacylated lactosylceramide were 38-58% relative to the starting lactosylceramide; the purity of each of the molecular species of lactosylceramide was greater than 95%. [ABSTRACT FROM AUTHOR]

Abstract: Compared to other M1 muscarinic acetylcholine receptor (M1 mAChR) agonists, xanomeline demonstrates both reversible and persistent modes of binding to the receptor. In our study, we investigated the long-term consequences of brief incubation of Chinese hamster ovary cells expressing M1 mAChR (M1-CHO) with low concentrations of xanomeline followed by washing off the free drug. Thus, M1-CHO cells were exposed to 100nM xanomeline for 1h then washed extensively. Washed cells were either used immediately for binding assays or incubated for 23h in the absence of free xanomeline. Only the latter treatment conditions resulted in marked attenuation of binding of the muscarinic radioligand [3H]N-methylscopolamine ([3H]NMS) to intact cells. Shortening the xanomeline pretreatment period to 1min had the same trends as the 1h pretreatment, implying that xanomeline binds instantly to the receptor to elicit long-term wash-resistant effects. Presence of atropine during the brief period of xanomeline pretreatment did not markedly modulate xanomeline''s long-term effects, which suggests that persistent anchoring of the xanomeline molecule to the M1 receptor takes place at a site distinct from the orthosteric binding domain. Our findings suggest the possibility of a time-dependent transition of the conformation of the muscarinic M1 receptor-xanomeline complex between states that vary in their ability to bind [3H]NMS. However, possible involvement of other mechanisms of long-term receptor regulation cannot be discounted. [Copyright &y& Elsevier]

The Ets1 transcription factor is a member of the Ets protein family, a group of evolutionarily related DNA-binding transcriptional factors. Ets proteins activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. FOXO1 is a member of the forkhead-box proteins (FOX proteins), which comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation and differentiation. The FOXO subgroup of FOX proteins regulates the transcription of genes that control metabolism, cell survival, cellular proliferation, DNA damage responses, stress resistance and longevity. The DNA-binding domains (DBDs) of Ets1 and FOXO1 were crystallized in complex with DNA containing a composite sequence for a noncanonical forkhead binding site (AATAACA) and an ETS site (GGAA), FOX:ETS, by the sitting-drop vapor-diffusion method. The FOX:ETS motif has been shown to be a conserved cis-acting element in several endothelial cell-specific genes, including Vegfr2, Tie2, Mef2c and ve-cadherin. Crystals were grown at 291 K using 30% polyethylene glycol 400, 50 mM Tris pH 8.5, 100 mM KCl, 10 mM MgCl2 as the reservoir solution. The crystals belonged to space group C222(1), with unit-cell parameters a = 68.7, b = 104.9, c = 136.3 Å. Diffraction data were collected to a resolution of 2.2 Å.

Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human alpha3(IV)NC1 domains bind autoantibodies. The alpha3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio alpha3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio alpha3(IV)NC1 domain. In contrast to recombinant human alpha3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio alpha3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human alpha3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years.

Neurotoxic assemblies of the amyloid beta-protein (Abeta) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Abeta assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Abeta(1-40) and Abeta(1-42). The results revealed a 10-residue, protease-resistant segment, Ala21-Ala30, in both peptides. Remarkably, the homologous decapeptide, Abeta(21-30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24-Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a beta-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24-Lys28 region of Abeta nucleates the intramolecular folding of Abeta monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Abeta(1-40) and Abeta(1-42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Abeta(1-42) production. Our results suggest that therapeutic approaches targeting the Val24-Lys28 turn or the Abeta(1-42)-specific C-terminal fold may hold promise.

Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca(2+) channels expressed in mouse pancreatic beta-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453-32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a D-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two gamma-carboxyglutamic acid (Gla) residues, formed by post-translational modification of glutamic acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional (1)H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys(11)-Cys(5)-Pro(6) region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla(2) and Ser(3), moves away from the His(8) and Trp(10) aromatic rings and the alignment of the D-Trp(7) and His(8) aromatic rings with respect to the Trp(10) rings is altered. The blockage of L-type voltage-gated Ca(2+) channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone C alpha conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 A, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation.