RESUMEN
The CED-4 homo-oligomer or apoptosome is required for initiation of programmed cell death in Caenorhabditis elegans by facilitating autocatalytic activation of the CED-3 caspase zymogen. How the CED-4 apoptosome assembles and activates CED-3 remains enigmatic. Here we report the crystal structure of the complete CED-4 apoptosome and show that it consists of eight CED-4 molecules, organized as a tetramer of an asymmetric dimer via a previously unreported interface among AAA(+) ATPases. These eight CED-4 molecules form a funnel-shaped structure. The mature CED-3 protease is monomeric in solution and forms an active holoenzyme with the CED-4 apoptosome, within which the protease activity of CED-3 is markedly stimulated. Unexpectedly, the octameric CED-4 apoptosome appears to bind only two, not eight, molecules of mature CED-3. The structure of the CED-4 apoptosome reveals shared principles for the NB-ARC family of AAA(+) ATPases and suggests a mechanism for the activation of CED-3.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio/química , Secuencia de Aminoácidos , Animales , Apoptosomas/metabolismo , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Caenorhabditis elegans/química , Caspasas/química , Cristalografía por Rayos X , Modelos Moleculares , Alineación de Secuencia , Difracción de Rayos XRESUMEN
NleC is one of the virulence factors that is injected into infected host cells by enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) via a needle-like protein complex called the type III secretion system (T3SS). The cytosolic NleC specifically cleaves the p65 subunit of NF-κB in the p65-p50 heterodimeric complex just after the Cys38 site in its N-terminal domain. The degradation of the remainder of the p65 C-terminal domain by the proteasome disrupts the NF-κB signalling pathway, thus dampening the host inflammatory response. Here, the crystal structure of NleC is reported at 1.55â Å resolution. In conjunction with biochemical analyses, the structure reveals that NleC is a member of the zincin zinc protease family and that the configuration of the NleC active site resembles that of the metzincin clan of metallopeptidases but without the canonical Met turn of astacin. The extended zinc-binding motif of NleC (HEXXHXXTXXXD) includes three metal ligands. The fifth zinc ligand, a conserved tyrosine (a bound water molecule is the fourth ligand), lies 45 residues downstream of the zincin motif. Furthermore, the electrostatic potential complementarity between NleC and p65 also contributes to the cleavage activity of the protease. These results not only provide important insights into the mechanism of how NleC recognizes its substrates, but also shed light on the design of new antibiotics for the food-borne diseases arising from EPEC and EHEC.
Asunto(s)
Escherichia coli Enteropatógena/química , Proteínas de Escherichia coli/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli Enteropatógena/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformación Proteica , Zinc/metabolismoRESUMEN
As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Binding to their target single-stranded RNAs (ssRNAs) in a modular and base-specific fashion, PPR proteins can serve as designable modules for gene manipulation. However, the structural basis for nucleotide-specific recognition by designer PPR (dPPR) proteins remains to be elucidated. Here, we report four crystal structures of dPPR proteins in complex with their respective ssRNA targets. The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA. Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G. These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.
Asunto(s)
Secuencias de Aminoácidos , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Sitios de Unión/genética , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN/química , ARN/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Homología de Secuencia de AminoácidoRESUMEN
Necroptosis is a cellular mechanism that mediates necrotic cell death. The receptor-interacting serine/threonine protein kinase 1 (RIP1) is an essential upstream signaling molecule in tumor-necrosis-factor-α-induced necroptosis. Necrostatins, a series of small-molecule inhibitors, suppress necroptosis by specifically inhibiting RIP1 kinase activity. Both RIP1 structure and the mechanisms by which necrostatins inhibit RIP1 remain unknown. Here, we report the crystal structures of the RIP1 kinase domain individually bound to necrostatin-1 analog, necrostatin-3 analog, and necrostatin-4. Necrostatin, caged in a hydrophobic pocket between the N- and C-lobes of the kinase domain, stabilizes RIP1 in an inactive conformation through interactions with highly conserved amino acids in the activation loop and the surrounding structural elements. Structural comparison of RIP1 with the inhibitor-bound oncogenic kinase B-RAF reveals partially overlapping binding sites for necrostatin and for the anticancer compound PLX4032. Our study provides a structural basis for RIP1 inhibition by necrostatins and offers insights into potential structure-based drug design.