Skip to main content
SpringerLink
Log in

Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment

  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

Diphtheria toxin translocation (T) domain inserts in lipid bilayers upon acidification of the environment. Computational and experimental studies have suggested that low pH triggers a conformational change of the T-domain in solution preceding membrane binding. The refolded membrane-competent state was modeled to be compact and mostly retain globular structure. In the present work, we investigate how this refolded state interacts with membrane interfaces in the early steps of T-domain’s membrane association. Coarse-grained molecular dynamics simulations suggest two distinct membrane-bound conformations of the T-domain in the presence of bilayers composed of a mixture of zwitteronic and anionic phospholipids (POPC:POPG with a 1:3 molar ratio). Both membrane-bound conformations show a common near parallel orientation of hydrophobic helices TH8–TH9 relative to the membrane plane. The most frequently observed membrane-bound conformation is stabilized by electrostatic interactions between the N-terminal segment of the protein and the membrane interface. The second membrane-bound conformation is stabilized by hydrophobic interactions between protein residues and lipid acyl chains, which facilitate deeper protein insertion in the membrane interface. A theoretical estimate of a free energy of binding of a membrane-competent T-domain to the membrane is provided.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

T-domain:

Diphtheria toxin translocation domain

CG:

Coarse-grained

MD:

Molecular dynamics

POPC:

1-Palmitoyl-2-oleoyl-phosphatidyl-choline

POPG:

1-Palmitoyl-2-oleoyl-phosphatidyl-glycerol

COM:

Center of mass

PMF:

Potential of mean force

K :

Binding constant

K x :

Partitioning coefficient

ΔG x :

Partitioning free energy

LUV:

Large unilamellar vesicles

W+ :

Membrane competent state in low pH solution

References

  • Bennett MJ, Eisenberg D (1994) Refined structure of monomeric diphtheria toxin at 2.3 A resolution. Protein Sci 3:1464–1475

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chenal A, Savarin P, Nizard P, Guillain F, Gillet D, Forge V (2002) Membrane protein insertion regulated by bringing electrostatic and hydrophobic interactions into play. A case study with the translocation domain of diphtheria toxin. J Biol Chem 277:43425–43432

    Article  CAS  PubMed  Google Scholar 

  • Chenal A, Prongidi-Fix L, Perier A, Aisenbrey C, Vernier G, Lambotte S, Haertlein M, Dauvergne MT, Fragneto G, Bechinger B, Gillet D, Forge V, Ferrand M (2009) Deciphering membrane insertion of the diphtheria toxin T domain by specular neutron reflectometry and solid-state NMR spectroscopy. J Mol Biol 391:872–883

    Article  CAS  PubMed  Google Scholar 

  • Choe S, Bennett MJ, Fujii G, Curmi PM, Kantardjieff KA, Collier RJ, Eisenberg D (1992) The crystal structure of diphtheria toxin. Nature 357:216–222

    Article  CAS  PubMed  Google Scholar 

  • Haugland RP (1996) Handbook of fluorescent probes and research chemicals. Molecular Probes Inc, Eugene

    Google Scholar 

  • Hess B, Kutzner C, van der Spoel D, Lindahl E (2008) GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4:435–447

    Article  CAS  Google Scholar 

  • Honjo T, Nishizuka Y, Hayaishi O (1968) Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J Biol Chem 243:3553–3555

    CAS  PubMed  Google Scholar 

  • Hope MJ, Bally MB, Mayer LD, Janoff AS, Cullis PR (1986) Generation of multilamellar and unilamellar phospholipid vesicles. Chem Phys Lipids 40:89–107

    Article  Google Scholar 

  • Hub JS, de Groot BL, van der Spoel D (2010) g_wham—a free weighted histogram analysis implementation including robust error and autocorrelation estimates. J Chem Theory Comput 6:3713–3720

    Article  CAS  Google Scholar 

  • Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph Model 14:33–38

    Article  CAS  Google Scholar 

  • Kalli AC, Wegener KL, Goult BT, Anthis NJ, Campbell ID, Sansom MSP (2010) The structure of the talin/integrin complex at a lipid bilayer: an NMR and MD simulation study. Structure 18:1280–1288

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kalli AC, Campbell ID, Sansom MS (2011) Multiscale simulations suggest a mechanism for integrin inside-out activation. Proc Natl Acad Sci USA 108:11890–11895

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kumar S, Bouzida D, Swendsen RH, Kollman PA, Rosenberg JM (1992) The weighted histogram analysis method for free-energy calculations on biomolecules. 1. The method. J Comput Chem 13:1011–1021

