Rational peptide design for regulating liquid-liquid phase separation on the basis of residue-residue contact energy

Abstract

Since liquid-liquid phase separation (LLPS) of proteins is governed by their intrinsically disordered regions (IDRs), it can be controlled by LLPS-regulators that bind to the IDRs. The artificial design of LLPS-regulators based on this mechanism can be leveraged in biological and therapeutic applications. However, the fabrication of artificial LLPS-regulators remains challenging. Peptides are promising candidates for artificial LLPS-regulators because of their ability to potentially bind to IDRs complementarily. In this study, we provide a rational peptide design methodology for targeting IDRs based on residue-residue contact energy obtained using molecular dynamics (MD) simulations. This methodology provides rational peptide sequences that function as LLPS regulators. The peptides designed with the MD-based contact energy showed dissociation constants of 35-280 nM for the N-terminal IDR of the tumor suppressor p53, which are significantly lower than the dissociation constants of peptides designed with the conventional 3D structure-based energy, demonstrating the validity of the present peptide design methodology. Importantly, all of the designed peptides enhanced p53 droplet formation. The droplet-forming peptides were converted to droplet-deforming peptides by fusing maltose-binding protein (a soluble tag) to the designed peptides. Thus, the present peptide design methodology for targeting IDRs is useful for regulating droplet formation.

Figure 1

Comparison between MD-based energy and 3D structure-based energy for residue pairs. (A) MD-based relative contact energy matrix for side chains. (B) 3D structure-based relative contact energy matrix obtained by Miyazawa and Jernigan.

Figure 2

Peptide design scheme and binding properties of the designed peptides to p53 N-terminal IDR. (A) Schematic diagram of peptide design targeting p53 N-terminal IDR. NT, Core, Tet, and CT in the primary sequence of p53 represent the N-terminal, core, tetramerization, and C-terminal domains, respectively (top left), where thick and thin bands represent folded and disordered regions, respectively. The NT sequence (1–94) was used for the present peptide design (bottom right). Blue and red characters denote positive and negative charged residues, respectively. The peptide sequences with the highest binding property were designed based on the calculation of one-by-one (OO) or one-by-three (OT) contact energy for each residue of the p53 sequence (right). Ten-residue peptides designed with the MD-based energy are shown as examples. (B) Sequence, complimentary target residue number, and dissociation constant (K_d) of designed peptides used in this study. The top and bottom four peptides were designed with the 3D structure-based and MD-based energies, respectively. For example, 3D-OO-10 and MD-OT-16 designate a 10-residue peptide designed with 3D structure-based OO contact energy and a 16-residue peptide designed with the MD-based OT contact energy, respectively. K_d was determined by titrating peptides against the target p53 N-terminal peptide fragment underlined in (A). Errors represent the fitting errors of titration data.

Figure 3

All designed peptides targeting p53 N-terminal IDR promote p53 droplet formation. (A) Effect of the 3D structure-based designed peptides targeting the N-terminal IDR as additives on p53 droplet formation detected as OD₃₅₀. (B) Effect of the MD-based designed peptides targeting the N-terminal IDR as additives on p53 droplet formation detected as OD₃₅₀. (C) DIC images of p53 solutions in the presence and absence of 250 µM designed peptides. “None” denotes the absence of designed peptides as control. Scale bar, 20 µm. In (A and B), error bars represent standard error (N ≥ 3).

Figure 4

Soluble MBP-tag converts the designed peptide from an accelerator to a suppressor for p53 droplet formation. (A) Schematic illustration of primary sequences of the MBP-fused designed peptides and MBP used in this study. TEV represents the TEV protease cleavage sequence. The thick and thin bands correspond to folded and disordered regions, respectively. (B) Effect of the MBP-fused designed peptides or MBP as additives on p53 droplet formation detected as OD₃₅₀. (C) DIC images of p53 solutions in the presence and absence of 125 µM MBP-fused designed peptides and MBP. Scale bar, 20 µm. These images were taken after 5 min of incubation of the p53 solutions with additives or without additive (none). (D) Time course of scattering (OD₃₅₀) from 12.5 µM p53 solutions after incubation in the presence of 125 µM MBP-fused designed peptides or MBP for 5 min and the subsequent addition of TEV protease. The data before adding TEV protease are plotted at 0 min. “None” denotes the absence of MBP-fused designed peptide or MBP additives as control. In (B and D), error bars represent standard error (N = 3).