S1 Text -

<p><b>Fig A. AlphaFold3 predicts the designs to induce unfavorable receptor orientations. (A–F)</b> AlphaFold3 predicts the expected parallel and antiparallel orientations induced by bop2 or boa2 indicate longer C-terminal spacing of the receptor subunits, and unfavored orientations with respect to membrane-embedded receptors. Representatives of the top 5 models are shown for bop (<b>A</b>) and boa (<b>D</b>). <b>(F–H)</b> In contrast, the 2:2 G-CSF:G-CSFR complex shows much more proximal inter-TMD spacing (Fig B in S1 Text). Shown are the first 2 models which represent the conformational diversity of the 5 predicted models. Prediction quality metrics for the structures (pLDDT: <b>B</b>, <b>D</b> and <b>H</b>; pAE: <b>C</b>, <b>F</b>, and <b>I</b>) are plotted next to the predicted structures. <b>Fig B. Inter-TMD spacing of the AlphaFold3 models of design:G-CSFR complexes predict a much larger inter-TMD spacing in comparison to the G-CSF:G-CSFR complex</b>. Boxplots of the spacing measured from the 5 models generated by each prediction. Numerical data for graphical items in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig C. AlphaFold2 models predictions of the bifaceted designs and the bv6 template show minimal deviation from the template. (A)</b> Monomeric design models. <b>(B)</b> pLDDT values for the predicted structures. <b>Fig D. The G-CSFR-binding site in all design models aligned more poorly to the secondary binding site compared to the primary binding site</b>. AlphaFold2 models of the designed G-CSFR binders bop1, bop2, boa1, and boa2. The corresponding residues of the primary binding site and secondary binding site were structurally aligned to the corresponding residues of G-CSF (from PBD:2D9Q) to determine their backbone atoms RMSD. Numerical data for graphical items in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig E. Oligomeric state of Boskar4 in solution dictates its effect on either activating or inhibiting G-CSFR. (A)</b> The illustration demonstrates how Boskar4 in a two-copy tandem connected by a short linker (Boskar4_st2) can dimerize the receptor subunits (left), while the monomeric fraction can inhibit G-CSF-induced receptor dimerization (right). <b>(B)</b> Analytical SEC shows the Boskar4 design to partition between a dimer and monomer in solution. Chromatography was performed using a Superdex 200 Increase 10/300 GL analytical column, where the data was obtained from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.ref031" target="_blank">31</a>]. <b>(C)</b> The monomeric fraction of Boskar4 could to some extent inhibit G-CSF-induced proliferation of NFS-60 cells. The figure shows the results of testing 10 μg/ml Boskar4 in a cell-based (NFS-60) competitive inhibition assay against 0 pg/ml, 100 pg/ml, and 1,000 pg/ml of G-CSF, where Boskar4 could outcompete 100 pg/ml G-CSF as indicated by an observed proliferation reduction. <b>(D)</b> Varying concentrations of Boskar4 titrated against 100 pg/ml G-CSF in the same cell assay explained also showed a clear reduction in proliferation at high concentration of monomeric Boskar4 (50 μg/ml). However, below non-inhibiting concentrations, an increase in proliferation was clear. Confluence values shown in (C, D) where obtained using the IncuCyte S3 Live-Cell Analysis System, where the bar plots show mean and standard deviation of 3 technical replicates. <b>(E)</b> Proliferative activity for non-inhibiting concentrations were also observed in NFS-60 proliferation assays for the affinity enhanced Boskar4 variant bv6 (dark gray circles). Shown is the fold change to untreated cells. The proliferation level of untreated cells is indicated by a gray box which represents mean and standard deviation of 12 parallel replicates. Numerical data for graphical items in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig F</b>. <b>NanoDSF shows the bifaceted designs to be highly thermostable</b>. Heating (left column) and cooling (middle column) show no unfolding or refolding transitions for all 4 designs (bop1, bop2, boa1, and boa2) within the temperature range from 25 °C to 110 °C. Ratio values indicate relative fluorescence intensity at 350 nm and 330 nm. Scattering signal during the heating phase (right column) also indicate no aggregation of the designs. At least 4 (at most 7) technical replicates are shown for each design. Numerical data for graphical items in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig G. SPR titrations of the bifaceted designs (bop/boa) compared to the starting template (bv6)</b>. SPR sensograms (black line) and the corresponding fit (green line) of a 2-fold dilution series starting at 20 nM of bv6, bop1, bop2, boa1, and boa2. Binding kinetic parameters that were calculated from the displayed fits (Table C in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s001" target="_blank">S1 Text</a>; indicated in blue). Numerical data for graphical items in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig H. Single molecule tracking of G-CSFR. (A)</b> Diffusion coefficients of G-CSFR under different treatments, as quantified by single-molecule tracking analysis on live HeLa cell membranes. <b>(B)</b> Receptor localization densities at the cell surface, indicating similar receptor expression of analyzed cells. Numerical data for graphical items in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig I. bop2 induces proliferation only partial across a narrower concentration range when compared than G-CSF. (A)</b> Three biological replicas of NFS-60 proliferation assays performed under the comparable conditions using either rhG-CSF (circles) or bop2 (squares). Color shades indicate the experiments performed on the same place. The plot shows the individual measurements of the averaged values shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.g002" target="_blank">Fig 2A</a>. <b>(B)</b> <i>E</i><sub><i>max</i></sub> values for bop2 and rhG-CSF show the latter to be significantly more active. Numerical data for graphical items in Figs I and R<i>D-G</i> can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig J. Tandemly repeating bop2 and boa2 yields agonists, with the parallel orientation induced by bop2 exhibiting higher cell activity</b>. (A) NFS-60 proliferation assays using short-tandems of bop2 (bop2_st2) or boa2 (boa2_st2). Mean and standard deviation of 3 independent replicates are shown as lines and shades, respectively. (B, C) Individual measurements of each construct fitted to derive the respective <i>EC</i><sub><i>50</i></sub> values (D), which show bop2_st2 to be significantly more active (compare Table C). Numerical data for graphical items in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig K. The bop1 design is the most potently inhibits G-CSF activity with no residual receptor activation. (A)</b> The half-maximal effective concentration (<i>EC</i><sub><i>50</i></sub>) of the bop2 was determined for non-inhibiting concentrations observed in NFS-60 activity assays (cf. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.g002" target="_blank">Fig 2A</a>). The fits of 3 independent experiments are shown. Mean and standard deviation values are provided in Table C. <b>(B)</b> The half-maximal inhibitory concentrations (<i>IC</i><sub><i>50</i></sub>, indicated by dotted lines) were obtained from the corresponding competitive inhibition NFS-60 assay (cf. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.g002" target="_blank">Fig 2B</a>) by fitting the individual independent replicates. Mean and standard deviation of the obtained <i>IC</i><sub><i>50</i></sub> values is presented in Table C. Numerical data for graphical items in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002883#pbio.3002883.s002" target="_blank">S1 Data</a>. <b>Fig L. Bop1 outcompetes G-CSF in a dose-dependent manner</b>. Competitive inhibition assays of NFS-60 proliferation using varying concentrations of bop1 against 3 concentrations of of G-CSF (200, 100, 50 pg/ml). The half-maximal inhibitory concentrations (<i>IC</i><sub><i>50</i></sub>) were obtai