De novo design of stable proteins that efficaciously inhibit oncogenic G proteins

Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs from a native partner into a host protein. Here, we develop and experimentally test a computational protocol for embedding binding motifs in de novo designed proteins. The protocol uses an “inside-out” approach: Starting with a structural model of the binding motif docked against the target protein, the de novo protein is built by growing new structural elements off the termini of the binding motif. During backbone assembly, a score function favors backbones that introduce new tertiary contacts within the designed protein and do not introduce clashes with the target binding partner. Final sequences are designed and optimized using the molecular modeling program Rosetta. To test our protocol, we designed small helical proteins to inhibit the interaction between Gαq and its effector PLC-β isozymes. Several of the designed proteins remain folded above 90°C and bind to Gαq with equilibrium dissociation constants tighter than 80 nM. In cellular assays with oncogenic variants of Gαq, the designed proteins inhibit activation of PLC-β isozymes and Dbl-family RhoGEFs. Our results demonstrate that computational protein design, in combination with motif grafting, can be used to directly generate potent inhibitors without further optimization via high throughput screening or selection. statement for broader audience Engineered proteins that bind to specific target proteins are useful as research reagents, diagnostics, and therapeutics. We used computational protein design to engineer de novo proteins that bind and competitively inhibit the G protein, Gαq, which is an oncogene for uveal melanomas. This computational method is a general approach that should be useful for designing competitive inhibitors against other proteins of interest. ### Competing Interest Statement The authors have declared no competing interest. * BLI : biolayer interferometry CD : circular dichroism DAG : diacyl glycerol FA : fluorescence anisotropy Gαq/i : Gαq G protein : guanine nucleotide-binding protein GuHCl : guanidine hydrochloride HTH : helix-turn-helix IP3 : inositol 1,4,5-trisphosphate PIP2 : phosphatidylinositol 4,5-bisphosphate PLC-β : phospholipase C-β RMSD : root mean square deviation SEWING : structure extension with native fragment graphs