A monodisperse transmembrane α-helical peptide barrel

Journal name:
Nature Chemistry
Volume:
9,
Pages:
411–419
Year published:
DOI:
doi:10.1038/nchem.2647
Received
Accepted
Published online

Abstract

The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.

At a glance

Figures

  1. Structure of E. coli Wza, peptide design and electrical properties of the cWza pore.
    Figure 1: Structure of E. coli Wza, peptide design and electrical properties of the cWza pore.

    a, Structure of native E. coli Wza (Protein Data Bank (PDB): 2J58). b, Structure of the Wza D4 domain. c, The WebLogo (http://weblogo.berkeley.edu/) representation of a BLAST search for the Wza D4 domain (top) shows the consensus Wza peptide (cWza) sequence (bottom). d, Electrical recording of multiple insertions of cWza peptide pores into a planar bilayer at +100 mV. Each step represents the appearance of an L state. e, Appearance of the precursor state P followed by conversion into the low-conductance pore L at +100 mV. The P state can also form reversibly (Supplementary Fig. 17). f, Histogram of the unitary conductance of L events at +100 mV. The mean unitary conductance was obtained by fitting the distribution to a Gaussian (n = 110). g, Interconversion of an L state and an H state at +200 mV. h, Histogram of the unitary conductance of H events at +200 mV. The mean unitary conductance was obtained by fitting the distribution to a Gaussian (n = 100). i, Conversion of an L state into the P state followed by closure at + 100 mV. Electrolyte, 1 M KCl, 10 mM HEPES, pH 7.4. The current signals were filtered at 2 kHz and sampled at 10 kHz. In all the experiments, cWza peptide pores were formed by adding the peptide to the cis compartment.

  2. Electrical properties of mutant cWza pores.
    Figure 2: Electrical properties of mutant cWza pores.

    a, Structural models of cWza-K375C peptides with two helices facing each other. The D4 α-helix barrel in the native Wza crystal structure (PDB: 2J58) was used as a template. b, P state of a cWza-K375C pore and its conversion into the L state at +100 mV. c, Histogram of the unitary conductance values of cWza-K375C L events at +100 mV. The mean unitary conductance was obtained by fitting the distribution to a Gaussian (n = 100). d, H state of a cWza-K375C pore and conversion to the L state at +200 mV. e, Histogram of the unitary conductance values of cWza-K375C H events at +200 mV. The mean unitary conductance was obtained by fitting the distribution to a Gaussian (n = 100). f, L state of a cWza-K375C pore and closure via the P state at +100 mV. g, Structural models of cWza-Y373C peptides with two helices facing each other. The D4 α-helix barrel in the native Wza crystal structure (PDB: 2J58) was used as a template. h, P state of the cWza-Y373C pore and conversion into the H state at +100 mV. i, Histogram of the unitary conductance values of cWza-Y373C H events at +100 mV. The mean unitary conductance was obtained by fitting the distribution to a Gaussian (n = 100). j, Single cWza-Y373C pore at +200 mV. k, Current–voltage curve obtained from a single cWza-Y373C pore. Electrolyte: 1 M KCl, 10 mM HEPES, pH 7.4. The current signals were filtered at 2 kHz and sampled at 10 kHz.

  3. Interaction of cWza peptide pores with cationic CDs.
    Figure 3: Interaction of cWza peptide pores with cationic CDs.

    a, Structure of am8γCD (left), interaction of the cWza-K375C H state with am8γCD (100 µM, trans) at +50 mV (middle left) and at +150 mV (middle right), and a plot of koff versus the applied potential (mean values (±s.d.) from three independent experiments are shown) (right). b, Structure of am6αCD (left), interaction of the cWza-K375C H state with am6αCD (100 µM, trans) at +50 mV (middle left) and at +150 mV (middle right), and a plot of koff versus the applied potential (mean values (±s.d.) from three independent experiments are shown) (right). c, Competitive interaction of the cWza-K375C H state with am6αCD and am8γCD (both 100 µM, trans) at +50 mV. The current signals were filtered at 10 kHz and sampled at 50 kHz. d, Interaction of the cWza-Y373C H state with am8γCD (100 µM, trans) at +50 mV (left) and at +100 mV (middle), and with am6αCD (100 µM, trans) at +100 mV (right). The current signals were filtered at 2 kHz and sampled at 10 kHz. Electrolyte: 1 M KCl, 10 mM HEPES, pH 7.4.

