Epitopes of inhibitory nanobodies Nd4 and Nd6. (A) Representative BLI sensorgrams of immobilized AIM-A1, A1-CAIM, NAIM-A1, or tAIM-A1 binding to a serial dilution of purified nanobody Nd4 (blue) or Nd6 (green). Nanobody concentration ranges are 64 to 1 nM for Nd4 binding to AIM-A1, tAIM-A1, 4 μM to 2 nM for Nd4 binding to A1-CAIM, 20 μM to 20 nM for Nd6 binding to AIM-A1, A1-CAIM and tAIM-A1, and 2 μM to 125 nM for Nd4 and Nd6 binding to NAIM-A1. The fitted affinity, when obtained, is shown in each panel. For binding, association was performed for 120 seconds, and dissociation for 240 seconds. The fitting trace for each concentration is shown in black. (B) Dot blots of immobilized NR and R AIM-A1 by Nd4 and Nd6. 6G1, which recognizes a linear epitope, and CR1, which is partially conformationally sensitive, were included for comparison. (C) Crystal structure of the AIM-A1/Nd6 complex, shown in ribbon diagram. The nanobody Nd6 is shown in green, the A1 domain in gray, NAIM residues in sky blue, and CAIM residues in orange. Dashed lines denote unresolved residues in AIM-A1. (D-E) Close-up diagrams showing specific contacts between AIM-A1 and Nd6. Hydrogen bonds between side chains or main chains of noted residues are marked by dashed lines. Note that CAIM residues beyond residue Ala1464 do not show any direct contacts with Nd6. The GalNAC group on residue 1468 is colored magenta. (F) Relative deuterium uptake displayed on the 3D structure of the A1 domain and partial AIM as in the Nd6 complex. Residues experiencing more exchange in the presence of Nd6 are colored in shades of red, no difference in exchange is displayed as white, and decreased exchange is shown in cyan. Note that many residues in the AIM were unresolved in this structure. (G-J) Uptake plots of representative peptides in AIM-A1 with (green) and without (black) bound 6D12 at 10, 30, 100, and 1000 seconds of exchange. Peptide 1240-1257 (G) is a NAIM-peptide. Peptide 1308-1320 (H) is in the α1β2 loop of the A1 domain, peptide 1330-1340 (I) is in the β3α2 loop, and peptide 1367-1383 (J) is in the α3β4 loop. For each time point, experiments were performed in triplicate. ∗∗∗P < .001 using a Student t test; ∗∗P < .01; ∗P < .05.

Epitopes of inhibitory nanobodies Nd4 and Nd6. (A) Representative BLI sensorgrams of immobilized AIM-A1, A1-CAIM, NAIM-A1, or tAIM-A1 binding to a serial dilution of purified nanobody Nd4 (blue) or Nd6 (green). Nanobody concentration ranges are 64 to 1 nM for Nd4 binding to AIM-A1, tAIM-A1, 4 μM to 2 nM for Nd4 binding to A1-CAIM, 20 μM to 20 nM for Nd6 binding to AIM-A1, A1-CAIM and tAIM-A1, and 2 μM to 125 nM for Nd4 and Nd6 binding to NAIM-A1. The fitted affinity, when obtained, is shown in each panel. For binding, association was performed for 120 seconds, and dissociation for 240 seconds. The fitting trace for each concentration is shown in black. (B) Dot blots of immobilized NR and R AIM-A1 by Nd4 and Nd6. 6G1, which recognizes a linear epitope, and CR1, which is partially conformationally sensitive, were included for comparison. (C) Crystal structure of the AIM-A1/Nd6 complex, shown in ribbon diagram. The nanobody Nd6 is shown in green, the A1 domain in gray, NAIM residues in sky blue, and CAIM residues in orange. Dashed lines denote unresolved residues in AIM-A1. (D-E) Close-up diagrams showing specific contacts between AIM-A1 and Nd6. Hydrogen bonds between side chains or main chains of noted residues are marked by dashed lines. Note that CAIM residues beyond residue Ala1464 do not show any direct contacts with Nd6. The GalNAC group on residue 1468 is colored magenta. (F) Relative deuterium uptake displayed on the 3D structure of the A1 domain and partial AIM as in the Nd6 complex. Residues experiencing more exchange in the presence of Nd6 are colored in shades of red, no difference in exchange is displayed as white, and decreased exchange is shown in cyan. Note that many residues in the AIM were unresolved in this structure. (G-J) Uptake plots of representative peptides in AIM-A1 with (green) and without (black) bound 6D12 at 10, 30, 100, and 1000 seconds of exchange. Peptide 1240-1257 (G) is a NAIM-peptide. Peptide 1308-1320 (H) is in the α1β2 loop of the A1 domain, peptide 1330-1340 (I) is in the β3α2 loop, and peptide 1367-1383 (J) is in the α3β4 loop. For each time point, experiments were performed in triplicate. ∗∗∗P < .001 using a Student t test; ∗∗P < .01; ∗P < .05.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal