Sequence-Specific Solution NMR Assignments of the β-Barrel Insertase Bam A to Monitor Its Conformational Ensemble at the Atomic Level

摘要

beta-barrel outer membrane proteins (Omps) are key functional components of the outer membranes of Gram-negative bacteria, mitochondria, and plastids. In bacteria, their biogenesis requires the beta-barrel-assembly machinery (Barn) with the central insertase BamA, but the exact translocation and insertion mechanism remains elusive. The BamA insertase features a loosely closed gating region between the first and last beta-strand 16. Here, we describe similar to 70% complete sequence specific NMR resonance assignments of the transmembrane region of the BamA beta-barrel in detergent micelles. On the basis of the assignments, NMR spectra show that the BamA barrel populates a conformational ensemble in slow exchange equilibrium, both in detergent micelles and lipid bilayer nanodiscs. Individual conformers can be selected from the ensemble by the introduction of a C-terminal strand extension, single-point mutations, or specific disulfide cross-linkings, and these modifications at the barrel seam are found to be allosterically coupled to sites at the entire barrel circumference. The resonance assignment provides a platform for mechanistic studies of BamA at atomic resolution, as well as for investigating interactions with potential antibiotic drugs and partner proteins.