De novo design of parallel and antiparallel A3B3 heterohexameric α-helical barrels

Joel J. Chubb, Katherine I. Albanese, Alison Rodger, Derek N. Woolfson

Research output: Working paperPreprint

Abstract

The de novo design of α-helical coiled-coil peptides is advanced. Using established sequence-to-structure relationships, it is possible to generate various coiled-coil assemblies with predictable numbers and orientations of helices. Here we target new assemblies, namely A3B3 heterohexamer α-helical barrels. These designs are based on pairs of sequences with 3-heptad repeats (abcdefg) programmed with a = Leu, d = Ile, e = Ala, and g = Ser, and b = c = Glu to make the acidic (A) chains and b = c = Lys in the basic (B) chains. These design rules ensure that the desired oligomeric state and stoichiometry are readily achieved. However, controlling the orientation of neighboring helices (parallel or anti-parallel) is less straightforward. Surprisingly, we find that assembly and helix orientation are sensitive to the starting position of the heptad repeats (the register) in the peptide sequences. Peptides starting at g (g-register) form a parallel 6-helix barrel in solution and in an X-ray crystal structure, whereas the b- and c-register peptides form an antiparallel complex. In lieu of experimental X-ray structures for b- and c-register peptides, AlphaFold-Multimer is used to predict atomistic models. However, considerably more sampling than the default value is required to match the models and the experimental data, as many confidently predicted and plausible models are generated with incorrect helix orientations. This work reveals the previously unknown influence of heptad register on the orientation of α-helical coiled-coil peptides and provides insights for the modeling of oligopeptide coiled-coil complexes with AlphaFold.

Original languageEnglish
DOIs
Publication statusSubmitted - 25 Sept 2024

Publication series

NamebioRxiv

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