Structure of the LGI1–ADAM22ECD complex

a Domain organizations of LGI1 and ADAM22. LGI1 consists of the LRR (blue) and EPTP (pink) domains. The N-terminal secretion signal peptide (SP, enclosed by dotted lines) is removed in the secreted LGI1. The shaded blue boxes represent the N- and C-terminal caps, whereas the filled blue boxes represent the LRRs. The premature form of ADAM22 contains the N-terminal prosequence (enclosed by dotted lines). The mature ADAM22 consists of the metalloprotease-like (magenta), disintegrin (salmon pink), cysteine-rich (brown), EGF-like (orange), transmembrane (TM; white), and cytoplasmic domains. The major ADAM22 isoform has a PDZ-binding motif in the C-terminal of the cytoplasmic domain. b Schematic diagram of the LGI1LRR– LGI1*EPTP–ADAM22ECD complex. The black circle indicates the location of this complex within the 3:3 LGI1-ADAM22 complex. * indicates a distinct molecule. c Cryo-EM map and structure of the LGI1LRR–LGI1*EPTP–ADAM22ECD complex at 2.78 Å resolution. d Schematic diagram of the 3:3 LGI1–ADAM22ECD complex. e Cryo-EM map and structure of the 3:3 LGI1– ADAM22ECD complex at 3.79 Å resolution.

Interactions between LGI1EPTP and ADAM22ECD

a Overall view of the cryo-EM structure of the LGI1LRR–LGI1*EPTP–ADAM22ECD complex and a magnified view of the interface between LGI1*EPTP and ADAM22ECD. The density map of the interface is shown as white surfaces. The previously reported X-ray structure of the LGI1EPTP– ADAM22ECD complex is superposed with distinct colors. Two boxes in the magnified view indicate the locations of the views shown in (b; left box) and (c; right box). b Close-up view of the interaction between Arg330 of LGI1*EPTP and Asp405 of ADAM22ECD. The structure (top) and corresponding map (bottom) are shown. Yellow and cyan dashed lines indicate a hydrogen bond observed in the cryo-EM and X-ray structures, respectively. c Close-up view of the interaction around Arg378 of LGI1. The structure (top) and corresponding map (bottom) are shown. Yellow and cyan dashed lines indicate hydrogen bonds observed in the cryo-EM and X-ray structures, respectively.

Structure and interaction of the higher-order LGI1–ADAM22ECD complex

a Overall view of the cryo-EM structure of the LGI1LRR–LGI1*EPTP–ADAM22ECD complex and a magnified view of the interface between LGI1LRR and LGI1*EPTP. The density map of the interface is shown as white surfaces. b Chain IDs of the individual LGI1 or ADAM22ECD molecules in the 3:3 LGI1–ADAM22ECD complex, assigned in this study. c Superposition of the three LGI1– ADAM22ECD complexes in the cryo-EM structure of the 3:3 LGI1-ADAM22ECD complex, using LGI1*EPTP–ADAM22ECD as the reference.

HS-AFM observations of LGI1–ADAM22ECD complexes

a A representative HS-AFM image of the 3:3 LGI1–ADAM22ECD complex. The color bars on the right indicate height in nanometers. White arrows indicate the 3:3 complex. The frame rate was 1.0 frames/s.

b, d Magnified HS-AFM images of the 3:3 (left in b) and 2:2 (left in d) LGI1–ADAM22ECD complexes. The simulated AFM images (middle) were derived from fitting to the experimental HS-AFM image (left). The well-fitting simulated AFM images and the coefficient of correlation (CC) are indicated. The cryo-EM structure of the 3:3 complex (right in b) and the X-ray structure of the 2:2 complex (right in d) are shown in the same orientation as the simulated AFM images. c, e, f Sequential HS-AFM images of the 3:3 (c; see also Movie S1) and 2:2 (e, f; see also Movie S2) LGI1–ADAM22ECD complexes. A schematic illustration of the interpretation of HS-AFM images is shown at the bottom. Imaging parameters: scanning area = 120 × 96 nm2 (240 × 192 pixels); frame rate = 3.3 frames/s. HS-AFM experiments were repeated independently at least 3 times with consistent results.