Design of protein-interaction specificity gives selective bZIP-binding peptides

Abstract

Interaction specificity is a required feature of biological networks and a necessary characteristic of protein or small-molecule reagents and therapeutics. The ability to alter or inhibit protein interactions selectively would advance basic and applied molecular science. Assessing or modelling interaction specificity requires treating multiple competing complexes, which presents computational and experimental challenges. Here we present a computational framework for designing protein-interaction specificity and use it to identify specific peptide partners for human basic-region leucine zipper (bZIP) transcription factors. Protein microarrays were used to characterize designed, synthetic ligands for all but one of 20 bZIP families. The bZIP proteins share strong sequence and structural similarities and thus are challenging targets to bind specifically. Nevertheless, many of the designs, including examples that bind the oncoproteins c-Jun, c-Fos and c-Maf (also called JUN, FOS and MAF, respectively), were selective for their targets over all 19 other families. Collectively, the designs exhibit a wide range of interaction profiles and demonstrate that human bZIPs have only sparsely sampled the possible interaction space accessible to them. Our computational method provides a way to systematically analyse trade-offs between stability and specificity and is suitable for use with many types of structure-scoring functions; thus, it may prove broadly useful as a tool for protein design.

Figure 1

Designing specific peptides using CLASSY. A) Specificity sweep scheme. A sequence (black) is sought that binds a target (red) but not several undesired partners (gray) or itself. Panels from left to right illustrate iterations of the CLASSY procedure, during which the specificity gap Δ is increased. B) and C) A specificity sweep with MafG as the target and all other human bZIP coiled coils (except MafK, in the same family as MafG) and the design homodimer as undesired complexes. The plot in B corresponds to the cartoon in A. Red dots, black bars and gray bars represent energies of the design•target, design•design, and design•other bZIP complexes, respectively. C plots design•target complex stability vs. specificity (Δ). Portions of several designed complexes are shown using helical wheels (orange highlights amino-acid changes from the previously shown sequence). The rightmost solution is anti-SMAF.

Figure 2

Experimental testing of anti-bZIP designs. A) Peptide array results for the most specific design identified for each human bZIP family. Columns show experiments using the indicated protein to probe an array. For the Specificity panel (left), designs in solution were used to probe human bZIPs and designs on the surface. In the Relative Stability panel (right), human bZIP targets were used to probe an array containing the cognate design of each target and 33 human bZIPs. Data are plotted as -log(F/F_max), with F the fluorescence signal on the array, such that the strongest interaction has a value of zero. Values of -log(F/F_max) above 1.0 were set to 1.0. Thick red circles – design•target; thin red circles – design interactions with siblings in the target family; grey squares – interactions with other human bZIPs; black squares – design•design. Designs are named using the family of their target. B) Solution testing of anti-SMAF complexes assayed using circular dichroism. In each panel, anti-SMAF alone is shown with dashed lines, the partner being tested with a solid line, the numerical average of these two signals with open circles (◦) and the mixture of the two peptides with closed circles (•). (B, C) Anti-SMAF interacts with target MafG (T_m ~ 38 °C). (D) Anti-SMAF interacts, at most, very weakly with cJun, the closest competitor according to microarray data. (E) There is no evidence for anti-SMAF interacting with MafB, a sequence closely related to the target. CD spectra in (B) were collected at 25 °C. Anti-SMAF unfolds with T_m ~12 °C. Similar data for other complexes are included in Supplemenatary Figures 3–8.

Figure 3

Properties of designed peptides compared to human bZIP leucine-zippers. A) Hierarchical clustering of interaction profiles for 33 human peptides and 48 designs; an interaction profile consists of the array signals for interactions with 33 surface-bound human peptides. Proteins on the surface are in columns and those in solution are in rows, with designed proteins and their interaction profiles in blue and human bZIP interaction profiles in yellow. B), C) Sequence logos for a, d, e, and g positions from the first 5 heptads of the 33 human bZIP leucine zippers in B) and the 48 designed peptides in C) (http://weblogo.berkeley.edu).