RESUMEN
The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport.
Asunto(s)
Actinomycetales/química , Proteínas Bacterianas/química , Proteínas de Transporte de Nucleobases/química , Simportadores/química , Actinomycetales/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cationes/química , Cationes/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Cristalografía por Rayos X , Hidantoínas/química , Hidantoínas/metabolismo , Transporte Iónico , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Transporte de Nucleobases/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Sodio/metabolismo , Simportadores/metabolismoRESUMEN
A genomic strategy for the overexpression of bacterial multidrug and antibiotic resistance membrane efflux proteins in Escherichia coli is described. Expression is amplified so that the encoded proteins from a range of Gram-positive and Gram-negative bacteria comprise 5% to 35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGS(His)(6)-tag or a Strep-tag at the C terminus. These tags facilitate the purification of the overexpressed proteins in milligram quantities for structural studies. The strategy is illustrated for the bicyclomycin resistance efflux protein, Bcr, of E. coli.
