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Genetic, biochemical, and molecular characterization of the polypeptide transport-associated domain of Escherichia coli BamA.

Journal of bacteriology (2012-05-01)
Patricia Workman, Kristina Heide, Nicolas Giuliano, Nanhee Lee, James Mar, Phu Vuong, Drew Bennion, Rajeev Misra
ABSTRACT

The BamA protein of Escherichia coli plays a central role in the assembly of β-barrel outer membrane proteins (OMPs). The C-terminal domain of BamA folds into an integral outer membrane β-barrel, and the N terminus forms a periplasmic polypeptide transport-associated (POTRA) domain for OMP reception and assembly. We show here that BamA misfolding, caused by the deletion of the R44 residue from the α2 helix of the POTRA 1 domain (ΔR44), can be overcome by the insertion of alanine 2 residues upstream or downstream from the ΔR44 site. This highlights the importance of the side chain orientation of the α2 helix residues for normal POTRA 1 activity. The ΔR44-mediated POTRA folding defect and its correction by the insertion of alanine were further demonstrated by using a construct expressing just the soluble POTRA domain. Besides misfolding, the expression of BamA(ΔR44) from a low-copy-number plasmid confers a severe drug hypersensitivity phenotype. A spontaneous drug-resistant revertant of BamA(ΔR44) was found to carry an A18S substitution in the α1 helix of POTRA 1. In the BamA(ΔR44, A18S) background, OMP biogenesis improved dramatically, and this correlated with improved BamA folding, BamA-SurA interactions, and LptD (lipopolysaccharide transporter) biogenesis. The presence of the A18S substitution in the wild-type BamA protein did not affect the activity of BamA. The discovery of the A18S substitution in the α1 helix of the POTRA 1 domain as a suppressor of the folding defect caused by ΔR44 underscores the importance of the helix 1 and 2 regions in BamA folding.

MATERIALS
Product Number
Brand
Product Description

Millipore
Protein G Immunoprecipitation Kit, sufficient for 50 assays