  4. Orientation and stoichiometry of the cWza-Y373C pore.
    Figure 4: Orientation and stoichiometry of the cWza-Y373C pore.

    a, Reaction of 1 mM MePEG-OPSS-5k (trans) with the cWza-Y373C H state at +50 mV. The structures of the PEG reagents are shown in Supplementary Fig. 12. b, Subsequent addition of 10 mM DTT (trans) results in the cleavage of PEG chains from the pore. c, Reaction of 1 mM MePEG-OPSS-5k (cis) with the cWza-Y373C H state at +50 mV. Pore blockade was not observed. d, It was deduced that cWza-Y373C added to the cis compartment formed a stable pore with all the peptides oriented so that the C termini faced the trans compartment. e, Reaction of 1 mM MePEG-OPSS-1k (trans) with the cWza-Y373C pore at +50 mV. Inset: the histogram shows the number of current steps seen after the addition of 1 mM MePEG-OPSS-1k (trans) to individual cWza-Y375C pores. f, Reaction of 1 mM MePEG-OPSS-1k (cis) with the cWza-Y373C pore at +50 mV. The displayed current signals af were filtered digitally at 200 Hz using an eight-pole Bessel digital filter. g, Formation of a K4-cWza-Y373C pore at +100 mV. h, The cWza-Y373C-K4 pore fluctuates between different conductance states at +100 mV. The current signals (g,h) were filtered at 2 kHz and sampled at 10 kHz. Electrolyte: 1 M KCl, 10 mM HEPES, pH 7.4.

  5. CD-templated barrel and its electrical properties.
    Figure 5: CD-templated barrel and its electrical properties.

    a, (PDPam)8γCD was coupled to cWza-S355C through disulfide bonds. The structure of the fully derivatized CD, (cWza-S355C-TPam)8γCD, is based on the D4 domain in the native Wza crystal structure (PDB: 2J58). b, The peptide–CD conjugates, peptide and protein markers (M) were run on an SDS–PAGE gel. The arrow indicates the presumed fully derivatized (cWza-S355C-TPam)8γCD. c, Electrical recording of a single (cWza-S355C-TPam)8γCD pore. d, Single (cWza-S355C-TPam)8γCD pore after treatment with 10 mM DTT (cis). For display, the current signals (c,d) were digitally filtered at 500 Hz using an eight-pole Bessel digital filter.

  6. Model for membrane insertion and pore formation by cWza peptides.
    Figure 6: Model for membrane insertion and pore formation by cWza peptides.

    The cWza peptide binds to the membrane to form a precursor state P, which converts into a low-conductance state, L. At high potentials, L is converted into a high-conductance state H, which can be blocked by cationic CDs. The pore can close, via the P state, by reversal of the assembly pathway. The L and H states most probably comprise eight helices (see text), whereas the structure of the noisy P state is probably less stable but with an octameric component that transitions into the L state. The structure of the H state is based on that of the D4 domain in native Wza. The structure of the L state is a coiled-coil octamer with dimensions that reflect the lower conductance of this state (Supplementary Fig. 1).

