RESUMEN
Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway.
Asunto(s)
NADH NADPH Oxidorreductasas/química , Proteína Transportadora de Acilo/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Acido Graso Sintasa Tipo I/química , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Humanos , Cinética , Mitocondrias/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold. Small-angle X-ray scattering experiments performed on filamin C tandem Ig-like domains 23 and 24 reveal a dimer that is formed by domain 24 and that domain 23 has little interactions with itself or with domain 24, while the analytical ultracentrifugation experiments showed that the filamin C domains 19-21 form elongated monomers in diluted solutions.
Asunto(s)
Proteínas Contráctiles/química , Proteínas de Microfilamentos/química , Modelos Moleculares , Pliegue de Proteína , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Filaminas , Humanos , Níquel/química , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , UltracentrifugaciónRESUMEN
Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.
Asunto(s)
Proteínas Contráctiles/química , Proteínas de Microfilamentos/química , Complejos Multiproteicos/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Proteínas Contráctiles/ultraestructura , Filaminas , Humanos , Cadenas beta de Integrinas/química , Cadenas beta de Integrinas/ultraestructura , Proteínas de Microfilamentos/ultraestructura , Complejos Multiproteicos/ultraestructura , Complejo GPIb-IX de Glicoproteína Plaquetaria/ultraestructura , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Filamins are essential in cell motility and many developmental processes. They are large actin cross linking proteins that contain actin binding domains in their N termini and a long rod region constructed from 24 tandem Ig domains. Dimerization is crucial for the actin crosslinking function of filamins and requires the most C-terminal Ig domain. We describe here the crystal structure of this 24th Ig domain (Ig24) of human filamin C and show how it mediates dimerization. The dimer interface is novel and quite different to that seen in the Dictyostelium discoideum filamin analog. The sequence signature of the dimerization interface suggests that the C-terminal domains of all vertebrate filamins share the same dimerization mechanism. Furthermore, we show that point mutations in the dimerization interface disrupt the dimer and that the dissociation constant for recombinant Ig24 is in the micromolar range.