Fig. 3: Structural comparison between AF4H1K1 and AF4H1L1.

a AF4H1L1 Fab is overlapped with AF4H1K1/H3 complex. H3 HA is labeled with cyan, AF4H1K1 Fab VH in complex with H3 HA is in light pink, and the VL is in yellow. AF4H1L1 VH is in gray color and VL is in marine. Circled area shows the conformation difference between AF4H1K1 and AF4H1L1, and the red dashed arrow line suggests the flipping over of AF4H1L1 HCDR3 toward A-helix comparing AF4H1K1 HCDR3. b VH and VL CDR3 loops from both AF4H1K1 and AF4H1L1 Fab fragments are highlighted. The labels of amino acids in the HA are in black color, the residues in the AF4H1K1 HCDR3 are in green, those in AF4H1L1 HCDR3 are in red, those in AF4H1K1 LCDR3 are in yellow, and the others in AF4H1L1 are in marine. It is clearly that HCDR3 of AF4H1L1 flippers over toward the HA A-helix and causes fierce clash between Asp108, Trp109 in HCDR3 and Ile45, Asp46 in the A-helix. c Hydrogen bonds between LCDR3 and HCDR3 of AF4H1K1. LCDR3 makes contacts with HCDR3 mainly by hydrogen bonds (yellow dashed line) between Ser111, Gly112, Gly113 of HCDR3 and Gln90, Gly93, Ser94, or Ser95 of LCDR3. d Contacts between HCDR3 and LCDR3 of AF4H1L1. HCDR3 and LCDR3 of AF4H1L1 mainly involved hydrophobic interactions and one hydrogen bond. Trp90, Val95 in LCDR3, and Leu105, Pro106, Phe107, Trp109, and Phe115 in HCDR3 are the amino residues for the hydrophobic interaction. e Unbound AF4H1K1 scFv molecule (green) is overlapped with Fab molecules from H3-bound (cyran) and H4-bound (magenta) AF4H1K1. All the CDR loops adopt similar conformations in both apo and HA-bound states. f Unbound AF4H1K1 scFv molecule (green) is overlapped with AF4H1L1 Fab molecules (gray). The different conformations of HCDR3 are highlighted by a red dashed oval