    Article  CAS  Google Scholar 

  • Kurnikov IV, Kyrychenko A, Flores-Canales JC, Rodnin MV, Simakov N, Vargas-Uribe M, Posokhov YO, Kurnikova M, Ladokhin AS (2013) pH-triggered conformational switching of the diphtheria toxin T-domain: the roles of N-terminal histidines. J Mol Biol 425:2752–2764

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kyrychenko A, Posokhov YO, Rodnin MV, Ladokhin AS (2009) Kinetic intermediate reveals staggered pH-dependent transitions along the membrane insertion pathway of the diphtheria toxin T-domain. Biochemistry 48:7584–7594

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ladokhin AS (2009) Fluorescence spectroscopy in thermodynamic and kinetic analysis of pH-dependent membrane protein insertion. Methods Enzymol 466:19–42

    Article  CAS  PubMed  Google Scholar 

  • Ladokhin AS (2013) pH-triggered conformational switching along the membrane insertion pathway of the diphtheria toxin T-domain. Toxins (Basel) 5:1362–1380

    Article  CAS  Google Scholar 

  • Ladokhin AS, Isas JM, Haigler HT, White SH (2002) Determining the membrane topology of proteins: insertion pathway of a transmembrane helix of annexin 12. Biochemistry 41:13617–13626

    Article  CAS  PubMed  Google Scholar 

  • Ladokhin AS, Legmann R, Collier RJ, White SH (2004) Reversible refolding of the diphtheria toxin T-domain on lipid membranes. Biochemistry 43:7451–7458

    Article  CAS  PubMed  Google Scholar 

  • Leka O, Vallese F, Pirazzini M, Berto P, Montecucco C, Zanotti G (2014) Diphtheria toxin conformational switching at acidic pH. FEBS J 281:2115–2122

    Article  CAS  PubMed  Google Scholar 

  • Marrink SJ, Tieleman DP (2013) Perspective on the Martini model. Chem Soc Rev 42:6801–6822

    Article  CAS  PubMed  Google Scholar 

  • Marrink SJ, Risselada HJ, Yefimov S, Tieleman DP, de Vries AH (2007) The MARTINI force field: coarse grained model for biomolecular simulations. J Phys Chem B 111:7812–7824

    Article  CAS  PubMed  Google Scholar 

  • Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161–168

    Article  CAS  PubMed  Google Scholar 

  • Montagner C, Perier A, Pichard S, Vernier G, Menez A, Gillet D, Forge V, Chenal A (2007) Behavior of the N-terminal helices of the diphtheria toxin T domain during the successive steps of membrane interaction. Biochemistry 46:1878–1887

    Article  CAS  PubMed  Google Scholar 

  • Monticelli L, Kandasamy SK, Periole X, Larson RG, Tieleman DP, Marrink SJ (2008) The MARTINI coarse-grained force field: extension to proteins. J Chem Theory Comput 4:819–834

    Article  CAS  Google Scholar 

  • Murphy JR (2011) Mechanism of diphtheria toxin catalytic domain delivery to the eukaryotic cell cytosol and the cellular factors that directly participate in the process. Toxins (Basel) 3:294–308

    Article  CAS  Google Scholar 

  • Palchevskyy SS, Posokhov YO, Olivier B, Popot JL, Pucci B, Ladokhin AS (2006) Chaperoning of insertion of membrane proteins into lipid bilayers by hemifluorinated surfactants: application to diphtheria toxin. Biochemistry 45:2629–2635

    Article  CAS  PubMed  Google Scholar 

  • Perier A, Chassaing A, Raffestin S, Pichard S, Masella M, Menez A, Forge V, Chenal A, Gillet D (2007) Concerted protonation of key histidines triggers membrane interaction of the diphtheria toxin T domain. J Biol Chem 282:24239–24245

    Article  CAS  PubMed  Google Scholar 

  • Periole X, Cavalli M, Marrink SJ, Ceruso MA (2009) Combining an elastic network with a coarse-grained molecular force field: structure, dynamics, and intermolecular recognition. J Chem Theory Comput 5:2531–2543

    Article  CAS  Google Scholar 

  • Posokhov YO, Ladokhin AS (2006) Lifetime fluorescence method for determining membrane topology of proteins. Anal Biochem 348:87–93

