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. 2017 Apr 25;6:e23043. doi: 10.7554/eLife.23043

Figure 1. The ternary complex of nAChR α1 ECD bound by Fab35 and α-Btx.

(a) Gel shift assay. Native PAGE showed the formation of the ternary complex of nAChR α1 ECD, α-Btx and Fab35. Lane 1: nAChR α1 ECD alone (labeled as α1 ECD), Lane 2: α-Btx alone, Lane 3: Fab35 alone, Lane 4: nAChR α1 ECD plus α-Btx, Lane 5: nAChR α1 ECD plus Fab35, and Lane 6: nAChR α1 ECD plus α-Btx plus Fab35. Note that α-Btx in Lane 2 and Fab35 in Lane 3 were not visible because both proteins migrated upward due to their net positive charges under the experimental condition. (b) Ribbon representation of nAChR α1 ECD (α1: cyan) bound by α-Btx (green) and Fab35 (heavy chain, H: yellow and light chain, L: magenta). The variable domains (VH and VL) and the constant domains (CH and CL) of Fab35 are indicated accordingly. This color scheme is kept the same throughout illustration unless noted otherwise. (c) Surface representation of the ternary complex. (d) Zoomed-in view of the binding interface. The complementarity determining regions (CDRs) of the heavy chain (CDR-H1, CDR-H2, and CDR-H3) are indicated as H1, H2, and H3, respectively. Those of the light chain (CDR-L1, CDR-L2 and CDR-L3) are indicated as, L1, L2, and L3, respectively.

DOI: http://dx.doi.org/10.7554/eLife.23043.003

Figure 1.

Figure 1—figure supplement 1. Key structural features of the human nAChR α1 ECD.

Figure 1—figure supplement 1.

Our study also generated the first atomic picture of the human nAChR α1 ECD, which appears very similar to the previously characterized mouse nAChR α1 ECD (PDB ID, 2QC1) (Lindstrom, 2000). Many functionally important structural features observed in the mouse nAChR α1 ECD are also conserved in the human α1 ECD as shown here. (a) The hydrophilic residues (Thr52 and Ser126) and a bound water molecule buried inside the beta sandwich core of the ECD. The 2Fo-Fc electron density map (shown in blue) is countered at one sigma level. The electron density of water is shown as the Fo-Fc omit map (shown in green) countered at three sigma level. (b) The N-linked glycan (at the Asn141) bridging the Cys-loop and the loop C. The 2Fo-Fc electron density map (shown in blue) is contoured at one sigma level.
DOI: http://dx.doi.org/10.7554/eLife.23043.004
Figure 1—figure supplement 2. Structural differences between the human and mouse nAChR α1 ECDs.

Figure 1—figure supplement 2.

A significant structural difference between the human and mouse nAChR α1 ECDs is the binding interface of α-Btx. (a) In the mouse nAChR α1 ECD, Phe189 is inserted into a surface pocket of α-Btx. (b) In the human nAChR α1 ECD, Thr189, is too small to fill in the α-Btx pocket. These observations are consistent with predictions from previous analyses of the nAChR α1/α-Btx complex (Vincent et al., 2001). The 2Fo-Fc electron density map (shown in blue) was contoured at one sigma level.
DOI: http://dx.doi.org/10.7554/eLife.23043.005
Figure 1—figure supplement 3. Structural comparison of mouse nAChR α1 ECDs in the ternary complex of Fab35/nAChR α1 ECD/α-Btx and the binary complex of nAChR α1 ECD/α-Btx.

Figure 1—figure supplement 3.

(a) Superposition of the mouse nAChR α1 ECD from the Fab35/nAChR α1 ECD/α-Btx complex (blue) and the nAChR α1 ECD/α-Btx complex (green) (PDB ID, 2QC1) (Lindstrom, 2000) using the Cα backbone of the nAChR α1 ECD. (b) Detailed comparison of side chain orientation of residues involved in Fab35 binding.
DOI: http://dx.doi.org/10.7554/eLife.23043.006