Limits...
Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

Ghatak C, Rodnin MV, Vargas-Uribe M, McCluskey AJ, Flores-Canales JC, Kurnikova M, Ladokhin AS - Toxins (Basel) (2015)

Bottom Line: Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts.Both mutants adopt a final functional state upon further acidification.We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. c.ghatak79@gmail.com.

ABSTRACT
The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

Show MeSH

Related in: MedlinePlus

Crystal structure of the diphtheria toxin translocation domain (T domain) in solution at neutral pH [10], highlighting hydrophobic helices TH8 and TH9 as dark yellow ribbons. Acidic residues mutated in this study are shown as CPK representations in blue and red.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4417968&req=5

toxins-07-01303-f001: Crystal structure of the diphtheria toxin translocation domain (T domain) in solution at neutral pH [10], highlighting hydrophobic helices TH8 and TH9 as dark yellow ribbons. Acidic residues mutated in this study are shown as CPK representations in blue and red.

Mentions: The crystal structure of the T domain in solution at neutral pH [10] consists of a globular protein of nine α-helices (named TH1-TH9) of various lengths (see Figure 1), with the most hydrophobic helices, TH8 and TH9 (Figure 1, dark yellow helices), forming the hydrophobic core of the protein. There is no high-resolution structure available for the T domain in its membrane-inserted state, but several studies have demonstrated that helices TH8–TH9 insert as a transmembrane hairpin, while other helices can adopt multiple conformations [11,12,13,14,15,16,17]. Previously, we have characterized the kinetic membrane insertion pathway [18], which comprises a series of conformational transitions occurring first in solution and then in the membrane. We have also determined the free energy stabilizing the different intermediates [19,20] along the pathway.


Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

Ghatak C, Rodnin MV, Vargas-Uribe M, McCluskey AJ, Flores-Canales JC, Kurnikova M, Ladokhin AS - Toxins (Basel) (2015)

Crystal structure of the diphtheria toxin translocation domain (T domain) in solution at neutral pH [10], highlighting hydrophobic helices TH8 and TH9 as dark yellow ribbons. Acidic residues mutated in this study are shown as CPK representations in blue and red.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4417968&req=5

toxins-07-01303-f001: Crystal structure of the diphtheria toxin translocation domain (T domain) in solution at neutral pH [10], highlighting hydrophobic helices TH8 and TH9 as dark yellow ribbons. Acidic residues mutated in this study are shown as CPK representations in blue and red.
Mentions: The crystal structure of the T domain in solution at neutral pH [10] consists of a globular protein of nine α-helices (named TH1-TH9) of various lengths (see Figure 1), with the most hydrophobic helices, TH8 and TH9 (Figure 1, dark yellow helices), forming the hydrophobic core of the protein. There is no high-resolution structure available for the T domain in its membrane-inserted state, but several studies have demonstrated that helices TH8–TH9 insert as a transmembrane hairpin, while other helices can adopt multiple conformations [11,12,13,14,15,16,17]. Previously, we have characterized the kinetic membrane insertion pathway [18], which comprises a series of conformational transitions occurring first in solution and then in the membrane. We have also determined the free energy stabilizing the different intermediates [19,20] along the pathway.

Bottom Line: Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts.Both mutants adopt a final functional state upon further acidification.We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. c.ghatak79@gmail.com.

ABSTRACT
The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

Show MeSH
Related in: MedlinePlus