Programmable design of orthogonal protein heterodimers

Abstract

Heterodimeric interaction specificity between two DNA strands, and between protein and DNA, is often achieved by varying side chains or bases coming off the protein or DNA backbone -- for example, the bases participating in Watson-Crick base pairing in the double helix, or the side chains of protein contacting DNA in TALEN-DNA complexes. This modularity enables the generation of an essentially unlimited number of orthogonal DNA-DNA and protein-DNA heterodimers. In contrast, protein-protein interaction specificity is often achieved through backbone shape complementarity 1, which is less modular and hence harder to generalize. Coiled coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions 2) limits the number of orthogonal pairs that can be created simply by varying sidechain interactions 3,4. Here we demonstrate that heterodimeric interaction specificity can be achieved using extensive and modular buried hydrogen bond networks. We used the Crick generating equations 5 to produce millions of four helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen bond networks, and used Rosetta to connect pairs of helices with short loops and optimize the remainder of the sequence. 65 of 97 such designs expressed in E. coli formed constitutive heterodimers, and crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen bond networks. In cells, a set of six heterodimers were found to be fully orthogonal, and in vitro, following mixing of 32 chains from sixteen heterodimer designs, denaturation in 5M GdnHCl and reannealing, the vast majority of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities.

Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering

“…Subsequent refinement of the protocol has led to the design of numerous structures with extended hydrogenbonding networks that have been faithfully recapitulated in high-resolution crystal structures, including pHresponsive hydrogen-bonding networks rooted by histidine residues. 25,29,30 Second, we applied the preliminary design protocol to an extremely limited design space. We searched only the docked configurations that we experimentally characterized in our previous report on twocomponent tetrahedral complexes, 7 and we did not allow backbone movement at any point during the HBNet or RosettaDesign portions of our design protocol.…”

Deep exploration networks for rapid engineering of functional DNA sequences

“…Moving on to the world of proteins, we imagine a vast number of application areas for deep exploration nets. Rational design of heterodimer pairs with orthogonal interaction has recently been demonstrated (Chen et. al., 2019).…”

Protein assembly systems in natural and synthetic biology

“…On the other hand, engineering protein-based systems, such as synthetic signaling circuits, has been more limited in part because of the inherent difficulty in making programmable protein-protein interactions [205,206]. To date, synthetic biologists have relied on a relatively limited set of wellcharacterized, folded interaction domains to accomplish this task, for example, using leucine zippers and PDZ domains to enable recruitment of signaling proteins and pathway modulators [207][208][209]. More recent work has begun to expand this protein toolkit to viral proteases [210,211] and even synthetic phospho-regulon motifs for building phosphorylation circuits [212].…”