BamA, the core component of the β-barrel assembly machinery complex, is an integral outer-membrane protein (OMP) in Gram-negative bacteria that catalyzes the folding and insertion of OMPs. A key feature of BamA relevant to its function is a lateral gate between its first and last β-strands. Opening of this lateral gate is one of the first steps in the asymmetric-hybrid-barrel model of BamA function. In this study, multiple hybrid-barrel folding intermediates of BamA and a substrate OMP, EspP, were constructed and simulated to better understand the model’s physical consequences. The hybrid-barrel intermediates consisted of the BamA β-barrel and its POTRA5 domain and either one, two, three, four, five, or six β-hairpins of EspP. The simulation results support an asymmetric-hybrid-barrel model in which the BamA N-terminal β-strand forms stronger interactions with the substrate OMP than the C-terminal β-strand. A consistent “B”-shaped conformation of the final folding intermediate was observed, and the shape of the substrate β-barrel within the hybrid matched the shape of the fully folded substrate. Upon further investigation, inward-facing glycines were found at sharp bends within the hybrid and fully folded β-barrels. Together, the data suggest an influence of sequence on shape of the substrate barrel throughout the OMP folding process and of the fully folded OMP.

BamA 是 β 桶组装机械复合体的核心成分，是革兰氏阴性菌中的一种完整外膜蛋白 (OMP)，可催化 OMP 的折叠和插入。BamA 与其功能相关的一个关键特征是其第一条和最后一条 β 链之间的侧向门。该侧门的打开是 BamA 函数的不对称混合桶模型的第一步。在这项研究中，构建并模拟了 BamA 和底物 OMP EspP 的多个混合桶折叠中间体，以更好地理解模型的物理后果。混合桶中间体由 BamA β-桶及其 POTRA5 结构域以及 EspP 的 1、2、3、4、5 或 6 个 β-发夹组成。模拟结果支持不对称混合桶模型，其中 BamA N 端 β 链与底物 OMP 形成比 C 端 β 链更强的相互作用。观察到最终折叠中间体具有一致的“B”形构象，并且杂化物内基底β-桶的形状与完全折叠的基底的形状相匹配。经过进一步研究，在混合和完全折叠的β-桶内的急弯处发现了向内的甘氨酸。总之，数据表明序列对整个 OMP 折叠过程中的基底筒形状以及完全折叠的 OMP 的形状有影响。