Accession codes

Referenced accessions

Protein Data Bank

References

  1. Woolfson, D. N. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 70, 79112 (2005).
  2. Woolfson, D. N. et al. De novo protein design: how do we expand into the universe of possible protein structures? Curr. Opin. Struct. Biol. 33, 1626 (2015).
  3. Woolfson, D. N., Bartlett, G. J., Bruning, M. & Thomson, A. R. New currency for old rope: from coiled-coil assemblies to alpha-helical barrels. Curr. Opin. Struct. Biol. 22, 432441 (2012).
  4. Lear, J. D., Wasserman, Z. R. & DeGrado, W. F. Synthetic amphiphilic peptide models for protein ion channels. Science 240, 11771181 (1988).
  5. Joh, N. H. et al. De novo design of a transmembrane Zn2+-transporting four-helix bundle. Science 346, 15201524 (2014).
  6. Franceschini, L., Soskine, M., Biesemans, A. & Maglia, G. A nanopore machine promotes the vectorial transport of DNA across membranes. Nat. Commun. 4, 2415 (2013).
  7. Bayley, H. Membrane-protein structure: piercing insights. Nature 459, 651652 (2009).
  8. Dong, C. et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature 444, 226229 (2006).
  9. Kong, L. et al. Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor. Nat. Chem. 5, 651659 (2013).
  10. Soskine, M. et al. An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry. Nano Lett. 12, 48954900 (2012).
  11. Soskine, M., Biesemans, A., De Maeyer, M. & Maglia, G. Tuning the size and properties of ClyA nanopores assisted by directed evolution. J. Am. Chem. Soc. 135, 1345613463 (2013).
  12. Tanaka, K., Caaveiro, J. M., Morante, K., González-Mañas, J. M. & Tsumoto, K. Structural basis for self-assembly of a cytolytic pore lined by protein and lipid. Nat. Commun. 6, 6337 (2015).
  13. Zaccai, N. R. et al. A de novo peptide hexamer with a mutable channel. Nat. Chem. Biol. 7, 935941 (2011).
  14. Thomson, A. R. et al. Computational design of water-soluble alpha-helical barrels. Science 346, 485488 (2014).
  15. Bayley, H. Designed membrane channels and pores. Curr. Opin. Biotechnol. 10, 94103 (1999).
  16. Bayley, H. & Jayasinghe, L. Functional engineered channels and pores (Review). Mol. Membr. Biol. 21, 209220 (2004).
  17. Majd, S. et al. Applications of biological pores in nanomedicine, sensing, and nanoelectronics. Curr. Opin. Biotechnol. 21, 439476 (2010).
  18. Braha, O. et al. Designed protein pores as components for biosensors. Chem. Biol. 4, 497505 (1997).
  19. Bayley, H. & Cremer, P. S. Stochastic sensors inspired by biology. Nature 413, 226230 (2001).
  20. Bayley, H. Nanopore sequencing: from imagination to reality. Clin. Chem. 61, 2531 (2015).
  21. Jain, M. et al. Improved data analysis for the MinION nanopore sequencer. Nat. Methods 12, 351356 (2015).
  22. Song, L. et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 18591866 (1996).
  23. Gu, L. Q., Braha, O., Conlan, S., Cheley, S. & Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686690 (1999).
  24. Banerjee, A. et al. Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores. Proc. Natl Acad. Sci. USA 107, 81658170 (2010).
  25. Walshaw, J. & Woolfson, D. N. Socket: a program for identifying and analysing coiled-coil motifs within protein structures. J. Mol. Biol. 307, 14271450 (2001).
  26. van den Berg, B., Prathyusha Bhamidimarri, S., Dahyabhai Prajapati, J., Kleinekathöfer, U. & Winterhalter, M. Outer-membrane translocation of bulky small molecules by passive diffusion. Proc. Natl Acad. Sci. USA 112, E2991E2999 (2015).
  27. Doyle, D. A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 6977 (1998).
  28. Mueller, M., Grauschopf, U., Maier, T., Glockshuber, R. & Ban, N. The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism. Nature 459, 726730 (2009).
  29. Miles, G., Movileanu, L. & Bayley, H. Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore. Protein Sci. 11, 894902 (2002).
  30. Smart, O. S., Breed, J., Smith, G. R. & Sansom, M. S. A novel method for structure-based prediction of ion channel conductance properties. Biophys. J. 72, 11091126 (1997).
  31. Sukharev, S., Betanzos, M., Chiang, C. S. & Guy, H. R. The gating mechanism of the large mechanosensitive channel MscL. Nature 409, 720724 (2001).