    Article  CAS  PubMed  Google Scholar 

  • Posokhov YO, Rodnin MV, Das SK, Pucci B, Ladokhin AS (2008a) FCS study of the thermodynamics of membrane protein insertion into the lipid bilayer chaperoned by fluorinated surfactants. Biophys J 95:L54–L56

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Posokhov YO, Rodnin MV, Lu L, Ladokhin AS (2008b) Membrane insertion pathway of annexin B12: thermodynamic and kinetic characterization by fluorescence correlation spectroscopy and fluorescence quenching. Biochemistry 47:5078–5087

    Article  CAS  PubMed  Google Scholar 

  • Rodnin MV, Posokhov YO, Contino-Pepin C, Brettmann J, Kyrychenko A, Palchevskyy SS, Pucci B, Ladokhin AS (2008) Interactions of fluorinated Surfactants with diphtheria toxin T-domain: testing new media for studies of membrane proteins. Biophys J 94:4348–4357

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rodnin MV, Kyrychenko A, Kienker P, Sharma O, Posokhov YO, Collier RJ, Finkelstein A, Ladokhin AS (2010) Conformational switching of the diphtheria toxin T domain. J Mol Biol 402:1–7

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rodnin MV, Kyrychenko A, Kienker P, Sharma O, Vargas-Uribe M, Collier RJ, Finkelstein A, Ladokhin AS (2011) Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain. Biophys J 101:L41–L43

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Roux B (1995) The calculation of the potential of mean force using computer-simulations. Comput Phys Commun 91:275–282

    Article  CAS  Google Scholar 

  • Stansfeld PJ, Sansom MS (2011) Molecular simulation approaches to membrane proteins. Structure 19:1562–1572

    Article  CAS  PubMed  Google Scholar 

  • Steere B (2001) Characterization of high-order oligomerization and energetics in diphtheria toxin. University of California, Los Angeles

    Google Scholar 

  • Vargas-Uribe M, Rodnin MV, Kienker P, Finkelstein A, Ladokhin AS (2013a) Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state. Biochemistry 52:3457–3463

    Article  CAS  PubMed  Google Scholar 

  • Vargas-Uribe M, Rodnin MV, Ladokhin AS (2013b) Comparison of membrane insertion pathways of the apoptotic regulator Bcl-xL and the diphtheria toxin translocation domain. Biochemistry 52:7901–7909

    Article  CAS  PubMed  Google Scholar 

  • Vargas-Uribe M, Rodnin MV, Öjemalm K, Holgado A, Kyrychenko A, Nilsson I, Posokhov YO, Makhatadze G, Von Heijne G, Ladokhin AS (2014) Thermodynamics of membrane insertion and refolding of the diphtheria toxin T-domain. J Membrane Biol. doi:10.1007/s00232-014-9734-0

  • Vivcharuk V, Tomberli B, Tolokh IS, Gray CG (2008) Prediction of binding free energy for adsorption of antimicrobial peptide lactoferricin B on a POPC membrane. Phys Rev E 77:031913

    Article  Google Scholar 

  • Wang Y, Malenbaum SE, Kachel K, Zhan H, Collier RJ, London E (1997) Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width. J Biol Chem 272:25091–25098

    Article  CAS  PubMed  Google Scholar 

  • Wee CL, Ulmschneider MB, Sansom MSP (2010) Membrane/toxin interaction energetics via serial multiscale molecular dynamics simulations. J Chem Theory Comput 6:966–976

    Article  CAS  Google Scholar 

  • Zhan HJ, Choe S, Huynh PD, Finkelstein A, Eisenberg D, Collier RJ (1994) Dynamic transitions of the transmembrane domain of diphtheria-toxin—disulfide trapping and fluorescence proximity studies. Biochemistry 33:11254–11263

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This research was supported by the National Institutes of Health Grant GM-069783. Computational work was performed under XSEDE Grant TGMCB040051N.

Author information

Authors and Affiliations

  1. Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, 15213, USA

    Jose C. Flores-Canales & Maria Kurnikova

  2. Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, Kansas City, KS, 66160, USA

    Mauricio Vargas-Uribe & Alexey S. Ladokhin

Authors
  1. Jose C. Flores-Canales
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mauricio Vargas-Uribe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexey S. Ladokhin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Kurnikova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria Kurnikova.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 2271 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Flores-Canales, J.C., Vargas-Uribe, M., Ladokhin, A.S. et al. Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment. J Membrane Biol 248, 529–543 (2015). https://doi.org/10.1007/s00232-015-9771-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00232-015-9771-3

Keywords

Search

Navigation