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Structural insight into the biogenesis of β-barrel membrane proteins

Abstract

β-barrel membrane proteins are essential for nutrient import, signalling, motility and survival. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β-barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Here we describe the structure of BamA, the central and essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features shed light on the mechanism by which BamA catalyses β-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the β-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane.

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Figure 1: The structure of BamA from the BAM complex.
Figure 2: HdBamA and NgBamA crystal structures reveal conformational changes.
Figure 3: Mutational analysis of E.coli BamA.
Figure 4: BamA primes the membrane for OMP insertion.

Accession codes

Accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for HdBamAΔ3 and NgBamA are deposited in the Protein Data Bank under accession codes 4K3C and 4K3B, respectively.

References

  1. 1

    Dalbey, R. E., Wang, P. & Kuhn, A. Assembly of bacterial inner membrane proteins. Annu. Rev. Biochem. 80, 161–187 (2011)

    CAS  Google Scholar 

  2. 2

    White, S. H. & von Heijne, G. How translocons select transmembrane helices. Annu. Rev. Biophys. 37, 23–42 (2008)

    CAS  PubMed  Google Scholar 

  3. 3

    du Plessis, D. J., Nouwen, N. & Driessen, A. J. The Sec translocase. Biochim. Biophys. Acta 1808, 851–865 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Osborne, A. R., Rapoport, T. A. & van den Berg, B. Protein translocation by the Sec61/SecY channel. Annu. Rev. Cell Dev. Biol. 21, 529–550 (2005)

    CAS  PubMed  Google Scholar 

  5. 5

    Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. Importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628–644 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Walther, D. M., Rapaport, D. & Tommassen, J. Biogenesis of β-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence. Cell. Mol. Life Sci. 66, 2789–2804 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Webb, C. T., Heinz, E. & Lithgow, T. Evolution of the β-barrel assembly machinery. Trends Microbiol. 20, 612–620 (2012)

    CAS  PubMed  Google Scholar 

  8. 8

    Paschen, S. A., Neupert, W. & Rapaport, D. Biogenesis of β-barrel membrane proteins of mitochondria. Trends Biochem. Sci. 30, 575–582 (2005)

    CAS  PubMed  Google Scholar 

  9. 9

    Jiang, J. H., Tong, J., Tan, K. S. & Gabriel, K. From evolution to pathogenesis: the link between β-barrel assembly machineries in the outer membrane of mitochondria and Gram-negative bacteria. Int. J. Mol. Sci. 13, 8038–8050 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Tommassen, J. Assembly of outer-membrane proteins in bacteria and mitochondria. Microbiology 156, 2587–2596 (2010)

    CAS  PubMed  Google Scholar 

  11. 11

    Pugsley, A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57, 50–108 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Knowles, T. J., Scott-Tucker, A., Overduin, M. & Henderson, I. R. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nature Rev. Microbiol. 7, 206–214 (2009)

    CAS  Google Scholar 

  13. 13

    Wu, T. et al. Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli. Cell 121, 235–245 (2005)

    CAS  Google Scholar 

  14. 14

    Hagan, C. L., Silhavy, T. J. & Kahne, D. β-Barrel membrane protein assembly by the Bam complex. Annu. Rev. Biochem. 80, 189–210 (2011)

    CAS  Google Scholar 

  15. 15

    Ricci, D. P. & Silhavy, T. J. The Bam machine: a molecular cooper. Biochim. Biophys. Acta 1818, 1067–1084 (2012)

    CAS  PubMed  Google Scholar 

  16. 16

    Rigel, N. W. & Silhavy, T. J. Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria. Curr. Opin. Microbiol. 15, 189–193 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Hagan, C. L., Kim, S. & Kahne, D. Reconstitution of outer membrane protein assembly from purified components. Science 328, 890–892 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Noinaj, N., Fairman, J. W. & Buchanan, S. K. The crystal structure of BamB suggests interactions with BamA and its role within the BAM complex. J. Mol. Biol. 407, 248–260 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Heuck, A., Schleiffer, A. & Clausen, T. Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins. J. Mol. Biol. 406, 659–666 (2011)

    CAS  PubMed  Google Scholar 

  20. 20

    Kim, K. H., Aulakh, S. & Paetzel, M. Crystal structure of β-barrel assembly machinery BamCD protein complex. J. Biol. Chem. 286, 39116–39121 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Albrecht, R. & Zeth, K. Structural basis of outer membrane protein biogenesis in bacteria. J. Biol. Chem. 286, 27792–27803 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Sandoval, C. M., Baker, S. L., Jansen, K., Metzner, S. I. & Sousa, M. C. Crystal structure of BamD: an essential component of the β-barrel assembly machinery of gram-negative bacteria. J. Mol. Biol. 409, 348–357 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Dong, C., Hou, H. F., Yang, X., Shen, Y. Q. & Dong, Y. H. Structure of Escherichia coli BamD and its functional implications in outer membrane protein assembly. Acta Crystallogr. D 68, 95–101 (2012)

