title: Conserved residues of the putative L6 loop of Escherichia coli BamA play a critical role in the assembly of β-barrel outer membrane proteins, including that of BamA itself.
abstract: Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved (641)RGF(643) residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved (641)RGF(643) residues of the BamA L6 loop are important for BamA folding and biogenesis.
authors: School of Life Sciences, Arizona State University, Tempe, Arizona, USA.
authors: Margaret
authors: M
authors: Leonard-Rivera
authors: Rajeev
authors: R
authors: Misra
mesh_terms: D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010641:Phenotype; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017384:Sequence Deletion
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins; C098052:fhaC protein, Bordetella pertussis