Conformational Plasticity of Cyclic Ras‐Inhibitor Peptides Defines Cell Permeabilization Activity

Abstract

Cyclorasins 9A5 and 9A54 are 11‐mer cyclic peptides that inhibit the Ras‐Raf protein interaction. The peptides share a cell‐penetrating peptide (CPP)‐like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell‐based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three‐dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino‐acid sidechains, Tle‐2 and dVal‐3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.

Flexibility and Cell Permeability of Cyclic Ras-Inhibitor Peptides Revealed by the Coupled Nosé–Hoover Equation

“…The overall FESs from the cNH simulations were found to include the NMR structures, 42 confirming the agreement between the present enhanced samplings and the experiment (a slight difference from the most probable structure at the bottom of the basin is discussed below). The FESs for 9A5 and 9A54 appeared similar but were found to include substantial differences by calculating the quantities that characterize the feature of each cluster (Table 1; see Section 2 for detail).…”

“…The cNH simulations were performed for two 11-mer cyclic peptides, cyclorasin 9A5 and 9A54. Initial structural models were taken from one of the NMR structures recently determined by Takeuchi et al 42 (deposited in Biological Magnetic Resonance Data Bank entries 50415/50417 and 50416/50418 for 9A5/ 9A54 in water and in DMSO, respectively). Four Arg residues were protonated in water and deprotonated in DMSO, depending on the dielectric constants of the two environments.…”

“…To examine similarity/dissimilarity between the 9A5/9A54 structural ensembles, principal component analysis was performed, which visualized the ring structures that were common to both the cyclic peptides. For this purpose, all of the Cα-atoms of each structure generated in the cNH simulations of both 9A5 and 9A54 were fitted to a reference coordinate (one of the 9A5 NMR structures 42 ) and used to calculate the variance− covariance matrix of the interatomic fluctuations, wherein the weight w BG,300 K was duly considered. The matrix thus obtained was diagonalized to characterize the variations of the structure data along the derived eigenvectors named principal compoments (PCs).…”

“…To do this, the inter-residue hydrogen distances were compared that were measured by the nuclear Overhauser effect (NOE) of the solution NMR experiment. 42 The upper bound of interatomic distance derived from the NOE intensity, ⟨r⟩ exp , was used as the experimental value, while the average of r −6 over the cNH trajectory together with the reweighting to the canonical ensemble (see Section 2) was used to calculate the MD simulation value, ⟨r⟩ MD . This was performed in accordance with the temperature of the NMR experiment (283 K in water and 298 K in DMSO).…”

“…Recently, the three-dimensional structures of both 9A5 and 9A54 were determined in aqueous and a less polar solvent dimethyl sulfoxide (DMSO) conditions, revealing the structural origin of the decreased cell permeability via such modifications from 9A5 to 9A54 as the decreased conformational plasticity due to the bulkiness of the modified side chains, Tle2 and dVal3. 42 Cyclorasin is characterized as having many long side-chain residues such as Trp and Arg, 42 compared to other cyclic peptides such as cyclosporine. 30,43 Since such side-chain atoms in cyclorasin are tightly packed within the restriction on the cyclic ring of the main-chain atoms, the conformational change of cyclorasin is a complicated puzzle: the change in the main chain is allowed by accompanying complicated combinations of the side-chain rotamer flips.…”