1. Volume 25, Issue 9, Pages 1391-1402.e3 (September 2017)
Epitope and Paratope Mapping Reveals Temperature-Dependent Alterations in the Dengue-Antibody Interface 
Xin-Xiang Lim, Arun Chandramohan, Xin-Ying Elisa Lim, James E. Crowe, Shee-Mei Lok, Ganesh S. Anand 
Structure 
Volume 25, Issue 9, Pages e3 (September 2017)
DOI: /j.str
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2. Structure 2017 25, 1391-1402.e3DOI: (10.1016/j.str.2017.07.007)
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3. Figure 1
Hydrogen/Deuterium Exchange Mass Spectrometric Analysis of 2D22-DENV2 Complexes at 28°C and 37°C
Top panel: mapping the epitope and paratope of 2D22 on 2D22 unexpanded DENV2 complex (2D22-DENV2UN) at a vector temperature of 28°C by HDXMS. Bottom panel: epitope and paratope mapping of 2D22 in complex with expanded DENV2 (2D22-DENV2EXP) at 37°C by HDXMS. Monitoring temperature-dependent changes accompanying expansion of 2D22 prebound DENV2 by increasing the temperature from 28°C to 37°C (2D22-DENV2UN-37°C). All structures of unexpanded (DENV2UN), expanded DENV2 (DENV2EXP), and Fab 2D22-DENV2 complexes are represented in cartoon. E proteins constituting the 5-, 3-, and 2-fold icosahedral vertices are represented in yellow, orange, and red, respectively. Heavy and light chain of Fab 2D22 are represented in cyan and pink, respectively.
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4. Figure 2
Epitope and Paratope Mapping of 2D22 Unexpanded DENV2 Complex (2D22-DENV2UN) at 28°C by HDXMS
(A) Isotopic mass envelope of representative E protein peptides that spanned the heavy and light chain epitopes from DENV2UN (black spectra) and 2D22-DENV2UN (purple spectra) at 28°C after 0 and 1 min of deuterium exchange. Amino acids of peptides spanning the heavy and light chain are indicated as cyan and pink letters, respectively. Red dashed lines represent the average centroid of the mass envelope.
(B) Differences in deuterium exchange in pepsin-proteolyzed peptides of DENV2 E protein between free DENV2UN and 2D22-DENV2UN after 1 min of deuterium exchange at 28°C are represented in a difference plot. For the x axis, each node represents a pepsin-proteolyzed peptide, and all peptides are listed from the N to C terminus. The y axis shows differences in number of deuterons between the two states compared. Domain organization of DENV2 E protein is indicated below the difference plot.
(C) Deuterium exchange differences in E protein peptides between free DENV2UN and 2D22-DENV2UN are mapped onto the cryo-EM structure of an E protein dimer (PDB: 4UIF) from the DENV2UN. The E-dimer is represented as a ribbon and one of the protomers is shaded in light gray. The three E protein ectodomains are labeled DI, DII, and DIII.
(D and E) The difference plot of the heavy chain that interacts with DENV2UN (D) and the difference plot of the light chain that interacts with DENV2UN (E). Differences in deuterium exchange in pepsin-proteolyzed peptides of 2D22 heavy and light chains after 1 min of deuterium exchange between free 2D22 and 2D22-DENV2UN at 28°C are represented in the difference plots. Peptides exhibiting decreased deuterium differences that spanned residues reported to be the 2D22 epitope (E protein) and the paratope (Fab 2D22) by a previous cryo-EM study (Fibriansah et al., 2015) are highlighted in regions of blue and pink, respectively. Differences in deuterium exchange lower than −0.5 D (greater than 0.5 D in magnitude) were considered significant (red dashed lines) across the two states compared. SE values for each peptide are shown as purple shaded regions along the x axis in the difference plots and represent the sum of the single sigma standard deviations of each of the two conditions being compared.
(F) Deuterium exchange differences between free 2D22 and 2D22-DENV2UN in the peptides of 2D22 heavy (H) and light (L) chains are mapped onto the cryo-EM structure of 2D22 (PDB: 4UIF). The 2D22 epitope and paratope reported in a previous cryo-EM study are indicated as spheres. Epitope and paratope residues of the heavy chain are indicated with a cyan circle. Epitope and paratope residues of the light chain are indicated with a pink circle. Regions with no peptide coverage are in gray.
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5. Figure 3
Expansion of DENV2 under a Human Host Temperature of 37°C Affects 2D22 Binding
(A) Isotopic mass envelope of representative E protein peptides that spanned the heavy and light chain epitopes from expanded DENV2 (DENV2EXP) (black spectra) and 2D22-expanded DENV2 complex (2D22-DENV2EXP) (green spectra) at 37°C after 0 and 1 min of deuterium exchange. Amino acid sequences of peptides spanning the heavy and light chain are indicated in cyan and pink letters, respectively. The red dashed line indicates the average centroid of the mass envelope.
(B) Differences in deuterium exchange in pepsin-proteolyzed peptides of DENV2 E protein after 1 min of deuterium exchange between free DENV2EXP and 2D22-DENV2EXP at 37°C are represented in a difference plot. For the x axis, each point represents a pepsin-proteolyzed peptide, and all peptides are listed from the N to the C terminus. The y axis shows differences in the number of deuterons between the two states compared. Domain organization of DENV2 E protein is indicated below the difference plot.
(C–E) Differences in deuterium uptake after 1 min of deuterium exchange between free DENV2EXP and 2D22-DENV2EXP at 37°C are mapped onto an E-dimer from the cryo-EM structure of expanded DENV2 (PDB: 3ZKO). The three E protein ectodomains are labeled DI, DII, and DIII. Difference plots representing the differences in deuterium exchange in pepsin-proteolyzed peptides of 2D22 (D) heavy and (E) light chain after 1 min of deuterium exchange between free 2D22 and 2D22-DENV2EXP at 37°C. Peptides exhibiting decreased deuterium differences that spanned residues reported to be 2D22 epitope (E protein) and paratope (Fab 2D22) by a previous cryo-EM study (Fibriansah et al., 2015) are indicated in blue and pink, respectively. Differences in deuterium exchange lower than −0.5 D (greater than 0.5 D in magnitude) were considered significant (red dashed lines) across the two states compared. SE for each peptide is represented as green shaded region along the x axis.
(F) Deuterium exchange differences in 2D22 heavy (H) and light (L) chain peptides between free Fab 2D22 and 2D22-DENV2EXP are mapped onto the cryo-EM structure of 2D22 (PDB: 4UIF). Regions with no peptide coverage are in gray. 2D22 epitope and paratope residues reported by cryo-EM are represented as spheres. Residues constituting the reciprocal epitope and paratope interaction interface mediated by the heavy chain and light chain of 2D22 are circled cyan and pink, respectively.
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6. Figure 4 2D22 Binding to DENV2 Limits Temperature-Dependent Expansion
(A) Isotopic mass envelope of representative E protein peptides that spanned the heavy and light chain epitopes from expanded DENV2 (DENV2EXP) (black spectra) at 37°C and 2D22-prebound DENV2 subjected to expansion (2D22-DENV2UN-37°C) (orange spectra) at 37°C after 0 and 1 min of deuterium exchange. Amino acid sequences of peptides spanning the heavy and light chain are indicated as cyan and pink letters, respectively. The red dashed lines indicate the average centroid of the mass envelope.
