Flexibility in the Periplasmic Domain of BamA Is Important for Function

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• The β-barrel assembly machine (BAM) complex is essential for the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria.
• BamA, a key component of BAM, possesses a unique structure with transmembrane and periplasmic POTRA domains, crucial for OMP folding and insertion.

Purpose of the Study:

• To investigate the role of conformational flexibility between the POTRA domains of BamA in OMP biogenesis.
• To determine if specific hinge motions within BamA are critical for its biological function.

Main Methods:

• Utilized Nuclear Magnetic Resonance (NMR) spectroscopy, including relaxation studies and residual dipolar coupling analysis.
• Assessed the functional consequences of mutations designed to restrict POTRA domain flexibility in vivo.

Main Results:

• NMR data revealed that POTRA1-2 is flexibly linked to POTRA3-5.
• Disulfide crosslinking mutants that reduced flexibility between POTRA2 and POTRA3 exhibited impaired BamA function in vivo.
• These findings support a model of conformational cycling involving hinge motions between POTRA domains.

Conclusions:

• Conformational flexibility, particularly hinge motions between POTRA2 and POTRA3 in BamA, is essential for the proper function of the β-barrel assembly machine.
• This flexibility likely plays a critical role in the catalytic cycle of OMP folding and insertion into the bacterial outer membrane.