    CAS  PubMed  Google Scholar 

  24. 24

    Knowles, T. J. et al. Structure and function of BamE within the outer membrane and the beta-barrel assembly machine. EMBO Rep. 12, 123–128 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Gatzeva-Topalova, P. Z., Walton, T. A. & Sousa, M. C. Crystal structure of YaeT: conformational flexibility and substrate recognition. Structure 16, 1873–1881 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Kim, S. et al. Structure and function of an essential component of the outer membrane protein assembly machine. Science 317, 961–964 (2007)

    ADS  CAS  PubMed  Google Scholar 

  27. 27

    Zhang, H. et al. High-resolution structure of a new crystal form of BamA POTRA4–5 from Escherichia coli. Acta Crystallogr. F 67, 734–738 (2011)

    CAS  Google Scholar 

  28. 28

    Gatzeva-Topalova, P. Z., Warner, L. R., Pardi, A. & Sousa, M. C. Structure and flexibility of the complete periplasmic domain of BamA: the protein insertion machine of the outer membrane. Structure 18, 1492–1501 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Knowles, T. J. et al. Fold and function of polypeptide transport-associated domains responsible for delivering unfolded proteins to membranes. Mol. Microbiol. 68, 1216–1227 (2008)

    CAS  PubMed  Google Scholar 

  30. 30

    Clantin, B. et al. Structure of the membrane protein FhaC: a member of the Omp85–TpsB transporter superfamily. Science 317, 957–961 (2007)

    ADS  CAS  PubMed  Google Scholar 

  31. 31

    Fan, E., Fiedler, S., Jacob-Dubuisson, F. & Muller, M. Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane. J. Biol. Chem. 287, 2591–2599 (2012)

    CAS  PubMed  Google Scholar 

  32. 32

    Rigel, N. W., Ricci, D. P. & Silhavy, T. J. Conformation-specific labeling of BamA and suppressor analysis suggest a cyclic mechanism for β-barrel assembly in Escherichia coli. Proc. Natl Acad. Sci. USA 110, 5151–5156 (2013)

    ADS  CAS  PubMed  Google Scholar 

  33. 33

    Leonard-Rivera, M. & Misra, R. Conserved residues of the putative L6 loop of Escherichia coli BamA play a critical role in the assembly of β-barrel outer membrane proteins, including that of BamA itself. J. Bacteriol. 194, 4662–4668 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Gumbart, J., Wiener, M. C. & Tajkhorshid, E. Coupling of calcium and substrate binding through loop alignment in the outer-membrane transporter BtuB. J. Mol. Biol. 393, 1129–1142 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Hearn, E. M., Patel, D. R., Lepore, B. W., Indic, M. & van den Berg, B. Transmembrane passage of hydrophobic compounds through a protein channel wall. Nature 458, 367–370 (2009)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Cuesta-Seijo, J. A. et al. PagP crystallized from SDS/cosolvent reveals the route for phospholipid access to the hydrocarbon ruler. Structure 18, 1210–1219 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Hong, H., Patel, D. R., Tamm, L. K. & van den Berg, B. The outer membrane protein OmpW forms an eight-stranded beta-barrel with a hydrophobic channel. J. Biol. Chem. 281, 7568–7577 (2006)

    CAS  PubMed  Google Scholar 

  38. 38

    Tamm, L. K., Hong, H. & Liang, B. Folding and assembly of beta-barrel membrane proteins. Biochim. Biophys. Acta 1666, 250–263 (2004)

    CAS  PubMed  Google Scholar 

  39. 39

    Tamm, L. K., Arora, A. & Kleinschmidt, J. H. Structure and assembly of beta-barrel membrane proteins. J. Biol. Chem. 276, 32399–32402 (2001)

    CAS  PubMed  Google Scholar 

  40. 40

    Agah, S. & Faham, S. Crystallization of membrane proteins in bicelles. Methods Mol. Biol. 914, 3–16 (2012)

    CAS  PubMed  Google Scholar 

  41. 41

    Ujwal, R. & Bowie, J. U. Crystallizing membrane proteins using lipidic bicelles. Methods 55, 337–341 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  43. 43

    Stein, N. CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallogr. 41, 641–643 (2008)

