Figure 1. Computational design methodology.

(a) The homo-trimeric scaffold was designed to bind amantadine such that the C₃ axes of the protein and the small molecule are aligned. Amantadine is colored orange and each monomer of ABP is colored magenta, green, or cyan. The amantadine binding site is highlighted by a dashed oval. (b) The binding pocket in ABP was designed to have polar serine residues (Ser-71) that hydrogen-bond (yellow dashed lines) to the amino group of amantadine and nonpolar residues (Ile-64, Leu-67, and Ala-68) to complement the shape of the hydrophobic moiety of amantadine. (c) The design model contains hydrogen-bond networks that specify the trimeric assembly of ABP. (d) A sequence alignment of ABP and 2L6HC3_13 is shown, with mutated regions shown in black boxes. Both sequences are preceded by a five-residue GHSMG pre-sequence (not shown) that result from the cloning strategy. The residues highlighted in (b) are annotated by green circles.

Figure 1—figure supplement 1. ABP variant designs.

(a) The original ABP design is shown colored in magenta, green, or cyan. (b) Ala/Ser mutations were introduced in the core of the helical bundle in order to reduce the trimeric interface. (c) HBNet was used to incorporate more polar residues at the monomer-monomer interfaces that could make hydrogen-bond interactions (yellow dashed lines). (d-f) The outer helices were truncated to various lengths and the identities of surface-exposed positions redesigned to be polar. (g) The helical bundle was shortened by removing a heptad repeat. (h) The helical bundle was shortened to just the amantadine-binding site, and helical repeats were fused onto the core bundle in order to stabilize the structure of the reduced core.