RESUMEN
A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotiana tabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward-facing forms. The overall structure has a bilobate V-shape with pseudo-symmetrical assembly of the N- and C-lobes. In one crystal structure, the C-lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2-specific conformational transitions imply that an unprecedented movement of the transmembrane α-helix 7 is related to the release of the substrate into the vacuolar lumen.
Asunto(s)
Nicotiana/metabolismo , Nicotina/química , Proteínas de Transporte de Catión Orgánico/química , Proteínas de Plantas/química , Vacuolas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Modelos Moleculares , Nicotina/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Células Vegetales/química , Células Vegetales/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Nicotiana/genética , Vacuolas/químicaRESUMEN
The ß-barrel assembly machinery (BAM) complex mediates the assembly of ß-barrel membrane proteins in the outer membrane. BepA, formerly known as YfgC, interacts with the BAM complex and functions as a protease/chaperone for the enhancement of the assembly and/or degradation of ß-barrel membrane proteins. To elucidate the molecular mechanism underlying the dual functions of BepA, its full-length three-dimensional structure is needed. Here, we report the crystal structure of full-length BepA at 2.6-Å resolution. BepA possesses an N-terminal protease domain and a C-terminal tetratricopeptide repeat domain, which interact with each other. Domain cross-linking by structure-guided introduction of disulfide bonds did not affect the activities of BepA in vivo, suggesting that the function of this protein does not involve domain rearrangement. The full-length BepA structure is compatible with the previously proposed docking model of BAM complex and tetratricopeptide repeat domain of BepA.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Metaloproteasas/química , Cristalografía por Rayos X/métodos , Escherichia coli/química , Modelos Moleculares , Dominios Proteicos , Pliegue de ProteínaRESUMEN
The multidrug and toxic compound extrusion (MATE) family of proteins consists of transporters responsible for multidrug resistance in prokaryotes. In plants, a number of MATE proteins were identified by recent genomic and functional studies, which imply that the proteins have substrate-specific transport functions instead of multidrug extrusion. The three-dimensional structure of eukaryotic MATE proteins, including those of plants, has not been reported, preventing a better understanding of the molecular mechanism of these proteins. Here, we describe the crystal structure of a MATE protein from the plant Camelina sativa at 2.9 Å resolution. Two sets of six transmembrane α helices, assembled pseudo-symmetrically, possess a negatively charged internal pocket with an outward-facing shape. The crystal structure provides insight into the diversity of plant MATE proteins and their substrate recognition and transport through the membrane.
Asunto(s)
Brassicaceae/metabolismo , Proteínas de Plantas/química , Sitios de Unión , Brassicaceae/química , Cristalografía por Rayos X , Resistencia a Múltiples Medicamentos , Evolución Molecular , Modelos Moleculares , Filogenia , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Protein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF.
