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)
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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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
Kyrychenko, Alexander; Rodnin, Mykola V; Ladokhin, Alexey S (2015) Calibration of Distribution Analysis of the Depth of Membrane Penetration Using Simulations and Depth-Dependent Fluorescence Quenching. J Membr Biol 248:583-94
Kyrychenko, Alexander; Freites, J Alfredo; He, Jing et al. (2014) Structural plasticity in the topology of the membrane-interacting domain of HIV-1 gp41. Biophys J 106:610-20
Kyrychenko, Alexander; Ladokhin, Alexey S (2014) Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching. Anal Biochem 446:19-21
Li, Jing; Rodnin, Mykola V; Ladokhin, Alexey S et al. (2014) Hydrogen-deuterium exchange and mass spectrometry reveal the pH-dependent conformational changes of diphtheria toxin T domain. Biochemistry 53:6849-56
Alhoshani, Ali; Vithayathil, Rosemarie; Bandong, Jonathan et al. (2014) Glutamate provides a key structural contact between reticulon-4 (Nogo-66) and phosphocholine. Biochim Biophys Acta 1838:2350-6
Ladokhin, Alexey S (2014) Measuring membrane penetration with depth-dependent fluorescence quenching: distribution analysis is coming of age. Biochim Biophys Acta 1838:2289-95
Kyrychenko, Alexander; Tobias, Douglas J; Ladokhin, Alexey S (2013) Validation of depth-dependent fluorescence quenching in membranes by molecular dynamics simulation of tryptophan octyl ester in POPC bilayer. J Phys Chem B 117:4770-8
Ladokhin, Alexey S (2013) pH-triggered conformational switching along the membrane insertion pathway of the diphtheria toxin T-domain. Toxins (Basel) 5:1362-80
Kyrychenko, Alexander; Ladokhin, Alexey S (2013) Molecular dynamics simulations of depth distribution of spin-labeled phospholipids within lipid bilayer. J Phys Chem B 117:5875-85

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