RESUMEN
Secretin is a gastrointestinal hormone that exerts multiple physiological functions via activation of the secretin receptor (SECR). SECR belongs to the class B G-protein-coupled receptors and is involved in various processes, such as regulation of the pH of the duodenal content, food intake, and water homeostasis. Here, we report a cryo-electron microscopy structure of human SECR bound to secretin and an engineered Gs heterotrimer. The structure revealed the basic architecture of SECR and the secretin binding mode. A structural comparison of the SECR and PAC1R transmembrane domains revealed that transmembrane helices 1 and 2 play a prominent role in secretin recognition. Moreover, the extracellular domain of SECR is perpendicular to the TMD, unlike that of PAC1R. This comparison revealed the diverged peptide recognition mechanisms of these receptors, which belong to the same subgroup. Our structural information will facilitate drug discovery research for clinical applications.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/química , Receptores Acoplados a Proteínas G/química , Receptores de la Hormona Gastrointestinal/química , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Ingeniería de Proteínas , Secretina/químicaRESUMEN
PIWI-interacting RNAs (piRNAs) silence transposons to maintain genome integrity in animal germ lines. piRNAs are classified as primary and secondary piRNAs, depending on their biogenesis machinery. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters in the genome through the primary processing pathway. Although the existence of a ribonuclease participating in this pathway has been predicted, its molecular identity remained unknown. Here we show that Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily member, is an endoribonuclease essential for primary piRNA biogenesis. We solved the crystal structure of Drosophila melanogaster Zuc (DmZuc) at 1.75 Å resolution. The structure revealed that DmZuc has a positively charged, narrow catalytic groove at the dimer interface, which could accommodate a single-stranded, but not a double-stranded, RNA. DmZuc and the mouse homologue MmZuc (also known as Pld6 and MitoPLD) showed endoribonuclease activity for single-stranded RNAs in vitro. The RNA cleavage products bear a 5'-monophosphate group, a hallmark of mature piRNAs. Mutational analyses revealed that the conserved active-site residues of DmZuc are critical for the ribonuclease activity in vitro, and for piRNA maturation and transposon silencing in vivo. We propose a model for piRNA biogenesis in animal germ lines, in which the Zuc endoribonuclease has a key role in primary piRNA maturation.
Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Endorribonucleasas/química , Endorribonucleasas/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Secuencia de Bases , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Silenciador del Gen , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Relación Estructura-ActividadRESUMEN
PIWI-interacting RNAs (piRNAs) bind PIWI proteins and silence transposons to maintain the genomic integrity of germ cells. Zucchini (Zuc), a phospholipase D superfamily member, is conserved among animals and is implicated in piRNA biogenesis. However, the underlying mechanism by which Zuc participates in piRNA biogenesis remains elusive. Drosophila melanogaster Zuc (DmZuc) was expressed in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 1.75â Å resolution. The crystal belonged to space group P2(1), with unit-cell parameters a=55.0, b=71.2, c=56.3â Å, ß=107.9°.
Asunto(s)
Proteínas de Drosophila/química , Drosophila melanogaster/enzimología , Endorribonucleasas/química , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/aislamiento & purificación , Endorribonucleasas/biosíntesis , Endorribonucleasas/aislamiento & purificación , Escherichia coli , Datos de Secuencia Molecular , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
The type VI secretion system (T6SS) comprises needle-shaped multisubunit complexes that play a role in the microbial defense systems of Gram-negative bacteria. Some Gram-negative bacteria harboring a T6SS deliver toxic effector proteins into the cytoplasm or periplasm of competing bacteria in order to lyse and kill them. To avoid self-cell disruption, these bacteria have cognate immunity proteins that inhibit their toxic effector proteins. T6SS amidase effector protein 4 (Tae4) and T6SS amidase immunity protein 4 (Tai4) are a representative of the toxic effector-immunity pairs of the T6SS. Here, the three-dimensional structures of Tai4 and the Tae4-Tai4 complex from Agrobacterium tumefaciens are reported at 1.55 and 1.9â Å resolution, respectively. A structural comparison with other Tae4-Tai4 homologs revealed similarities and differences in the catalytic and inhibitory mechanisms among the Tae4 and Tai4 family proteins.