title: Structural and functional analysis of the β-barrel domain of BamA from Escherichia coli.
abstract: In gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved component. To elucidate the mechanism of BamA-mediated OMP biogenesis, we determined the crystal structure of the C-terminal transmembrane domain of BamA from Escherichia coli (EcBamA) at 2.6 Å resolution. The structure reveals 2 distinct features. First, a portion of the extracellular side of the β barrel is composed of 5 markedly short β strands, and the loops stemming from these β strands form a potential surface cavity, filled by a portion of the L6 loop that includes the conserved VRGF/Y motif found in the Omp85 family. Second, the 4 extracellular loops L3, L4, L6, and L7 of EcBamA form a dome over the barrel, stabilized by a salt-bridge interaction network. Functional data show that hydrophilic-to-hydrophobic mutations of the potential hydrophilic surface cavity and a single Arg547Ala point mutation that may destabilize the dome severely affect the function of EcBamA. Our structure of the EcBamA β barrel and structure-based mutagenesis studies suggest that the transmembrane β strands of OMP substrates may integrate into the outer membrane at the interface of the first and last β strands of the EcBamA barrel, whereas the soluble loops or domains may be transported out of the cell via the hydrophilic surface cavity on dislocation of the VRGF/Y motif of L6. In addition, the dome over the barrel may play an important role in maintaining the efficiency of OMP biogenesis.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; School of Public Health, Tianjin Medical University, Tianjin, China;
authors.forename: Dongchun
authors.initials: D
authors.lastname: Ni
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China; and.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China;
authors.forename: Xu
authors.initials: X
authors.lastname: Yang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China;
authors.forename: Haizhen
authors.initials: H
authors.lastname: Zhou
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China;
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Hou
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China;
authors.forename: Baohua
authors.initials: B
authors.lastname: Cao
authors.affiliation: Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species and Department of Chemical Biology, College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center, Peking University, Beijing, China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species and Department of Chemical Biology, College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center, Peking University, Beijing, China.
authors.forename: Xinsheng
authors.initials: X
authors.lastname: Zhao
authors.affiliation: School of Public Health, Tianjin Medical University, Tianjin, China;
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; yihuahuang@sun5.ibp.ac.cn.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D017434:Protein Structure, Tertiary
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins
keywords: Omp85 protein family; outer membrane protein; outer membrane protein biogenesis