Conformational Changes That Coordinate the Activity of BamA and BamD Allowing β-Barrel Assembly. Author Anne McCabe, Dante Ricci, Modupe Adetunji, Thomas Silhavy Publication Year 2017 Type Journal Article Abstract Most integral outer membrane proteins (OMPs) of Gram-negative bacteria, such as , assume a β-barrel structure. The β-barrel assembly machine (Bam), a five-member complex composed of β-barrel OMP BamA and four associated lipoproteins, BamB, BamC, BamD, and BamE, folds and inserts OMPs into the outer membrane. The two essential proteins BamA and BamD interact to stabilize two subcomplexes, BamAB and BamCDE, and genetic and structural evidence suggests that interactions between BamA and BamD occur via an electrostatic interaction between a conserved aspartate residue in a periplasmic domain of BamA and a conserved arginine in BamD. In this work, we characterize charge-change mutations at these key BamA and BamD residues and nearby charged residues in BamA with respect to OMP assembly and Bam complex stability. We show that Bam complex stability does not correlate with function, that BamA and BamD must adopt at least two active conformational states during OMP assembly, and that these charged residues are not required for function. Rather, these charged residues are important for coordinating the activities of BamA and BamD to allow efficient OMP assembly. We present a model of OMP assembly wherein recognition and binding of unfolded OMP substrate by BamA and BamD induce a signaling interaction between the two proteins, causing conformational changes necessary for the assembly reaction to proceed. By analogy to signal sequence recognition by SecYEG, we believe these BamA-BamD interactions ensure that both substrate and complex are competent for OMP assembly before the assembly reaction commences. Conformational changes in the proteins of the β-barrel assembly machine (Bam complex) are associated with the folding and assembly of outer membrane proteins (OMPs) in Gram-negative bacteria. We show that electrostatic interactions between the two essential proteins BamA and BamD coordinate conformational changes upon binding of unfolded substrate that allow the assembly reaction to proceed. Mutations affecting this interaction are lethal not because they destabilize the Bam complex but rather because they disrupt this coordination. Our model of BamA-BamD interactions regulating conformation in response to proper substrate interaction is reminiscent of conformational changes the secretory (Sec) machinery undergoes after signal sequence recognition that ensure protein quality control. Keywords Escherichia coli, Models, Biological, DNA Mutational Analysis, Models, Molecular, Protein Conformation, Escherichia coli Proteins, Bacterial Outer Membrane Proteins, Protein Multimerization Journal J Bacteriol Volume 199 Issue 20 Date Published 2017 Oct 15 ISSN Number 1098-5530 DOI 10.1128/JB.00373-17 Alternate Journal J Bacteriol PMCID PMC5637172 PMID 28760846 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML