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The Structure of a BamA-BamD Fusion Illuminates the Architecture of the ?-Barrel Assembly Machine Core.

ABSTRACT: The ?-barrel assembly machine (BAM) mediates folding and insertion of integral ?-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Of the five BAM subunits, only BamA and BamD are essential for cell viability. Here we present the crystal structure of a fusion between BamA POTRA4-5 and BamD from Rhodothermus marinus. The POTRA5 domain binds BamD between its tetratricopeptide repeats 3 and 4. The interface structural elements are conserved in the Escherichia coli proteins, which allowed structure validation by mutagenesis and disulfide crosslinking in E. coli. Furthermore, the interface is consistent with previously reported mutations that impair BamA-BamD binding. The structure serves as a linchpin to generate a BAM model where POTRA domains and BamD form an elongated periplasmic ring adjacent to the membrane with a central cavity approximately 30 × 60 Å wide. We propose that nascent OMPs bind this periplasmic ring prior to insertion and folding by BAM.

SUBMITTER: Bergal HT

PROVIDER: S-EPMC4740259 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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The Structure of a BamA-BamD Fusion Illuminates the Architecture of the β-Barrel Assembly Machine Core.

Bergal Hans Thor HT Hopkins Alex Hunt AH Metzner Sandra Ines SI Sousa Marcelo Carlos MC

Structure (London, England : 1993) 20151231 2

The β-barrel assembly machine (BAM) mediates folding and insertion of integral β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Of the five BAM subunits, only BamA and BamD are essential for cell viability. Here we present the crystal structure of a fusion between BamA POTRA4-5 and BamD from Rhodothermus marinus. The POTRA5 domain binds BamD between its tetratricopeptide repeats 3 and 4. The interface structural elements are conserved in the Escherichia coli proteins, which all ...[more]

PMID: 26749448

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Project description:Most integral outer membrane proteins (OMPs) of Gram-negative bacteria, such as Escherichia coli, 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.IMPORTANCE 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.

Dataset Information

The Structure of a BamA-BamD Fusion Illuminates the Architecture of the ?-Barrel Assembly Machine Core.

Publications

The Structure of a BamA-BamD Fusion Illuminates the Architecture of the β-Barrel Assembly Machine Core.

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