(B–D) Differences in deuterium exchange in pepsin-proteolyzed peptides of DENV2 E protein after 1 min of deuterium exchange between free DENV2EXP and 2D22-DENV2UN-37°C are represented in a difference plot. For the x axis, each point represents a pepsin-proteolyzed peptide, and all peptides are listed from the N to the C terminus. The y axis shows differences in the number of deuterons between the two states compared. The black dashed lines represent absolute differences in deuterium exchange across all the peptides from the E protein between 28°C and 37°C mapped from a previous HDXMS study (Lim et al., 2017). These temperature-dependent differences in deuterium exchange were determined by subtracting the deuterium uptake of the peptide at 28°C from that at 37°C. Hence, previously mapped temperature-dependent changes were represented as negative magnitude of deuterium-exchange differences. Domain organization of the DENV2 E protein is indicated below the difference plot. Differences in deuterium exchange in pepsin-proteolyzed peptides of 2D22 (C) heavy and (D) light chain after 1 min of deuterium exchange between free 2D22 and 2D22-DENV2UN-37°C are represented in difference plots. Peptides exhibiting decreased deuterium differences that spanned residues reported by a previous cryo-EM study (Fibriansah et al., 2015) to be 2D22 epitope (E protein) and paratope (2D22) are highlighted in regions of blue and pink, respectively. Differences in deuterium exchange lower than −0.5 D (greater than 0.5 D in magnitude) were considered significant (red dashed line) between the two states compared. SE values for each peptide are represented as orange shaded regions along the x axis.
(E and F) Differences in deuterium exchange at 1 min are mapped onto the structure of E dimers from the cryo-EM structure of 2D22-DENV2UN-37°C intermediate (PDB: 4UIH) and displayed in orthogonal views. The three E protein ectodomains, stem helices, and transmembrane helices are labeled DI, DII, DIII, S, and TM, respectively.
(G) Deuterium exchange differences in 2D22 heavy (H) and light (L) chain peptides between free 2D22 and 2D22-DENV2UN-37°C are mapped onto the cryo-EM structure of 2D22 (PDB: 4UIF). Regions with no peptide coverage are in gray. 2D22 epitope and paratope residues reported by a previous cryo-EM study (Fibriansah et al., 2015) are represented as spheres. Residues constituting the reciprocal epitope and paratope interaction interface mediated by the heavy chain and light chain of 2D22 are circled cyan and pink, respectively.
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7. Figure 5
Proposed Mechanism of 2D22 Action on Unexpanded (DENV2UN) and Expanded DENV2 (DENV2EXP)
Residues constituting the 2D22 heavy and light chain epitopes are represented on structures of E-dimers from DENV2UN (A) surface and (B) side view and DENV2EXP (C) surface and (D) side view at 28°C and 37°C, respectively. One E protein protomer is represented in light blue and the other in red. Loci of heavy and light chain epitopes are indicated by cyan and pink circles, respectively. Specific residues constituting the heavy and light chain epitopes are indicated and represented as spheres. The measured distances between heavy and light chain residues are indicated and represented with black dashed lines.
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8. Figure 6
Host-Specific Temperature Perturbation Alters Mode of 2D22 Binding to DENV2
Top panel: cryo-EM structures of (A) DENV2UN (PDB: 3J27) and (B) 2D22-DENV2UN (PDB: 4UIF). Deuterium exchange difference across all E protein peptides after 1 min of deuterium exchange at 28°C between free DENV2UN and 2D22-DENV2UN is mapped onto the structure of 2D22-DENV2UN. Bottom panel: cryo-EM structures of (C and D) DENV2EXP (PDB: 3ZKO) and (E) expansion intermediate of 2D22-DENV2EXP-37°C (PDB: 4UIH). Deuterium exchange differences across all E protein peptides after 1 min of deuterium exchange at 37°C between DENV2EXP and 2D22-DENV2EXP, 2D22-DENV2UN-37°C are mapped onto the structures of (D) DENV2EXP (PDB: 3ZKO) and (E) expansion intermediate 2D22-DENV2EXP-37°C (PDB: 4UIH), respectively. Fab 2D22 is represented in cartoon with the heavy and light chains colored cyan and pink, respectively.
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