SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains

Abstract

The synthetic biology toolkit contains a growing number of parts for regulating transcription and translation, but very few that can be used to control protein association. Here we report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. We present biophysical analysis of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the number of available protein interaction parts for synthetic biology and should facilitate a wide range of molecular engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/].

Figure 1

Biophysical properties of SYNZIPs. (a) SEC elution traces. The left panel shows SYNZIP1 in green, SYNZIP2 in blue, the SYNZIP1:SYNZIP2 mixture in red, and the GCN4-pIqI trimer control in black. The right panel shows SYNZIP21 in blue, SYNZIP19 in green, the SYNZIP19:SYNZIP21 mixture in red, and the GCN4-pIqI trimer control in black. (b) The cartoon shows a schematic of the FRET assay, and the bar graph shows FRET efficiencies for selected pairs. Hatched bars are the N-terminal donor/N-terminal acceptor mixes, and gray bars are the C-terminal donor/N-terminal acceptor mixes. (c) Representative FP titrations plotted as the fraction of the fluorescein labeled protein bound. Each plot shows reciprocal measurements, with each interaction partner used in turn as the labeled species. Raw data, showing millipolarization, is available in Supplementary Figure 3. (d) Competition between strong and weak SYNZIP pairs. Observed interactions are summarized in graphs at the top of each plot, where circles represent SYNZIPs, bold lines indicate strong interactions, and dotted lines indicate weak interactions observed in this assay. The fluorescence polarization of different mixtures is shown in the bar graphs. “F” and “R” in the legend of each plot designates the species labeled with fluorescein or rhodamine, and the curves show the best fit to the data (see Methods). Error bars show ±1 SD over three (b and c) or four (d) replicates.

Figure 2

SYNZIP interactions detected by Y2H. (a) Histidine selection with 100 mM 3-AT, 12 days growth, represented in greyscale with white as no growth and black as strongest growth. (b) Heat map comparing Y2H data with coiled-coil microarray data, with Y2H in the lower left and microarray in the upper right. Red ×'s indicate an interaction observed in Y2H but not seen on the microarray or vice versa. Y2H data is shown as black, strong interaction; gray, undetermined interaction; white, no interaction; blue, two autoactivators (see Methods). Microarray data is the maximum of the two reciprocal measurements ranging from no interaction (white, arrayscore > 1) to strong interaction (black, arrayscore = 0). (c, d) Uracil selection, 12 days growth and heat map comparison with microarray, as in panel b.

Figure 3

Cell fluorescence as a measure of MAPK pathway modulation by SYNZIP pairs. SYNZIP pairs are rank ordered, left-to-right, by the relative mean cell fluorescence induced when the pair was used to recruit Msg5 to Ste5 (inset). The first SYNZIP listed was fused to the Ste5 scaffold protein and the second was fused to Msg5 phosphatase. Green bars indicate interacting pairs as determined by Y2H and coiled-coil microarray. Two instances where SYNZIPs that interacted on the array showed little MAPK pathway down-regulation are highlighted in yellow. Average signals from 4 replicates are reported relative to the average signal for a Ste5-SYNZIPX:Msg5-nozipper control, with X the corresponding SYNZIP for the Ste5 fusion. Error bars show 1 SD of 4 measurements.

Figure 4

SYNZIP interaction specification sheet for pair SYNZIP1:SYNZIP2.

Figure 5

Network motifs constructed from SYNZIP pair interactions: (a) linear, (b) ring, (c) hub, and (d) orthogonal-pair motifs. Proteins are denoted as circles, with the SYNZIP number indicated. Strong interactions are shown with solid lines, and weak interactions with dashed lines. SYNZIPs that are not connected by an edge did not interact in the Y2H or previous coiled-coil microarray assays. All interactions and some non-interactions were confirmed in vitro in this work. The asterisk indicates an antiparallel interaction between SYNZIP17 and SYNZIP18.