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. 2019 Mar 18;9:4735. doi: 10.1038/s41598-019-41179-0

Figure 2.

Figure 2

Binding of Fab CR6261 induces discrete local changes in deuterium incorporation that pinpoint the epitope. (A) Bar plot representing differences in deuterium uptake between free- and Fab CR6261-bound HA, with each bar corresponding to a unique HA peptide from the peptide library (ordered from N- to C- terminus based on the first amino acid of each peptide). The Y-axis represents the total difference in D uptake for a given peptide, calculated as the sum of the mass differences between free and bound HA at each time point. Positive bars indicate protection of the corresponding peptide upon binding and reveal that two distinct regions are affected by Fab CR6261. Deuterium uptake differences are shown for each peptide and labeling time point: 0.5 min (orange), 2 min (red), 10 min (blue) and 60 min (black). Light grey shades show the differential uptake errors for each peptide, and a dashed black horizontal line represents the threshold above which deuterium uptake differences between free and bound HA were considered significant. This threshold corresponds to three times the average standard deviation between triplicate repeats, and is approximately 0.3 Da for all measured peptides. (B) Deuterium uptake differences for each labeling time point projected on the monomer of HA A/California/07/2009 (PDB: 3UBQ) in a rainbow color scheme, where blue corresponds to HA regions that are not affected by Fab CR6261 binding, while red corresponds to the highest observed differences. Regions encompassing residues 18–21 and 39–54 in HA2 show protection upon Fab binding, and are therefore identified as the epitope by HDX-MS. (C) Atomic model of HA with the epitope determined by HDX-MS highlighted in cyan, and the uncovered regions shown in red. The close-up view shows the atomic details of the epitope determined in the co-crystal structure of HA SC1918/H1 in complex with Fab CR6261 (PDB: 3GBN), and HA residues involved in direct contacts are shown as sticks. The residues identified by HDX-MS are colored cyan, while those not detected are highlighted in red. Residues identified by both HDX-MS and X-Ray crystallography are labeled in green, and residues buried within the HA structure are labeled in italic. The 19 residues identified by HDX-MS as the epitope contain 16 residues seen to contact Fab CR6261 in the X-Ray structure, and the only 5 residues missed by HDX-MS localize to uncovered regions around glycosylation sites of HA1, which are shown as red spheres.