Portability of a Small-Molecule Binding Site between Disordered Proteins

Abstract

Intrinsically disordered proteins (IDPs) are important in both normal and disease states. Small molecules can be targeted to disordered regions, but we currently have only a limited understanding of the nature of small-molecule binding sites in IDPs. Here, we show that a minimal small-molecule binding sequence of eight contiguous residues derived from the Myc protein can be ported into a different disordered protein and recapitulate small-molecule binding activity in the new context. We also find that the residue immediately flanking the binding site can have opposing effects on small-molecule binding in the different disordered protein contexts. The results demonstrate that small-molecule binding sites can act modularly and are portable between disordered protein contexts but that residues outside of the minimal binding site can modulate binding affinity.

Keywords: Myc; SLiM; drug targets; intrinsically disordered proteins; protein-protein interaction; small-molecule inhibitors.

Figure 1

(A) Structure of 34RH. (B) Inner filter corrected fluorescence emission spectrum of 1 μM Myc_353–437 with (black circles) and without (white circles) 50 μM 34RH in 1xPBS at 25 °C, pH 7.4. (C) Equilibrium titration of 1 μM Myc_353–437 with excess 34RH fit to a Langmuir binding isotherm, K_D = 3.9 ± 1.3 µM. Error bars represent the standard error of three independent trials. (D) Circular dichroism of 2.5 µM Myc_353–437 with (black circles) and without (white circles) 50 µM 34RH in 1xCD buffer.

Figure 2

(A) Inner filter corrected fluorescence emission spectrum of 1 µM Myc_402–412 peptide with (black circles) and without (white circles) 50 µM 34RH. (B) Fraction quenched titration curve for 1 µM Myc_402–412 peptide and 34RH fitted to a Langmuir binding isotherm yielding a K_D = 11.5 ± 1.2 µM. Error bars represent the standard error of three independent trials. (C) CD spectrum of 2.5 µM Myc_402–412 peptide with (black circles) and without (white circles) 50 µM 34RH.

Figure 3

Alignment of the binding site region of Myc_353–437 with MaxRH and Max. Outlined residues are identical. Underlined residues denote the Myc_402–412 sequence. Highlighted green residues represent the overlap of the minimal binding site between the proteins.

Figure 4

(A) Inner filter corrected fluorescence emission spectrum of 1 µM MaxRH with (black circles) and without (white circles) 50 µM 34RH. (B) Fraction quenched titration curve for 1 µM MaxRH with 34RH fit to a Langmuir binding isotherm, K_D = 23.4 ± 1.1 µM (C) CD spectrum of 1 µM MaxRH with (black circles) with without (white circles) 50 µM 34RH. (D) Inner filter corrected fluorescence emission spectrum of 1 µM MaxRH-Y115F/Y123F with (black circles) and without (white circles) 50 µM 34RH. (E) Fraction quenched titration curve for 1 µM MaxRH-Y115F/Y123F with 34RH fit to a Langmuir binding isotherm, K_D = 14.9 ± 1.9 µM (F) CD spectrum of 1 µM MaxRH-Y115F/Y123F with (black circles) with without (white circles) 50 µM 34RH. (G) Inner filter corrected fluorescence emission spectrum of 1 µM P22 Max with (black circles) and without (white circles) 50 µM 34RH. (H) Fraction quenched titration curve for 1 µM P22 Max with 34RH (I) CD spectrum of 1 µM P22 Max with (black circles) with without (white circles) 50 µM 34RH at pH 7.4.

Figure 5

(A) CD of 1 µM P22 Max at pH 6 with (black circles) and without (white circles) 50 µM 34RH. (B) CD of 1 µM MaxRH at pH 6 with (black circles) and without (white circles) 50 µM 34RH. (C) CD of 4 µM P21 Max at pH 7.4 with (black circles) and without (white circles) 50 µM 34RH. (D) Fraction quenched titration curve for 1 µM P22 Max with 34RH at pH 6. (E) Fraction quenched titration curve for 1 µM MaxRH with 34RH at pH 6 fitted to a Langmuir binding isotherm, K_D = 9.1 ± 3.9 µM (F) Fraction quenched titration curve for 1 µM P21 Max with 34RH at pH 7.4. Error bars represent the standard error of three independent trials.

Figure 6

(A) MaxRH-N78E fluorescence quenching titration curve (black circles) overlaid with MaxRH curve (white circles). (B) Myc_353–437 E410N fluorescence quenching titration curve (black circles) overlaid with Myc_353–437 curve (white circles). (C) Myc_353–437 A401E fluorescence quenching titration curve (black circles) overlaid with Myc_353–437 curve (white circles). Error bars represent the standard error of three independent trials.