The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

• Published in: The Journal of biological chemistry Vol. 298; no. 4; p. 101802
• Main Authors: Wang, Xu, Bernstein, Harris D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2022; American Society for Biochemistry and Molecular Biology
• Subjects: bacterial outer membrane; Bacterial Outer Membrane Proteins - chemical synthesis; Bacterial Outer Membrane Proteins - metabolism; Bam complex; Cell Membrane - metabolism; disulfide bonds; Disulfides - metabolism; Escherichia coli - metabolism; Protein Conformation, beta-Strand; Protein Folding; Protein Structure, Secondary; β barrel; disulfide bonds; β barrel; protein folding; bacterial outer membrane; Bam complex
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2022.101802

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.