All-atom model for stabilization of alpha-helical structure in peptides by hydrocarbon staples

Abstract

Recent work has shown that the incorporation of an all-hydrocarbon "staple" into peptides can greatly increase their alpha-helix propensity, leading to an improvement in pharmaceutical properties such as proteolytic stability, receptor affinity, and cell permeability. Stapled peptides thus show promise as a new class of drugs capable of accessing intractable targets such as those that engage in intracellular protein-protein interactions. The extent of alpha-helix stabilization provided by stapling has proven to be substantially context dependent, requiring cumbersome screening to identify the optimal site for staple incorporation. In certain cases, a staple encompassing one turn of the helix (attached at residues i and i+4) furnishes greater helix stabilization than one encompassing two turns (i,i+7 staple), which runs counter to expectation based on polymer theory. These findings highlight the need for a more thorough understanding of the forces that underlie helix stabilization by hydrocarbon staples. Here we report all-atom Monte Carlo folding simulations comparing unmodified peptides derived from RNase A and BID BH3 with various i,i+4 and i,i+7 stapled versions thereof. The results of these simulations were found to be in quantitative agreement with experimentally determined helix propensities. We also discovered that staples can stabilize quasi-stable decoy conformations, and that the removal of these states plays a major role in determining the helix stability of stapled peptides. Finally, we critically investigate why our method works, exposing the underlying physical forces that stabilize stapled peptides.

Figure 1

(A) Experimental (grey) and simulation (black, blue, and red, T=0.72) percent helicities for WT and stapled peptides. Simulation results for alanine mutants (yellow or green, T=0.72) are also depicted. (B) Peptide sequences used in this study. Astericks (*) denote unnatural amino acids connecting the staple to the peptide. Residues mutated to alanine in control peptides are colored green.

Figure 2

Probability of each residue in RNAse A (A) or BID BH3 (B) peptides to reside in a helical region during simulations (T=0.72). Probabilities of finding RNAse A (C) or BID BH3 (D) peptides in α-helical (α-H), denatured (D), or decoy (DY) state during simulations (T=0.72).

Figure 3

Representative decoy structures for the BID BH3 WT (left), i,i+4 stapled (middle), and i,i+7 stapled (right) peptides. The N-termini are at the top of the image and the staples are rendered blue (i,i+4) or red (i,i+7).

Figure 4

RNAse A (A) and BID BH3 (B) changes in hydrogen bonding δH(hb) and sequence-dependent backbone torsions δH(bbtor) of denatured (D) and helical (α-H) states of stapled and Ala mutant peptides relative to the corresponding WT peptide. Probability that a residue is classified as a bend for RNAse A (C) and BID BH3 (D). A cartoon depicting the staple is above the plot. E: Probability of finding conformations with a certain number of “bend” residues in the denatured state for RNAse A peptides. F: The average backbone triplet energy H(bbtor) of conformations in the denatured state versus number of “bend” residues. RNAse A and BID BH3 simulations were carried out at T=0.78 and T=0.70, respectively. Colors of peptides in C, D, E, and F are as follows: WT (black), i,i+4 stapled (blue), i,i+7 stapled (red), i,i+4 Ala (yellow), i,i+7 stapled (green).