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Structure of the BAM complex and its implications for biogenesis of outer-membrane proteins

Nature Structural & Molecular Biology volume 23, pages 192–196 (2016)Cite this article

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Abstract

In Gram-negative bacteria, the assembly of β-barrel outer-membrane proteins (OMPs) requires the β-barrel–assembly machinery (BAM) complex. We determined the crystal structure of the 200-kDa BAM complex from Escherichia coli at 3.55-Å resolution. The structure revealed that the BAM complex assembles into a hat-like shape, in which the BamA β-barrel domain forms the hat's crown embedded in the outer membrane, and its five polypeptide transport–associated (POTRA) domains interact with the four lipoproteins BamB, BamC, BamD and BamE, thus forming the hat's brim in the periplasm. The assembly of the BAM complex creates a ring-like apparatus beneath the BamA β-barrel in the periplasm and a potential substrate-exit pore located at the outer membrane–periplasm interface. The complex structure suggests that the chaperone-bound OMP substrates may feed into the chamber of the ring-like apparatus and insert into the outer membrane via the potential substrate-exit pore in an energy-independent manner.

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Figure 1: General architecture of the BAM complex.
Figure 2: The overall conformation of BamA in the BAM complex.
Figure 3: The potential substrate-exit pore of the BAM complex.
Figure 4: Close-up view of intercomponent interactions in the BAM complex.

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Acknowledgements

We are grateful to T. Silhavy (Princeton University) for generously providing the E. coli strain JCM320. We also thank X. Zhang and H. Wu for valuable discussions; R. Zhang and Shanghai Synchrotron Radiation Facility beamline scientists for scheduling beamline time; F. Yang, L. Niu, M. Zhang, L. Shu, Z. Xie and X. Ding from the Mass Spectrometry Core Facility of the Institute of Biophysics, Chinese Academy of Sciences, for help with mass spectrometry analysis of the BAM complex; and the National Supercomputing Center Tianjin Center (Tianhe), China, for computational resources. This work was supported by grants from the Ministry of Science and Technology (2012CB917302 and 2013CB910603 to Y.H.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB080202 to Y.H.) and the National Natural Science Foundation of China (31170698 and 31470743 to Y.H.).

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Authors and Affiliations

  1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Long Han, Jiangge Zheng, Yan Wang, Xu Yang, Yanqing Liu, Chuanqi Sun, Baohua Cao, Haizhen Zhou, Dongchun Ni, Yongfang Zhao & Yihua Huang

  2. University of Chinese Academy of Sciences, Beijing, China

    Long Han, Yanqing Liu & Chuanqi Sun

  3. Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Jizhong Lou

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Contributions

Y.H. supervised the project. L.H., J.Z., Y.W., X.Y., H.Z. and D.N. performed protein purification, crystallization and diffraction data collection. Y.H. and B.C. determined the structure and built the model. Y.H. wrote the manuscript. All authors contributed to data analysis.

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Correspondence to Yihua Huang.

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Integrated supplementary information

Supplementary Figure 1 Schematic domain organization of the BAM complex and characterization of the purified BAM complex.

a. Schematic domain structures of the mature proteins of BamA, BamB, BamC, BamD and BamE. b. Size exclusion chromatographic profile of the purified BAM complex from bacterial OMs on Superose 6 10/300 GL column. c. 15% SDS-PAGE analysis of fractions from the elution peak (left) and of the BAM complex crystals (dissolved from 50 crystals) to show that BamC remained intact in the crystals (right).

Supplementary Figure 2 Overlay of isolated POTRA domains or β-barrel–attached POTRA domains of BamA.

a. Overlay of POTRA4 domains from BamA in the BAM complex (BAM-POTRA1-5), POTRA4-5 (PDB: 3Q6B), POTRA1-4 (PDBs: 3EFC and 2QCZ) to show the remarkably different inter-POTRA interfaces and orientations. b. Structural overlay of the β-barrel domains of BamA from the BAM complex (BAM-BamA), BamA lacking POTRA1-4 domains (BamA_POTRA5, PDB: 4C4V) and HdBamA lacking POTRA1-3 domains (HdBamA_POTRA4-5, PDB: 4K3C) to show the remarkably different interdomain orientation between BamA β barrel and the POTRA5 domain.

Supplementary Figure 3 Strand β1 of the BamA β-barrel might participate in β-augmentation.

Residues T243, S324, D399 (or T400) and G429 of BamA are located at the middle point of a β strand of POTRA3, POTRA4, POTRA5 and strand β1 of BamA β barrel, respectively. These β strands are located within the chamber of the BAM complex structure and have a potential role in participating β-augmentation for OMP substrates. a. G429P mutant was lethal to the bacteria while other mutants were not. b. G429P mutant was lethal in the presence of 10 μM IPTG. c. Western blot showing that G429P mutant protein was expressed in a similar level to that of the wild-type in the cell. d. Purified G429P mutant protein was well-folded. Purified G429P protein from membranes exhibited apparently heat-modifiable mobility on a 15% SDS-PAGE gel.

Supplementary Figure 4 Structural comparisons of BamE and BamCD complexes.

a. Overlay of BamE in the BAM complex with the isolated BamE structures from crystal structure (BamE-X-ray, PDB: 2YH9) and NMR structure (BamE-NMR, PDB: 2KXX). Overlay of the three β strands of BamE structures reveals an RMSD of 2.5 Å (35 aligned Cα atom pairs) and of 2.2 Å (35 aligned Cα atom pairs) between BamE in the BAM complex and BamE crystal structure or BamE NMR structure. b. Structural comparison of BamCD complex with that in the BAM complex. Neither the BamC_N domain nor the BamC_C domain was visible in the BAM complex structure.

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Supplementary Figures 1–4 (PDF 665 kb)

Supplementary Data Set 1

Uncropped images of western blots for Figure 2d, Figure 4e and Figure 4f (PDF 647 kb)

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Han, L., Zheng, J., Wang, Y. et al. Structure of the BAM complex and its implications for biogenesis of outer-membrane proteins. Nat Struct Mol Biol 23, 192–196 (2016). https://doi.org/10.1038/nsmb.3181

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