    CAS  Google Scholar 

  44. 44

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Google Scholar 

  45. 45

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Ramachandran, S., Kota, P., Ding, F. & Dokholyan, N. V. Automated minimization of steric clashes in protein structures. Proteins 79, 261–270 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Shaw, D. E. et al. Anton, a special-purpose machine for molecular dynamics simulation. Commun. ACM 51, 91–97 (2008)

    Google Scholar 

  48. 48

    Fairman, J. W. et al. Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis. Structure 20, 1233–1243 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    PubMed  PubMed Central  Google Scholar 

  50. 50

    Robert, V. et al. Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif. PLoS Biol. 4, e377 (2006)

    PubMed  PubMed Central  Google Scholar 

  51. 51

    Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Barnard, T. J. et al. Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter. J. Mol. Biol. 415, 128–142 (2012)

    CAS  PubMed  Google Scholar 

  55. 55

    Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Mackerell, A. D., Jr, Feig, M. & Brooks, C. L., III Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 25, 1400–1415 (2004)

    CAS  Google Scholar 

  57. 57

    Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983)

    ADS  CAS  Google Scholar 

  58. 58

    Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Ulmschneider, J. P., Smith, J. C., White, S. H. & Ulmschneider, M. B. In silico partitioning and transmembrane insertion of hydrophobic peptides under equilibrium conditions. J. Am. Chem. Soc. 133, 15487–15495 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Elbing, K. & Brent, R. Media preparation and bacteriological tools. Curr. Protoc. Mol. Biol. Chapter 1, Unit 1.1 (2002)

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Acknowledgements

We thank H. Bernstein and R. Ieva for providing JCM-166 cells, J. Beckwith and R. Misra for providing antibodies, and A. M. Stanley and T. Barnard for discussions and comments on the manuscript. N.N., A.J.K., N.C.E., H.C. and S.K.B. are supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Diabetes and Digestive and Kidney Diseases. J.C.G. acknowledges support from the NIH under grants K22-AI100927 and R01-GM67887. P.L. is supported by a postdoctoral fellowship through the Diamond Light Source. T.L. is an Australian Research Council (ARC) Federation Fellow and acknowledges support from ARC Discovery Project (DP120101878) and ARC Linkage International Grant (LX0776170). We thank the respective staffs at the Southeast Regional Collaborative Access Team (SER-CAT) and General Medicine and Cancer Institute’s Collaborative Access Team (GM/CA-CAT) beamlines at the Advanced Photon Source, Argonne National Laboratory and the Diamond Light Source for their assistance during data collection. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38 (SER-CAT), and by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357 (GM/CA-CAT). Anton computer time was provided by the National Resource for Biomedical Supercomputing and the Pittsburgh Supercomputing Center through Grant RC2GM093307 from the NIH, using a machine donated by DE Shaw Research.

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Contributions

N.N., H.C., N.C.E. and S.K.B. cloned, expressed and purified HdBamAΔ3 and NgBamA. P.L. performed data collection for experimental phasing. N.N. crystallized and solved the HdBamAΔ3 and NgBamA crystal structures. N.N. and A.J.K. performed the homology modelling and functional assays. J.C.G. designed, conducted and analysed the molecular dynamics simulations. N.N., A.J.K. and S.K.B. analysed and discussed all data. T.L. and S.K.B. conceived and designed the original project. N.N., A.J.K., T.L. and S.K.B. wrote the manuscript.

Corresponding author

Correspondence to Susan K. Buchanan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2, Supplementary Figures 1-18, Supplementary Models 1-2, details of Supplementary Videos 1-2 and additional references. (PDF 4312 kb)

Supplementary Data

This file contains the data for Supplementary Models 1-2. (ZIP 169 kb)

Molecular dynamics (MD) simulations of FhaC and NgBamA

MD simulations were used to probe the stability of the barrel domains of FhaC and NgBamA. This video illustrates the changes that occur within the barrel domain (opening and closing) in NgBamA as the simulation progresses (see also Figure 4 and Supplementary Figure 16). (MOV 10499 kb)

Proposed mechanisms for the role of BamA in the biogenesis of β-barrel membrane proteins.

Animated video summarizing the two proposed mechanisms believed to be involved in recognizing, folding, and inserting β-barrel membrane proteins into the outer membrane. These mechanisms are based on studies (structural biology, computational, and functional) reported here, as well as, previously reported studies about the function of BamA. (MOV 5810 kb)

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Noinaj, N., Kuszak, A., Gumbart, J. et al. Structural insight into the biogenesis of β-barrel membrane proteins. Nature 501, 385–390 (2013). https://doi.org/10.1038/nature12521

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