  32. Wang, Y. et al. Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel. eLife 3, e01834 (2014).
  33. Pliotas, C. et al. The role of lipids in mechanosensation. Nat. Struct. Mol. Biol. 22, 991–8 (2015).
  34. Walker, B., Krishnasastry, M., Zorn, L. & Bayley, H. Assembly of the oligomeric membrane pore formed by staphylococcal alpha-hemolysin examined by truncation mutagenesis. J. Biol. Chem. 267, 217826 (1992).
  35. Walker, B., Braha, O., Cheley, S. & Bayley, H. An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch. Chem. Biol. 2, 99105 (1995).
  36. Dunstone, M. A. & Tweten, R. K. Packing a punch: the mechanism of pore formation by cholesterol dependent cytolysins and membrane attack complex/perforin-like proteins. Curr. Opin. Struct. Biol. 22, 342349 (2012).
  37. Leung, C. et al. Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin. eLife 3, e04247 (2014).
  38. Stoddart, D. et al. Functional truncated membrane pores. Proc. Natl Acad. Sci. USA 111, 24252430 (2014).
  39. Karginov, V. A. Cyclodextrin derivatives as anti-infectives. Curr. Opin. Pharmacol. 13, 717725 (2013).
  40. Brogden, K. A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3, 238250 (2005).
  41. Cirac, A. D. et al. The molecular basis for antimicrobial activity of pore-forming cyclic peptides. Biophys. J. 100, 24222431 (2011).
  42. Song, C. et al. Crystal structure and functional mechanism of a human antimicrobial membrane channel. Proc. Natl Acad. Sci. USA 110, 45864591 (2013).
  43. Haswell, E. S., Phillips, R. & Rees, D. C. Mechanosensitive channels: what can they do and how do they do it? Structure 19, 13561369 (2011).
  44. Naismith, J. H. & Booth, I. R. Bacterial mechanosensitive channels—MscS: evolution's solution to creating sensitivity in function. Annu. Rev. Biophys. 41, 157177 (2012).
  45. Lee, J. & Bayley, H. Semisynthetic protein nanoreactor for single-molecule chemistry. Proc. Natl Acad. Sci. USA 112, 1376813773 (2015).
  46. Fernandez-Lopez, S. et al. Antibacterial agents based on the cyclic D,L-alpha-peptide architecture. Nature 412, 452455 (2001).
  47. Fjell, C. D., Hiss, J. A., Hancock, R. E. & Schneider, G. Designing antimicrobial peptides: form follows function. Nat. Rev. Drug. Discov. 11, 3751 (2012).
  48. Hoskin, D. W. & Ramamoorthy, A. Studies on anticancer activities of antimicrobial peptides. Biochim. Biophys. Acta 1778, 357375 (2008).
  49. Gaspar, D., Veiga, A. S. & Castanho, M. A. From antimicrobial to anticancer peptides. A review. Front. Microbiol. 4, 294 (2013).
  50. Mantri, S., Tanuj Sapra, K., Cheley, S., Sharp, T. H. & Bayley, H. An engineered dimeric protein pore that spans adjacent lipid bilayers. Nat. Commun. 4, 1725 (2013).
  51. Gutsmann, T., Heimburg, T., Keyser, U., Mahendran, K. R. & Winterhalter, M. Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization. Nat. Protoc. 10, 188198 (2015).

Download references

Author information

  1. These authors contributed equally to this work

    • Kozhinjampara R. Mahendran &
    • Ai Niitsu

Affiliations

  1. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA UK

    • Kozhinjampara R. Mahendran,
    • Lingbing Kong &
    • Hagan Bayley
  2. School of Chemistry, Cantock's Close, University of Bristol, Bristol BS8 1TS, UK

    • Ai Niitsu,
    • Andrew R. Thomson &
    • Derek N. Woolfson
  3. School of Biochemistry, Biomedical Sciences Building, University of Bristol, Bristol BS8 1TD, UK

    • Richard B. Sessions &
    • Derek N. Woolfson
  4. BrisSynBio, Life Sciences Building, Tyndall Avenue, University of Bristol, Bristol BS8 1TQ, UK

    • Richard B. Sessions &
    • Derek N. Woolfson

Contributions

K.R.M. performed and analysed the current recordings. A.N. synthesized peptides and determined their biophysical properties. L.K. produced the Wza protein and synthesized the CD derivatives. A.N. and A.R.T. performed the molecular modelling. A.N. and R.B.S. performed the molecular dynamics simulations. K.R.M., A.N., D.N.W. and H.B. designed experiments and wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary information (3,917 KB)

    Supplementary information

Additional data