Fig. 6: Design of binders to disordered regions of endogenous human proteins.
From: De novo design of modular peptide-binding proteins by superhelical matching
a, Schematic model of the human PAXT complex composed of a heterotetramer of ZFC3H1 and MTR4. CC, coiled-coil ___domain; ZN, Zn-finger ___domain. Inset shows the sequence environment of the target sequence. b, Surface shape complementarity between the target peptide from ZFC3H1 (sphere) and the highest-affinity cognate binder, αZFC-high. c, Fluorescence polarization binding curves between the indicated ZFC3H1 binders and the target ZFC3H1 peptide (PLP)4PEDPEQPPKPP. As a negative control, we used the (PLP)x6 binder, RPB_PLP3_R6 (see Fig. 4). αZFC-high shows a higher binding affinity to the target peptide than αZFC-low, in contrast with RPB_PLP3_R6, which shows negligible binding. d, Superdex 200 10/300 GL SEC profiles of purified αZFC-high, a fusion between GFP and a 103-amino-acid fragment of the disordered region of ZFC3H1 containing the target sequence (see a), or a 1:1 mix of the two after two hours of incubation. OD280 nm, optical density at 280 nm. e, Top, HeLa cell extracts were subjected to pull-down using the indicated binders bound to Ni-NTA agarose beads, or naked beads as a control. Recovered proteins were processed for western blot against endogenous ZFC3H1 (or tubulin as a loading control). Bottom, Coomassie-stained SDS–PAGE gel of the samples analysed at the top. These panels are representative of n = 3 experiments. f, Proteomic analysis of the His-pull-down samples shown in e. Top, overlap between the proteins identified, setting a threshold of five peptides for correct identification. Bottom, examples of proteins identified (number indicates exclusive peptide count; protein coverage is indicated in parentheses). See Source Data for the full dataset. For gel source data, see Supplementary Fig. 1.
