This project is focused on deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the diphtheria toxin T-domain (DTT), which is considered to be a paradigm for cell entry of other toxins (e.g., tetanus and botulinum) and has a potential for targeted delivery of anti-cancer therapies. The pH-triggered insertion of DTT will also reveal general physicochemical principles underlying membrane protein assembly and signalyng on membrane interfaces. This first competing renewal of the project will capitalize on our progress in identifying key intermediate states along the insertion pathway, in establishing the concept of conformational switching for DTT action and in developing new methodologies for structural, kinetic and thermodynamic characterization of membrane protein refolding/insertion. The innovation of this proposal resides in the unique way that molecular dynamics (MD) simulations and sophisticated spectroscopic experiments will be brought together in order to understand molecular mechanisms which will bring clarity to a complex field. MD simulations will be used for (a) building atomic models consistent with low resolution spectroscopic data, and (b) guiding the experimental design to further verify them. Site-specific labeling of single-cysteine mutants and a battery of spectroscopic approaches (including FCS, fluorescence lifetime quenching, FRET, stopped-flow kinetic measurements) will be utilized to test the interface-directed refolding/insertion hypothesis, which assigns a special role to the bilayer interfacial region in modulating transmembrane insertion by assisting the formation of key intermediate states, shifting the balance of electrostatic and hydrophobic interactions and altering protonation properties of titratable residues. The nature of the conformational switching resulting in refolding, insertion and translocation transitions of DTT will be established through mutagenesis of His, Asp and Glu residues, guided by Thermodynamic Integration calculations. Various DTT mutants will be used to ascertain whether protonation of histidines assists in the unfolding of the protein in solution and promotes formation of a previously identified insertion-competent intermediate on the membrane interface, through electrostatic interactions with anionic lipids, while protonation of acidic residues enables transmembrane insertion. To gain insights into the pH-triggered membrane action of DTT, thus establishing the general physicochemical principles of membrane-protein interactions, we will pursue the following goals: (1) determine molecular details of the structural organization of key intermediate and final inserted states;(2) determine the free energy profile of transitions along the insertion pathway and determine how the properties of the bilayer modulate structural, thermodynamic and kinetic parameters of the DTT insertion;and (3) identify key residues responsible for pH-triggered functional conformational switching.

Public Health Relevance

The project deals with the mechanism of bacterial toxin insertion into and translocation across the membranes, which are fundamental unanswered questions in cell biology. It also relates to (a) potential targeted cellular delivery of molecular therapy and (b) deciphering the membrane protein folding and stability problem, related to our understanding of molecular mechanisms of misfolding-associated diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
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University of Kansas
Schools of Medicine
Kansas City
United States
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Gnanasambandam, Radhakrishnan; Ghatak, Chiranjib; Yasmann, Anthony et al. (2017) GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels. Biophys J 112:31-45
Rodnin, Mykola V; Li, Jing; Gross, Michael L et al. (2016) The pH-Dependent Trigger in Diphtheria Toxin T Domain Comes with a Safety Latch. Biophys J 111:1946-1953
Ladokhin, Alexey S (2015) Membrane Protein Folding & Lipid Interactions: Theory & Experiment. J Membr Biol 248:369-70
Ghatak, Chiranjib; Rodnin, Mykola V; Vargas-Uribe, Mauricio et al. (2015) Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain. Toxins (Basel) 7:1303-23
Vargas-Uribe, Mauricio; Rodnin, Mykola V; Öjemalm, Karin et al. (2015) Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain. J Membr Biol 248:383-94
Flores-Canales, Jose C; Vargas-Uribe, Mauricio; Ladokhin, Alexey S et al. (2015) Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment. J Membr Biol 248:529-43
Flores-Canales, Jose C; Kurnikova, Maria (2015) Targeting electrostatic interactions in accelerated molecular dynamics with application to protein partial unfolding. J Chem Theory Comput 11:2550-9
Flores-Canales, Jose C; Kurnikova, Maria (2015) Microsecond Simulations of the Diphtheria Toxin Translocation Domain in Association with Anionic Lipid Bilayers. J Phys Chem B 119:12074-85
Kyrychenko, Alexander; Vasquez-Montes, Victor; Ulmschneider, Martin B et al. (2015) Lipid headgroups modulate membrane insertion of pHLIP peptide. Biophys J 108:791-4
Rodnin, Mykola V; Ladokhin, Alexey S (2015) Membrane translocation assay based on proteolytic cleavage: application to diphtheria toxin T domain. Biochim Biophys Acta 1848:35-40

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