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. 2016 Dec 16;354(6318):1441-1444.
doi: 10.1126/science.aah3404.

Engineering Extrinsic Disorder to Control Protein Activity in Living Cells

Free PMC article

Engineering Extrinsic Disorder to Control Protein Activity in Living Cells

Onur Dagliyan et al. Science. .
Free PMC article


Optogenetic and chemogenetic control of proteins has revealed otherwise inaccessible facets of signaling dynamics. Here, we use light- or ligand-sensitive domains to modulate the structural disorder of diverse proteins, thereby generating robust allosteric switches. Sensory domains were inserted into nonconserved, surface-exposed loops that were tight and identified computationally as allosterically coupled to active sites. Allosteric switches introduced into motility signaling proteins (kinases, guanosine triphosphatases, and guanine exchange factors) controlled conversion between conformations closely resembling natural active and inactive states, as well as modulated the morphodynamics of living cells. Our results illustrate a broadly applicable approach to design physiological protein switches.


Fig. 1
Fig. 1. Design concept and PI-Src
(A) Domains conferring either photo-inhibition (LOV2) or activation induced by small molecules (uniRapR) function at the same allosteric site. (B) The termini of LOV2 and uniRapR domains are closely spaced for insertion. (C) Paxillin phosphorylation assays show that PI-Src’s catalytic activity is inhibited upon irradiation. SYF cells expressing PI-Src(YF) show reduced phosphoylation of cell lysates blotted with anti-pTyr antibody. Blue denotes irradiation. Error bars show s.e.m (n=3). (D) Inactive (blue) and active (gray) conformations of WT Src. The red circle (L) is the insertion site. Conformational changes were quantified by displaying the pairwise distance changes (Δd) between all residues as a heat map. The upper left triangle shows distances for WT Src, computed from published crystal structures. The lower triangle shows distances for PI-Src, determined using molecular dynamics simulations of the dark and lit states. Blue = decreased distance, red = increased distance. (E) In SYF cells, irradiation causes PI-Src(WT) to translocate to focal adhesions (FA, red arrows), edge movements to increase, and cells to polarize and translocate. When cells are returned to the dark, FA translocation is reversed but effects on morphodynamics persist. (blue = irradiation, n= 18 cells, quantitation in fig. S7)
Fig. 2
Fig. 2. Designing PI-GTPases
(A) Sequence conservation, surface exposure, loop “tightness”, and contact maps were used to select insertion loops (fig. S9). Orange filled boxes indicate loops fulfilling selection criteria (red dashed lines = thresholds, SS = secondary structure). Lines extending perpendicular to the diagonal indicate loops (L) that connect tightly interacting elements of secondary structure. When these lines reached the active site (green bands) the loop was selected for testing. For PI-Rac1, we selected L1, which connects strands of the β-pleated sheet in the interswitch region. (B) GTPase activity in HEK293T cells reported using biosensors fused to PI-GTPases in a high throughput assay. gray = dark state mutant, blue = lit state mutant, red = T17N (Rac1 and Cdc42) and T19N (RhoA) dominant negative mutants, green = wild type GTPase positive control. CFP x axis indicates expression level of biosensor-GTPase fusion. Error bars show s.e.m (n=3). (C) (Left) Crystal structure of PI-Rac1 with interacting proteins (gray mesh); (right) structures of wild type Rac1 (gray) and PI-Rac1 (red) are in excellent agreement. L1 loop is the LOV2 insertion loop. (D) Map showing inter-residue distances for WT Rac1 versus PI-Rac1, suggesting that these molecules undergo similar conformational changes.
Fig. 3
Fig. 3. Designing PI-GEFs
(A) Computational analysis of the Vav2 catalytic DH domain. (Left) black and red boxes indicate local and non-local interactions (fig. S9) that mediate coupling between loops and the active site. (Right) structural model of the Vav2 DH domain showing insertion loops and the active site (green). (B) In living cells, PI-Vav2 was inhibited in the lit state. DM and LM = dark and lit mutants. EA = E200A/K333A dominant negative mutant. Error bars show s.e.m (n=3). (C) (Left) Reversible retraction induced by irradiation of PI-Vav2 in HeLa cells (n=9); retraction = red arrow, protrusion = black arrow. (D) High content live cell imaging showed that PI-ITSN was inhibited in the lit state. EA = E1244A dominant negative mutant. Error bars show s.e.m (n=3). (E) (Left) Crystal structure of PI-ITSN (L2) in complex with Cdc42 superimposed on the wild type ITSN:Cdc42 complex. (F) Comparison of deuterium exchange (HD/X) results and dynamic coupling computed using molecular dynamics simulations. CL2,I corresponds to the correlation coefficient between the motion of the L2 loop and the motions of each residue. DI-d corresponds to the differences in relative deuteration levels in the dark and light.
Fig. 4
Fig. 4. Designing PA-Vav2 and multiplexed control in living cells
(A) (Left) Computational analysis of Vav2’s AID indicated that loops L1 and L2 are coupled to the active site (green) through non-local (red box) and local (black box) interactions. CH and AC denote calponin-homology and acidic motifs. (Right) A structural model of the AID showing the connection of L1 and L2 to the active site. (B) PA-Vav2 is activated in the lit state, assayed as in Fig. 2B. Error bars show s.e.m (n=3). (C) Effects of irradiation and cessation of irradiation on cells expressing PA-Vav2 alone, PI-Rac1 alone, or both in the same cell. Blue box denotes irradiation, ea = edge activity, and envelopes show s.e.m (n= 15 for PA-Vav2, n= 18 for PI-Rac1, n= 17 for PA-Vav2+PI-Rac1).

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