Multi-input chemical control of protein dimerization for programming graded cellular responses

Abstract

Chemical and optogenetic methods for post-translationally controlling protein function have enabled modulation and engineering of cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of post-translational behaviors that can be programmed, we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics. Our system translates drug inputs into diverse outputs using a suite of engineered reader proteins to provide variable dimerization states of the receiver protein. We show that our single receiver protein architecture can be used to program a variety of cellular responses, including graded and proportional dual-output control of transcription and mammalian cell signaling. We apply our tools to titrate the competing activities of the Rac and Rho GTPases to control cell morphology. Our versatile tool set will enable researchers to post-translationally program mammalian cellular processes and to engineer cell therapies.

Fig. 1 |. Design of a danoprevir:NS3a complex reader.

a, Schematic of the PROCISiR system. Multiple NS3a-targeting drugs are used as inputs that are interpreted by designed readers to generate multiple outputs. b, Goal and process for designing and optimizing drug:NS3a complex readers, starting from docking of several scaffold classes on a drug/NS3a complex, Rosetta design of the reader interface, filtering based on Rosetta interface scoring metrics, and finally testing and optimization via yeast surface display. c, Rosetta model for D5 (left) and binding of 1 μM NS3a with avidity to yeast-displayed D5 in the presence or absence of 10 μM danoprevir. A point mutant of the D5 interface, W177D, and the original DHR79 scaffold show no binding. Technical triplicates and means from one experiment. d, A co-crystal structure of the DNCR2:danoprevir:NS3a complex aligned with the D5:danoprevir:NS3a model via NS3a. Measurements indicate shifts of DNCR2 relative to D5. e, Residues within 4 Å of NS3a:danoprevir are highlighted in dark blue on the surface of DNCR2. Residues at the interface in the D5 model are outlined in black.

Fig. 2 |. Design and testing of a grazoprevir:NS3a complex reader.

a, Rosetta model (left) and binding (right) of 1 μM NS3a with avidity to yeast-displayed G3 in the presence or absence of 10 μM grazoprevir. Point mutants at the G3 interface, M112E and A175Q, and the original DHR18 scaffold show no binding. Technical triplicates and means from one experiment. b, Colocalization of DNCR2-EGFP with Tom20-mCherry-NS3a after treatment with the drug indicated or DMSO. c, Colocalization of NS3a-mCherry with GNCR1-BFP-CAAX or Tom20-DNCR2-EGFP after treatment with danoprevir, grazoprevir, or DMSO. d, Colocalization of NS3a-mCherry with ANR-BFP-CAAX or NLS-DNCR2-EGFP after treatment with danoprevir, grazoprevir, or DMSO. The mean (marked by dot) and standard deviation (error bars) of the Pearson’s r of red/blue or red/green pixel intensities for the number of cells stated in Supplementary Table 3 is given for each condition in (b-d), along with the distributions of Pearson’s r. See Supplementary Table 3 for sample sizes and P values.

Fig. 3 |. Temporal, graded, and proportional transcriptional control using PROCISiR.

a, Reversibility of CXCR4 induction from danoprevir-promoted recruitment of DNCR2-VPR to NS3a-dCas9. “OFF” conditions indicate replacement of danoprevir-containing media with DMSO- (gray) or grazoprevir-containing media (yellow). b,c, Co-titrating grazoprevir as a competitor in the presence of a uniform titration of danoprevir inducer in cells expressing the constructs shown in (a) extends the linear range of the CXCR4 (b) or CD95 (c) expression response resulting from DNCR2-VPR recruitment. The curves with higher proportions of the competitive inhibitor grazoprevir (darker gray) were created by performing lower fold-dilutions of grazoprevir (“low” (light gray): 2-fold, “medium” (medium gray): 1.5-fold, and “high” (dark gray): 1.25-fold serial dilutions). See Supplemental Table 4 for the exact drug concentrations used for each condition. (a,b,c) Mean and standard deviation of three biological replicates relative to DMSO-baseline subtracted values. d, Schematic of the transcriptional activation system used in (e,f) to simultaneously modulate expression of CXCR4 and GFP in cells co-expressing an MS2 scRNA targeting CXCR4, a PP7 scRNA targeting a GFP reporter, GNCR1-MCP, DNCR2-PCP, NS3a-VPR, and dCas9. e, Data from (f) (points, mean, standard deviation) overlaid with modeling (lines) of the fraction of NS3a bound to grazoprevir (top) or danoprevir (bottom) at the drug concentrations used in (f), as described in the Supplementary Notes. f, Expression of CXCR4 and GFP after co-treatment with danoprevir and grazoprevir. Matrices are means of two biological replicates; single-drug titrations are means of three biological replicates. Raw median fluorescence values for (e,f) are shown in Fig. S8e,f.

Fig. 4 |. Graded and proportional control of GTPase-driven signaling pathways.

a, Schematic of the colocalization system used in (c-g) for GTPase-driven signaling activation. Combinations of danoprevir and grazoprevir were used to control the proportions of DNCR2 and GNCR1 colocalizing with NS3a at the plasma membrane. b, Colocalization of EGFP-DNCR2 (green) or BFP-GNCR1 (blue) with mCherry-NS3a-CAAX quantified by Pearson’s r from confocal images of HeLa cells. The mean (marked by dot) and standard deviation (error bars) of the Pearson’s r of red/blue or red/green pixel intensities for the number of cells stated in Supplementary Table 3 is given for each condition, along with the distributions of Pearson’s r. c, Representative images from two experiments of HeLa cells co-expressing EGFP-DNCR2-TIAM, BFP-GNCR1-LARG, NS3a-CAAX, and Lifeact-mCherry treated with danoprevir (left) or grazoprevir (right) for the times indicated. Fluorescent signal for Lifeact-mCherry, which stains F-actin, is shown. A red outline illustrating the cell boundary at −10 min is overlaid on each panel to illustrate the change in cell size. d,f, Change in normalized area (d) or solidity (f) (DMSO baseline subtracted) over time (drug addition at 0 min) in Hela cells expressing NS3a-CAAX with either DNCR2-TIAM and GNCR1-LARG (top), DNCR2-TIAM alone (middle), or GNCR1-LARG alone (bottom). Line colors correspond to the drug conditions in (b). e,g, Change in normalized area (e) or solidity (g) (average last 10 min-first 10 min). (d-g) Mean and s.e.m. of the number cells per condition listed in Supplementary Table 3 from four independent wells.