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Hydrogen-deuterium exchange and mass spectrometry reveal the pH-dependent conformational changes of diphtheria toxin T domain.

Li J, Rodnin MV, Ladokhin AS, Gross ML - Biochemistry (2014)

Bottom Line: Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form.At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8-9, leading to subsequent aggregation in the absence of membranes.This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Washington University , St. Louis, Missouri 63130, United States.

ABSTRACT
The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our "standard condition" (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W(+)-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8-9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8-9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain.

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HDX kinetic curves of peptides HDX under all pH conditions.HDXkinetic curves show three characteristic patterns: (1) Low D% at pH ≥ 6.5, high D% at pH ≤5.5, and intermediate D% at pH 6, (a–h, j–k);(2) no differences for all pH conditions, (i,o); (3) increase in D% when increasing pH to 6.5, protection in HDX at pH ≤5.5, (l–n). All time points were corrected to standard conditionat pH 7.5 and 4 °C. Secondary features were added to all kineticcurves: for example, a) TH1 represents helix1 in T domain; (i) solidline represents loop between helix 7 and 8.
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fig2: HDX kinetic curves of peptides HDX under all pH conditions.HDXkinetic curves show three characteristic patterns: (1) Low D% at pH ≥ 6.5, high D% at pH ≤5.5, and intermediate D% at pH 6, (a–h, j–k);(2) no differences for all pH conditions, (i,o); (3) increase in D% when increasing pH to 6.5, protection in HDX at pH ≤5.5, (l–n). All time points were corrected to standard conditionat pH 7.5 and 4 °C. Secondary features were added to all kineticcurves: for example, a) TH1 represents helix1 in T domain; (i) solidline represents loop between helix 7 and 8.

Mentions: The shifts in the centroid mass of isotopicpatterns of peptic peptides at all on-exchange time points under sixpH conditions were measured, and the deuterium incorporation percentagewas plotted as a function of incubation time (after applying the pH-dependenttime correction). A complete set of deuterium incorporation plotsare shown in Supporting Information Figure S1, and selected deuterium incorporation plots are shown in Figure 2. Upon close inspection of the selected pH-dependentdeuterium incorporation plots, we found three distinct HDX patterns:(1) N-terminal and middle region helices TH1–8 (Figure 2a–h,j–k), (2) C-terminal interhelicalloop TL8–9 and hydrophobic TH9 (Figure 2l–n), and (3) unstructured loops TL7–8 and TL9 (Figure 2i and o). For the first pattern, all helices remainfolded in their native state (W-state) between pH 7.5 and pH 6.5 (blue,cyan, and green curves), but become flexible and undergo two conformationaltransitions at pH 6.0 (yellow curve) and pH 5.5 (orange curves), characterizedby two major increases in HDX. Other studies reported that, betweenpH 7.5 and pH 6.0, the T domain undergoes conformational rearrangementinto a more solvent-exposed membrane competent (W+) state,4,15,34 and our results are consistentwith those outcomes. Interestingly, the transition midpoint betweenthe native and the membrane-competent state was calculated to be pH6.2,2,4,15 a pH conditionclose to one used in this study (pH 6.0). Thus, one may infer thatthe extent of HDX represents the combined structural dynamics of bothnative conformation (W) and the membrane-competent (W+)conformation.


Hydrogen-deuterium exchange and mass spectrometry reveal the pH-dependent conformational changes of diphtheria toxin T domain.

Li J, Rodnin MV, Ladokhin AS, Gross ML - Biochemistry (2014)

HDX kinetic curves of peptides HDX under all pH conditions.HDXkinetic curves show three characteristic patterns: (1) Low D% at pH ≥ 6.5, high D% at pH ≤5.5, and intermediate D% at pH 6, (a–h, j–k);(2) no differences for all pH conditions, (i,o); (3) increase in D% when increasing pH to 6.5, protection in HDX at pH ≤5.5, (l–n). All time points were corrected to standard conditionat pH 7.5 and 4 °C. Secondary features were added to all kineticcurves: for example, a) TH1 represents helix1 in T domain; (i) solidline represents loop between helix 7 and 8.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222528&req=5

fig2: HDX kinetic curves of peptides HDX under all pH conditions.HDXkinetic curves show three characteristic patterns: (1) Low D% at pH ≥ 6.5, high D% at pH ≤5.5, and intermediate D% at pH 6, (a–h, j–k);(2) no differences for all pH conditions, (i,o); (3) increase in D% when increasing pH to 6.5, protection in HDX at pH ≤5.5, (l–n). All time points were corrected to standard conditionat pH 7.5 and 4 °C. Secondary features were added to all kineticcurves: for example, a) TH1 represents helix1 in T domain; (i) solidline represents loop between helix 7 and 8.
Mentions: The shifts in the centroid mass of isotopicpatterns of peptic peptides at all on-exchange time points under sixpH conditions were measured, and the deuterium incorporation percentagewas plotted as a function of incubation time (after applying the pH-dependenttime correction). A complete set of deuterium incorporation plotsare shown in Supporting Information Figure S1, and selected deuterium incorporation plots are shown in Figure 2. Upon close inspection of the selected pH-dependentdeuterium incorporation plots, we found three distinct HDX patterns:(1) N-terminal and middle region helices TH1–8 (Figure 2a–h,j–k), (2) C-terminal interhelicalloop TL8–9 and hydrophobic TH9 (Figure 2l–n), and (3) unstructured loops TL7–8 and TL9 (Figure 2i and o). For the first pattern, all helices remainfolded in their native state (W-state) between pH 7.5 and pH 6.5 (blue,cyan, and green curves), but become flexible and undergo two conformationaltransitions at pH 6.0 (yellow curve) and pH 5.5 (orange curves), characterizedby two major increases in HDX. Other studies reported that, betweenpH 7.5 and pH 6.0, the T domain undergoes conformational rearrangementinto a more solvent-exposed membrane competent (W+) state,4,15,34 and our results are consistentwith those outcomes. Interestingly, the transition midpoint betweenthe native and the membrane-competent state was calculated to be pH6.2,2,4,15 a pH conditionclose to one used in this study (pH 6.0). Thus, one may infer thatthe extent of HDX represents the combined structural dynamics of bothnative conformation (W) and the membrane-competent (W+)conformation.

Bottom Line: Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form.At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8-9, leading to subsequent aggregation in the absence of membranes.This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Washington University , St. Louis, Missouri 63130, United States.

ABSTRACT
The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our "standard condition" (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W(+)-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8-9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8-9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain.

Show MeSH
Related in: MedlinePlus