Functional and structural study of the dimeric inner membrane protein SbmA

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17, J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino and peptide nucleic acids into the Escherichia coli cytoplasm. sbmA homologue was found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G and R385G showed indistinguishable transport capacities compared to strains harbouring a wild type SbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a negative-dominant phenotype. Electron microscopy data and in silico 3D homology modelling support SbmA to form a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport abilitity.