RESUMEN
The tetracycline resistance proteins (TetA) of gram-negative bacteria are secondary active transport proteins that contain buried charged amino acids that are important for tetracycline transport. Earlier studies have shown that insertion of TetA proteins into the cytoplasmic membrane is mediated by helical hairpin pairs of transmembrane (TM) segments. However, whether helical hairpins direct spontaneous insertion of TetA or are required instead for its interaction with the cellular secretion (Sec) machinery is unknown. To gain insight into how TetA proteins are inserted into the membrane, we have investigated how tolerant the class C TetA protein encoded by plasmid pBR322 is to placement of charged residues in TM segments. The results show that the great majority of charge substitutions do not interfere with insertion even when placed at locations that cannot be shielded internally within helical hairpins. The only mutations that frequently block insertion are proline substitutions, which may interfere with helical hairpin folding. The ability of TetA to broadly tolerate charge substitutions indicates that the Sec machinery assists in its insertion into the membrane. The results also demonstrate that it is feasible to engineer charged residues into the interior of TetA proteins for the purpose of structure-function analysis.
Asunto(s)
Antiportadores/genética , Antiportadores/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Resistencia a la Tetraciclina/genética , Secuencia de Aminoácidos , Antiportadores/química , Proteínas Bacterianas/química , Membrana Celular/química , Cisteína/química , Cisteína/genética , Análisis Mutacional de ADN , Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis , Plásmidos/genética , Estructura Secundaria de Proteína/fisiología , Relación Estructura-Actividad , Reactivos de Sulfhidrilo/químicaRESUMEN
The packing orientations of the 8 transmembrane (TM) segments that line the central, aqueous transport channel within tetracycline resistance proteins (TetA) have been established. However, the orientations of the remaining 4 segments, TMs 3, 6, 9, and 12, located at the periphery, and away from the transport channel, have not yet been determined. In this study, the packing orientation of TM6 within the class C TetA protein encoded by plasmid pBR322 was evaluated by substitution mutagenesis and analysis of sequence conservation and amphipathicity. The combined data support a model in which the conserved and polar face of the TM6 alpha-helix containing Asn170 and Asn173 orients towards channel-lining TM segments, and the relatively non-conserved and hydrophobic face of TM6 points towards membrane lipids.