CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones

Abstract

The detection of small molecules in living cells using genetically encoded FRET sensors has revolutionized our understanding of signaling pathways at the sub-cellular level. However, engineering fluorescent proteins and specific binding domains to create new sensors remains challenging because of the difficulties associated with the large size of the polypeptides involved, and their intrinsically huge conformational variability. Indeed, FRET sensors' design still relies on vague structural notions, and trial and error combinations of linkers and protein modules. We recently designed a FRET sensor for the second messenger cAMP named CUTie (Cyclic nucleotide Universal Tag for imaging experiments), which granted sub-micrometer resolution in living cells. Here we apply a combination of sequence/structure analysis to produce a new-generation FRET sensor for the second messenger cGMP based on Protein kinase G I (PKGI), which we named CUTie2. Coarse-grained molecular dynamics simulations achieved an exhaustive sampling of the relevant spatio-temporal coordinates providing a quasi-quantitative prediction of the FRET efficiency, as confirmed by in vitro experiments. Moreover, biochemical characterization showed that the cGMP binding module maintains virtually the same affinity and selectivity for its ligand thant the full-length protein. The computational approach proposed here is easily generalizable to other allosteric protein modules, providing a cost effective-strategy for the custom design of FRET sensors.

Keywords: CUTie; FRET; Sirah; biosensor; coarse-grained; cyclic nucleotide; molecular dynamics; signaling.

FIGURE 1

Design of CUTie2. (A) Global secondary structure of a CNBD and multiple sequence alignment of related domains. Rectangles and arrows are used to indicate α-helices and β-strands. Sequences 1 to 12, and 13 to 24 correspond to cGMP and cAMP binding proteins, respectively. These correspond to the UNIPROT codes, for the cGMP binding domains: 1) Q13976, 2) Q13237, 3) A0A444U9Q8, 4) A0A443SJC3, 5) B0X970, 6) A0A4Y2N1F1, 7) A0A4Y2BIM3, 8) A0A419PY19, 9) A0A2P8Y1R0, 10) A0A3R7JPE0, 11) A0A444UAH2, 12) A0A0M4ENX8 and the cAMP binding modules: 13 and 14) P00514, 15 and 16) P12369, 17) A0A2J8TG41, 18) E0VRT6, 19 and 20) A0A091CJC2, 21) A0A556UFG6, 22) A9QQ52, 23 and 24) A0A384A0R8. Aminoacids are colored by physicochemical character according to CLUSTAL. (B) Cartoon representation and domain organization of the CUTie2 sensor. Notice that the N-terminal of the protein remains free to be eventually fused to arbitrary targeting domains. The schematic architecture of the sensor and definition of the geometric factors and equations used to calculate the FRET signal are provided on the right bottom.

FIGURE 2

CG simulation of the CUTie2 sensor. (A) Instantaneous RMSD of one randomly chosen conformer in cGMP-bound conformation. Black, gray, yellow, and cyan correspond to the whole molecule, the CNBD, the YFP, and the CFP modules, respectively. (B–D) Distance between chromophores, κ², and FRET efficiency. The orange line in each panel corresponds to the average over the entire trajectory.

FIGURE 3

FRET efficiency and experimental validation. (A) Averaged FRET values calculated from different conformers vs. simulated time for cGMP-bound and -free sets of simulations. (B) Assessment of the completeness of the conformational sampling for the cGMP-bound simulations. The FRET efficiency is reported as a function of D and κ² for each of the conformers (indicated by different colors). Six particular conformers taken from three trajectories are shown in cartoon representation to illustrate the conformational dispersion achieved during the simulations. Gray spheres indicate the cGMP binding sites. The letters in lower case indicate the position of the corresponding conformer in the graph. (C) Representative concentration-response plot for recombinant CUTie2 titrated with different concentrations of cGMP and cAMP (0–10 mM). The data were fitted to the Boltzmann equation (R ² ≥ 0.98).