Figure 1.

HDX-MS reveals the conformational dynamics of free- and Fab-bound HA trimers in solution. (A) Atomic model of the HA trimer (PDB: 3UBQ) showing the HA head and stem, which mainly correspond to HA1 (orange) and HA2 (blue), respectively. In HA2, we highlight the CD Helix (residues 75–126) that forms a trimeric coiled coil driving oligomerization, and the highly conserved components of the fusion machinery required for HA function: The A Helix (residues 38–58, shown in green) and the fusion peptide (residues 1–10, shown in purple). (B) Atomic model of a single protomer of HA (PDB: 3UBQ) with the areas covered by our list of highly reproducible peptides shown in grey, whereas uncovered regions are colored red. All uncovered regions are found around the 7 glycosylation sites of HA1, which are highlighted as red spheres. (C) The residues of four representative peptides from our peptide library are shown as spheres in the atomic model of the HA monomer: Peptide 1 (¹¹⁹ERFEIF¹²⁴, yellow) corresponds to an exposed β strand in HA1, Peptide 2 (⁵⁸KMNTQF⁶³, pink) is a flexible loop in the HA2 subunit, Peptide 3 (³⁹KSTQNAIDEITNKVNSVIE⁵⁷, green) covers the entire A Helix, and Peptide 4 (¹⁷MVDGW²¹, blue) is directly adjacent to the N-terminal fusion peptide (D) The deuterium uptake graphs for Peptides 1–4 show the relative deuterium incorporation of each peptide as a function of exposure time (calculated as the difference between the mass centroid at t = 0 and the different labeling time points). Below, we show the mass spectra of each peptide for a selected charge state for each labeling time point in the bound and unbound states. Peptides originating from free HA show distinct profiles of deuterium incorporation that closely match the expected behavior based on the atomic model of HA. While binding of Fab CR6261 does not alter deuterium uptake profiles of Peptides 1 and 2, deuterium incorporation of Peptides 3 and 4 is significantly lower at all labeling time points in bound HA.