In nature, structural specificity in DNA and proteins is encoded quite differently: in DNA, specificity arises from modular hydrogen bonds in the core of the double helix, whereas in proteins, specificity arises largely from buried hydrophobic packing complemented by irregular peripheral polar interactions. Here we describe a general approach for designing a wide range of protein homo-oligomers with specificity determined by modular arrays of central hydrogen bond networks. We use the approach to design dimers, trimers, and tetramers consisting of two concentric rings of helices, including previously not seen triangular, square, and supercoiled topologies. X-ray crystallography confirms that the structures overall, and the hydrogen bond networks in particular, are nearly identical to the design models, and the networks confer interaction specificity in vivo. The ability to design extensive hydrogen bond networks with atomic accuracy is a milestone for protein design and enables the programming of protein interaction specificity for a broad range of synthetic biology applications.

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.

Author Contributions RAL, AHN, and SEB contributed equally to this publication. RAL, SEB, ZC, WRPN, and DB conceived of the idea and initial steps for designing protein switches from de novo designed helical bundles. RAL and DB developed the thermodynamic model and the code upon which it works. RAL, SEB, and WRPN designed and biophysically characterized LOCKR scaffolds and BimLOCKR. RAL performed mutagenesis and Bio-layer interferometry experiments. SB characterized Bim interactions to Bcl2 homologs and aided experimental design. RAL performed design calculations for orthogonal LOCKR designs using code from SEB and VKM. AHN and RAL conceived of caging cODC. RAL performed design calculations to cage cODC and tune degronLOCKR. AHN conceived of and contributed to all experiments with degronLOCKR. THN performed dynamic measurement of degronLOCKR. AMW tested degronLOCKR in HEK293T cells. MJL, SEB, and RAL performed design calculations for asymmetric LOCKR. GD performed experiments with degronLOCKR and dCas9. GD contributed to plasmid and strain construction. RAL, SEB, and MJL conceived of caging sequences to control subcellular location and RAL performed design calculations for nesLOCKR. JAS and AHN performed all experiments for nesLOCKR. RAL, SEB, AHN, HE-S, and DB wrote the manuscript, all authors edited and approved.

This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.