De novo design of protein logic gates

Abstract

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo-designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.

Figure 1.. Cooperativity of CIPHR logic gates.

(A) Left: Backbone structure of A:A’ heterodimer building block, with its hydrogen bond network in inset. Bottom: Shorthand representations used throughout figures. (B) Thermodynamic cycle describing the induced dimerization system. (C) Simulation of the induced dimerization system under thermodynamic equilibrium. A and B’ monomers were held constant at 10 μM each while titrating in various initial amounts of the A’-B dimerizer proteins. If binding is not cooperative (small c), the final amount of trimeric complexes decreases when the dimerizer protein is in excess. (D) Equilibrium denaturation experiments monitored by CD for designs with 6- and 12- amino acid (AA) linkers. Circles represent experimental data, and lines are fits to the 3-state unimolecular unfolding model. (E) Experimental SAXS profile of 1’−2’ with a 6-residue linker (in black), fitted to the calculated profile of 1:1’ heterodimer. (F) An induced dimerization system using a 6-residue linker. (G) Native MS titration of 2 against 1’−2’ in the presence (red) or absence (blue) of 1. (H) Native MS titration of 1’−2’ against 1 and 2. Dimer 1 and 2 refer to partial dimeric complexes consisting of the dimerizer binding to either of the monomers. For comparison, the thermodynamic model result with c = 991,000 is shown in cyan. (I) Schematic of testing of the induced dimerization system in yeast, with in vivo results in (J). Pg, progesterone. (K) A two-input AND gate schematic, with native MS titration results in (L). Trimer 1 and 2 refer to partial trimeric complexes of the two dimerizer proteins binding to either one of the monomers. (M) A three-input AND gate, with native MS titration results in (N). Tetramer 1 and 2 refer to partial tetrameric complexes of the three dimerizer proteins binding to either one of the monomers. All error bars are reported as standard deviations of n=3 independent replicates.

Figure 2.. CIPHR two input logic gates.

(A) CIPHR gates are built from DHDs (top) with monomers or covalently connected monomers as inputs (left); some gates utilize only the designed cognate interactions (left side of middle panel), while others take advantage of observed binding affinity hierarchies (right side of middle panel). (B-C) Two-input AND (B) and OR (C) CIPHR logic gates based on orthogonal DHD interactions. (D-G) NOT (D), NOR (E), XNOR (F), and NAND (G) CIPHR logic gates made from multispecific and competitive protein binding. For each gate, black dots represent individual Y2H growth measurement corrected over background growth, with their average values shown in green bars. * indicates no yeast growth over background. 0s and 1s in the middle and right blocks represent different input states and expected outputs, respectively.

Figure 3.. Three-input CIPHR logic gates.

(A) Schematic of a three-input AND gate. (B) Native MS results indicate proper activation of the 3-input AND gate only in the presence of all three inputs. (C) Schematic of a three-input OR gate. (D) Y2H results confirmed activation of the 3-input OR gate with either of the inputs. (E) Schematic of a DNF gate. (F) Y2H results confirmed proper activation of the gate. For each gate, black dots represent individual measurements with their average values shown in green bars. For Y2H-based measurements (D,F), the growth measurements are corrected over background growth.

Figure 4.. Transferability of CIPHR logic gates.

(A) Four pairs of DHDs were modularly combined to construct CIPHR logic gates that can be used to control different functions: (B-E) catalytic activity of split luciferase, and (F-G) gene expression in primary human T cells. (B) Induced dimerization system, (C) AND gate, and (D) NOR gate coupled to NanoBiT split luciferase system, tested by in vitro translation and monitoring luminescence. (E) In vitro titration of the two inputs of the NOR gate in D while keeping 1’-smBiT and 2’-lgBiT fixed at 5 nM. (F) NOT gate and (G) OR gate using a split TALE-KRAB repression system to control expression of TIM3 proteins in primary human T cells, tested by flow cytometry.