Disulphide bonds are crucial structural and functional elements in many virulence proteins found in pathogenic Gram-negative bacteria. The periplasmic oxidase DsbA mediates the formation of disulphide bonds (Dsb) in unfolded substrate proteins as part of a widely conserved disulphide bond formation system. DsbA’s targets include proteins essential for bacterial fitness and pathogenicity, such as secreted toxins, secretion systems, adhesion molecules, and components of the flagellar motor. DsbA has the ability to fold a diverse range of substrates; however, the precise mechanisms by which it recognises, and folds structurally varied targets remain unclear.
We determined the crystal structure of Escherichia coli DsbA (EcDsbA) in complex with peptides derived from the target protein LptD, a lipopolysaccharide (LPS) transport protein in E. coli. Comparison with the only published EcDsbA-substrate complex revealed that different target peptides bind to EcDsbA in varying orientations. We observed that the binding of LptD peptides introduces unexpected structural flexibility into EcDsbA, enabling it to accommodate a wider range of substrates.
Further analysis of EcDsbA-peptide complex structures identified the cis-proline loop (containing residues G149, V150, and P151) as displaying conserved rigidity across all structures. Previous mutational studies on the cis-proline residues revealed their significance, with mutations altering EcDsbA’s oxidative potential and disrupting substrate binding. We introduced mutations at P151 and G149 and, through structural and functional analysis, demonstrated the crucial role of these residues in EcDsbA activity. In particular, P151 is essential for efficient substrate binding and for enabling EcDsbA to fully resolve substrate oxidation.
Collectively, this work demonstrates that flexibility around the active site may explain EcDsbA's versatile role as a ‘jack of all trades’ in interacting with and folding diverse unfolded proteins. Additionally, the cis-proline loop, conserved across all Dsb proteins, appears to function as a platform, facilitating the efficient binding and positioning of substrates near the catalytic cysteines to promote effective thiol-disulphide exchange reactions. Overall, this study lays a strong foundation for understanding substrate interactions in DsbA proteins across pathogenic Gram-negative bacteria, opening new avenues for the rational design of structure-based inhibitors aimed at disrupting DsbA-substrate interactions, offering a potential strategy to combat antibiotic resistance.