Fig. S8.

Design of the two-loop DesAb variant. (A–D) structures of three nanobodies (A–C) and the HEL4 human single domain antibody (D) used for the design of the two-loop construct; loops containing the CDR3 and CDR2 are colored in dark gray, and disulphide bridges stabilizing the CDR3 are highlighted in cyan when present. (A) Structure of a nanobody (1RI8) with the ligand that binds between the CDR3 and the CDR2 shown in transparent orange. (B) Structure of a nanobody (2X6M) bound to the C-terminal peptide of α-synuclein (orange), Lys105 and Asn52 are colored in magenta, and their hydrogen bond network with the backbone of the CDR3 loop is drawn (formed hydrogen bonds are in dark blue, possible ones in light blue). (C) Structure of a nanobody (4KRP) showing Asn52 in magenta, Ala33 in yellow and Tyr122-Asp123-Tyr124 on the stem of the CDR3 in green. (E) Structural integrity of the two-loop DesAb assessed with far-UV CD. (F) Alignment of the four template sequences with the two-loop DesAb sequence (named 2Loops) with the grafted CDR sequences underlined and the complementary peptides colored in green; the sequence of the one-loop DesAb scaffold is also reported (1Loop), and the residues in the two-loop DesAb sequence that differ from those in the one-loop DesAb outside the CDR loops are highlighted in yellow. These residues are also those colored in the corresponding template structures (A–C) with the exception of Glu130, which was selected from ref. 40. (G) Representation of the pincer-like binding to α-synuclein of the complementary peptides grafted in the CDR2 and CDR3 loops of the two-loop DesAb construct; equal signs mark residues predicted to be involved in backbone-backbone hydrogen bonding and arrows denote the N to C terminus direction. (H) Results from the cascade method for the two peptides grafted in the two-loop DesAb scaffold; grafted sequences are shown in bold.