Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state

Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI

Factor XI FXI , the zymogen of activated FXI FXIa , is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa BAY 1213790 complex demonstrated that the tyrosine rich complementarity determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain 2.9 . The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration dependent manne

Seroprevalencia de la Leishmaniasis Visceral Canina Mediante Elisa con rK39 en Focos Endémicos de Venezuela

En el presente trabajo se presenta la seroprevalencia de la leishmaniasis visceral canina (LVC), zoonosis causada por Leishmania infantum/chagasi. Se realizaron pruebas serológicasy examen clínico a 15.822 perros de 13 entidades federales endémicas para la leishmaniasis visceral en Venezuela, en el periodo 2004-2012. Los sueros fueron analizados mediante ELISA con el antígeno recombinante rK39. Los resultados muestran que 14,8% de la población de caninos son positivos para LV. Los estados Lara (19%) y Guárico (18%) mostraron una mayor prevalencia de la enfermedad. Sin embargo, para los años 2010-2012, la prevalencia de la LVC para las entidades federales como Anzoátegui, Aragua, Carabobo, Cojedes, Nueva Esparta y Sucre se mantuvieron entre un 3% y un 31%. Los caninos seropositivos (67,1% machos y 32,9% hembras) tenían edades promedio de 4,8±2,9 años. El 81% de los caninos seropositivos encontrados en estas zonas, no presentó signos clínicos característicos de LVC, mientras que la clínica presentada por el resto fueron ulceraciones cutáneas (8,5%), alopecia (9,4%) y onicogrifosis (19,2%). Este reporte muestra la distribución geográfica (tanto en zonas rurales como urbanas) y características clínicas más resaltantes de perros parasitados en las diferentes regiones endémicas del país, con el fin de tomar medidas estratégicas que fortalezcan los programas de control y prevención de esta zoonosis problema de salud pública.AbstractThis study discloses the seroprevalence of canine visceral leishmaniasis (CVL), a zoonotic disease caused by Leishmania infantum/chagasi. In this study, serological tests and clinical examinations were performed in 15,822 dogs from 13 federal entities endemic for visceral leishmaniasis in Venezuela, during the period 2004-2012. Serum samples were analysed by ELISA against the recombinant antigen rK39. Results demonstrateda prevalence of 14.8% of positive dogs for VL. Lara (19%) and Guárico (18%) states showed the highest seroprevalence of the disease. However, for the years 2010-2012, the prevalence of CVL for federal entities as Anzoátegui, Aragua, Carabobo, Cojedes, Nueva Esparta, and Sucre remained between 3% and 31%. The seropositive canines (67.1% males and 32.9% females) average 4.8±2.9 years of age and 81% of the dogs found in these endemic areas did not show clinical signs characteristic of LVC, while clinical symptoms presented by the rest were skin ulceration (8.5%), alopecia (9.4%) and onychogryphosis (19.2%). This report demonstrates the geographical distribution (both rural and urban) and most striking clinical features of parasitized dogs in different endemic regions of the country, in order to take strategic actions to strengthen the control and prevention programs of this public health problem

Combinatorial recognition of clustered RNA elements by the multidomain RNA-binding protein IMP3.

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation

Crystal structure of the ectodomain of Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-Å resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily β-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features