De novo design of obligate ABC heterotrimeric proteins

Abstract

The self-association of three distinct proteins, or essentially an ABC heterotrimer, is seen in a few instances in nature but the challenge of designing cooperative 3-sided interfaces has not been tackled yet in the field of protein design. There has been increasing interest towards designing much more diverse higher-order architectures for downstream applications that require the presentation or recruitment of multiple proteins, which could be aided by the specific assembly of a heterotrimer. Therefore, we sought to design cooperative ABC heterotrimeric proteins such that chains A, B, and C are unique and different with respect to amino acid sequence. Inspired by the ease of parametric sampling for coiled coils and previous success of burying hydrogen bonds to mediate homo-oligomer and heterodimer specificity, we strategically searched for buried networks accommodating nonpolar aromatic residues like tyrosine and tryptophan, while also enhancing for phenylalanine during core packing to create de novo ABC heterotrimeric coiled coils and helical bundles. We hypothesized that the inclusion of these three large aromatic residues, supplemented by tight aliphatic packing, could help drive the desired ABC assembly. These designs were experimentally characterized through size exclusion chromatography, native mass spectrometry, and small angle x-ray scattering. Two of these successful “base” constructs were subsequently parsed through a library of monomeric designed helical repeat (DHR) proteins to ensure that these heterotrimers could be extended while preserving their cooperative specificity. These single stranded and helical bundle heterotrimers DHR extensions or “arms” were then used to design cyclic geometries to highlight the different stoichiometries that can result from the same starting ABC interface, enabling these heterotrimers to serve as potential building blocks for higher-order assembly or as scaffolds for multivalent display.