Flexibility in the Periplasmic Domain of BamA Is Important for Function

• Published in: Structure (London) 2017-01, Vol.25 (1), p.94-106
• Main Authors: Warner, Lisa R., Gatzeva-Topalova, Petia Z., Doerner, Pamela A., Pardi, Arthur, Sousa, Marcelo C.
• Format: Article
• Language: English
• Subjects: Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; BamA; disulfide crosslinking; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; interdomain flexibility; Models, Molecular; Mutation; Nuclear Magnetic Resonance, Biomolecular; Periplasm; POTRA; Protein Domains; Protein Folding; Protein Structure, Secondary; residual dipolar couplings
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2016.11.013

The β-barrel assembly machine (BAM) mediates the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria. BamA, the central BAM subunit composed of a transmembrane β-barrel domain linked to five polypeptide transport-associated (POTRA) periplasmic domains, is thought to bind nascent OMPs and undergo conformational cycling to catalyze OMP folding and insertion. One model is that conformational flexibility between POTRA domains is part of this conformational cycling. Nuclear magnetic resonance (NMR) spectroscopy was used here to study the flexibility of the POTRA domains 1–5 in solution. NMR relaxation studies defined effective rotational correlational times and together with residual dipolar coupling data showed that POTRA1–2 is flexibly linked to POTRA3–5. Mutants of BamA that restrict flexibility between POTRA2 and POTRA3 by disulfide crosslinking displayed impaired function in vivo. Together these data strongly support a model in which conformational cycling of hinge motions between POTRA2 and POTRA3 in BamA is required for biological function. [Display omitted] •BamA with a transmembrane β-barrel and soluble POTRA motifs mediates OMP biogenesis•NMR relaxation studies show POTRA1–2 is flexibly linked to POTRA3–5 in BamA•Residual dipolar couplings indicate POTRA1–2 and POTRA4–5 behave as rigid species•Disulfides that restrict POTRA2–3 flexibility impair in vivo function of BamA BamA is essential for folding and insertion of β-barrel outer membrane proteins in Gram-negative bacteria. Warner et al. show that the five POTRA domains of BamA feature a hinge between POTRA2 and POTRA3 and that its flexibility is important for BamA in vivo function.