Abstract
Dynamic disorder of the lipid bilayer presents a challenge for establishing structure–function relationships in membranous systems. The resulting structural heterogeneity is especially evident for peripheral and spontaneously inserting membrane proteins, which are not constrained by the well-defined transmembrane topology and exert their action in the context of intimate interaction with lipids. Here, we propose a concerted approach combining depth-dependent fluorescence quenching with Molecular Dynamics simulation to decipher dynamic interactions of membrane proteins with the lipid bilayers. We apply this approach to characterize membrane-mediated action of the diphtheria toxin translocation domain. First, we use a combination of the steady-state and time-resolved fluorescence spectroscopy to characterize bilayer penetration of the NBD probe selectively attached to different sites of the protein into membranes containing lipid-attached nitroxyl quenching groups. The constructed quenching profiles are analyzed with the Distribution Analysis methodology allowing for accurate determination of transverse distribution of the probe. The results obtained for 12 NBD-labeled single-Cys mutants are consistent with the so-called Open-Channel topology model. The experimentally determined quenching profiles for labeling sites corresponding to L350, N373, and P378 were used as initial constraints for positioning TH8–9 hairpin into the lipid bilayer for Molecular Dynamics simulation. Finally, we used alchemical free energy calculations to characterize protonation of E362 in soluble translocation domain and membrane-inserted conformation of its TH8–9 fragment. Our results indicate that membrane partitioning of the neutral E362 is more favorable energetically (by ~ 6 kcal/mol), but causes stronger perturbation of the bilayer, than the charged E362.
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Abbreviations
- Tempo-PC:
-
1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho(TEMPO)choline
- n-Doxyl-PC:
-
1-Palmitoyl-2-stearoyl-(n-Doxyl)-sn-glycero-3-phosphocholine
- POPC:
-
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
- POPG:
-
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
- LUV:
-
Large unillamelar vesicles
- MD:
-
Molecular dynamics
- DA:
-
Distribution analysis
- QP:
-
Quenching profile
References
Bartlett GR (1959) Phosphorus assay in column chromatography. J Biol Chem 234:466–468
Bennett CH (1976) Efficient estimation of free energy differences from Monte Carlo data. J Comput Phys 22:245–268
Bennett MJ, Choe S, Eisenberg D (1994) Refined structure of dimeric diphtheria toxin at 2.0 Å resolution. Protein Sci 3:1444–1463
Chodera JD, Swope WC, Pitera JW, Seok C, Dill KA (2007) Use of the weighted histogram analysis method for the analysis of simulated and parallel tempering simulations. J Chem Theor Comput 3:26–41
Choe S, Bennett MJ, Fujii G, Curmi PMG, Kantardjieff KA, Collier RJ, Eisenberg D (1992) The crystal structure of diphtheria toxin. Nature 357:216–222
Delbridge AR, Grabow S, Strasser A, Vaux DL (2016) Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies. Nat Rev Cancer 16:99–109
Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103:8577–8593
Feller SE, Zhang Y, Pastor RW, Brooks BR (1995) Constant pressure molecular dynamics simulation: the Langevin piston method. J Chem Phys 103:4613–4621
Ghatak C, Rodnin MV, Vargas-Uribe M, McCluskey AJ, Flores-Canales JC, Kurnikova M, Ladokhin AS (2015) Role of Acidic residues in helices TH8–TH9 in membrane interactions of the diphtheria toxin T domain. Toxins 7:1303–1323
Grubmüller H, Heller H, Windemuth A, Schulten K (1991) Generalized Verlet algorithm for efficient molecular dynamics simulations with long-range interactions. Mol Simul 6:121–142
Gumbart J, Roux B (2012) Determination of membrane-insertion free energies by molecular dynamics simulations. Biophys J 102:795–801
Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14:33–38
Jo S, Lim JB, Klauda JB, Im W (2009) CHARMM-GUI membrane builder for mixed bilayers and its application to yeast membranes. Biophys J 97:50–58
Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926–935
Kachel K, Ren JH, Collier RJ, London E (1998) Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain. J Biol Chem 273:22950–22956
Klauda JB, Venable RM, Freites JA, O’Connor JW, Tobias DJ, Mondragon-Ramirez C, Vorobyov I, MacKerell AD Jr, Pastor RW (2010) Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J Phys Chem B 114:7830–7843
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
Kyrychenko A, Freites JA, He J, Tobias DJ, Wimley WC, Ladokhin AS (2014a) Structural plasticity in the topology of the membrane-interacting domain of HIV-1 gp41. Biophys J 106:610–620
Kyrychenko A, Ladokhin AS (2013) Molecular dynamics simulations of depth distribution of spin-labeled phospholipids within lipid bilayer. J Phys Chem B 117:5875–5885
Kyrychenko A, Ladokhin AS (2014) Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching. Anal Biochem 446:19–21
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
Kyrychenko A, Posokhov YO, Vargas-Uribe M, Ghatak C, Rodnin MV, Ladokhin AS (2017) Fluorescence applications for structural and thermodynamic studies of membrane protein insertion. In: Geddes CD (ed) Reviews in fluorescence 2016. Springer, New York, pp 243–274
