Conformational Flexibility of the Protein Insertase BamA in the Native Asymmetric Bilayer Elucidated by ESR Spectroscopy

Abstract

The b-barrel assembly machinery(BAM) consisting of the central b-barrel BamA and four other lipoproteins mediates the folding of the majority of the outer membrane proteins.B amA is placed in an asymmetric bilayer and its lateral gate is suggested to be the functional hotspot. Here we used in situ pulsed electron-electron double resonance spectroscopytoc haracterize BamA in the native outer membrane. In the detergent micelles,the data is consistent with mainly an inward-open conformation of BamA. The native membrane considerably enhanced the conformational heterogeneity.T he lateral gate and the extracellular loop 3exist in an equilibrium between different conformations.T he outer membrane provides af avorable environment for occupying multiple conformational states independent of the lipoproteins.Our results reveal ahighly dynamic behavior of the lateral gate and other key structural elements and provide direct evidence for the conformational modulation of amembrane protein in situ.

Lateral gating mechanism and plasticity of the BAM complex in micelles andE. coli

“…Solid-state NMR and single-molecule force spectroscopy techniques are complementary tools for such investigations, in particular for isolated outer membranes (58,59). Using this approach, we showed that BamA displays an increased heterogeneity, in particular at the lateral gate when observed in the native outer membranes (35). Here we extended similar experiments for many other interloop pairs of BamABCDE full complex in intact E. coli cells (see Figs.…”

“…Under laboratory environment, BamA and the lipoproteins are expressed at a few thousand (1.5 -6.0 x 10 3 ) copies per cell (62). In our case, overexpression leads to a 100-fold increase in the copy number for BamA (35), which is comparable to the native expression level of some of the OMPs (such as OmpA, OmpC and OmpF). As for membrane protein overexpression in general, the cells may increase the surface area to accommodate the additional copies of BAM molecules (63).…”

“…The L3 might be sensitive to the surrounding environment and the presence of the substrate, which also might explain its varying conformations between different structures. Notably, when BamA was expressed without any lipoproteins, L3 displayed a dynamic behavior spanning the range of both IO and LO conformations in the native outer membranes (35). A rather narrow interloop distance distribution against the rigid L3 reveals a limited flexibility for L7 and L8 as well (Figs.…”

“…Structures captured distinct states from this broad conformational space. Interestingly, when observed in the native outer membranes, BamA showed a heterogenous conformation spanning multiple states (35). A systematic observation of BamAB, BamACDE and BamABCDE complexes under identical conditions is necessary to exclude the differential effects of the environments and to elucidate the gating mechanism.…”

Selective Translocation of Cyclic Sugars through Dynamic Bacterial Transporter

“…At the same time, cam 8 γCD8+ did not produce any ion current blockages through the truncated CymA similar to native CymA due to their much larger 8-fold symmetric structure (Figure S10). In the future, we will address molecular mechanisms associated with the discrete movement of the N terminus within the pore by targeting specific N terminus mutations using advanced structural methods. − Finally, we performed liposome permeation assays to determine the relative permeation rates of neutral cyclic sugars such as αCD and γCD through native and truncated CymA , (Figure c). Under isotonic conditions, αCD diffused through native and truncated CymA pores, whereas γCD did not show any diffusion establishing the importance of molecular size and symmetry for permeation (Figure c).…”