Kyrychenko A, Rodnin MV, Ladokhin AS (2014b) Calibration of distribution analysis of the depth of membrane penetration using simulations and depth-dependent fluorescence quenching. J Membr Biol 248(3):583–594
Kyrychenko A, Tobias DJ, Ladokhin AS (2013) Validation of depth-dependent fluorescence quenching in membranes by molecular dynamics simulation of tryptophan octyl ester in POPC bilayer. J Phys Chem B 117:4770–4778
Ladokhin AS (1997) Distribution analysis of depth-dependent fluorescence quenching in membranes: a practical guide. Methods Enzymol 278:462–473
Ladokhin AS (1999) Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: theoretical considerations. Biophys J 76:946–955
Ladokhin AS (2013) pH-triggered conformational switching along the membrane insertion pathway of the diphtheria toxin T-domain. Toxins 5:1362–1380
Ladokhin AS (2014) Measuring membrane penetration with depth-dependent fluorescence quenching: distribution analysis is coming of age. Biochim et Biophys Acta 1838:2289–2295
Ladokhin SA, Vargas-Uribe M, Rodnin VM, Ghatak C, Sharma O (2017) Cellular entry of the diphtheria toxin does not require the formation of the open-channel state by its translocation domain. Toxins 9(10):299
Leber B, Lin J, Andrews DW (2010) Still embedded together binding to membranes regulates Bcl-2 protein interactions. Oncogene 29:5221–5230
MacKerell AD, Feig M, Brooks CL (2004) 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
MacKerell AD Jr, Bashford D, Bellott M, Dunbrack RL Jr, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph-McCarthy D, Kuchnir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Reiher WE III, Roux B, Schlenkrich M, Smith JC, Stote R, Straub J, Watanabe M, Wiórkiewicz-Kuczera J, Yin D, Karplus M (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102:3586–3616
Malenbaum SE, Collier RJ, London E (1998) Membrane topography of the T domain of diphtheria toxin probed with single tryptophan mutants. Biochemistry 37:17915–17922
Mansoor SE, DeWitt MA, Farrens DL (2010) Distance mapping in proteins using fluorescence spectroscopy: the tryptophan-induced quenching (TrIQ) method. Biochemistry 49:9722–9731
Martyna GJ, Tobias DJ, Klein ML (1994) Constant-pressure molecular-dynamics algorithms. J Chem Phys 101:4177–4189
Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161–168
McGibbon RT, Beauchamp KA, Harrigan MP, Klein C, Swails JM, Hernández CX, Schwantes CR, Wang L-P, Lane TJ, Pande VS (2015) MDTraj: a modern open library for the analysis of molecular dynamics trajectories. Biophys J 109:1528–1532
Miyamoto S, Kollman PA (1992) Settle: an analytical version of the SHAKE and RATTLE algorithm for rigid water models. J Comput Chem 13:952–962
Moldoveanu T, Follis AV, Kriwacki RW, Green DR (2014) Many players in BCL-2 family affairs. Trends Biochem Sci 39:101–111
Montgomery DC, Peck EA (1982) Introduction to linear regression analysis. Wiley, New York
Oh KJ, Zhan H, Cui C, Hideg K, Collier RJ, Hubbell WL (1996) Organization of diphtheria toxin T domain in bilayers: a site-directed spin labeling study. Science 273:810–812
Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kalé L, Schulten K (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26:1781–1802
Posokhov YO, Ladokhin AS (2006) Lifetime fluorescence method for determining membrane topology of proteins. Anal Biochem 348:87–93
Raunest M, Kandt C (2011) dxTuber: detecting protein cavities, tunnels and clefts based on protein and solvent dynamics. J Mol Graph Model 29:895–905
Rocklin GJ, Mobley DL, Dill KA, Hunenberger PH (2013) Calculating the binding free energies of charged species based on explicit-solvent simulations employing lattice-sum methods: an accurate correction scheme for electrostatic finite-size effects. J Chem Phys 139:184103
Rodnin MV, Li J, Gross ML, Ladokhin AS (2016) The pH-dependent trigger in diphtheria toxin T domain comes with a safety latch. Biophys J 111:1946–1953
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
Rosconi MP, London E (2002) Topography of helices 5–7 in membrane-inserted diphtheria toxin T domain: identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin. J Biol Chem 277:16517–16527
Ryckaert J-P, Ciccotti G, Berendsen HJC (1977) Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys 23:327–341
Senzel L, Gordon M, Blaustein RO, Oh KJ, Collier RJ, Finkelstein A (2000) Topography of diphtheria toxin’s T domain in the open channel state. J Gen Physiol 115:421–434
Shirts MR, Chodera JD (2008) Statistically optimal analysis of samples from multiple equilibrium states. J Chem Phys 129:124105
Vargas-Uribe M, Rodnin MV, Kienker P, Finkelstein A, Ladokhin AS (2013) Crucial role of H322 in folding of the diphtheria toxin T-Domain into the open-channel state. Biochemistry 52:3457–3463
Wang Y, Malenbaum SE, Kachel K, Zhan HJ, 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
Acknowledgements
This research was supported in part by National Institutes of Health Grant P30-GM110761. A.K. also acknowledges support of Grant 0116U000835 of Ministry of Education and Science of Ukraine.
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Kyrychenko, A., Lim, N.M., Vasquez-Montes, V. et al. Refining Protein Penetration into the Lipid Bilayer Using Fluorescence Quenching and Molecular Dynamics Simulations: The Case of Diphtheria Toxin Translocation Domain. J Membrane Biol 251, 379–391 (2018). https://doi.org/10.1007/s00232-018-0030-2
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DOI: https://doi.org/10.1007/s00232-018-0030-2