Conformational flexibility of the complete catalytic domain of Cdc25B phosphatases

Abstract

Cdc25B phosphatases are involved in cell cycle checkpoints and have become a possible target for developing new anticancer drugs. A more rational design of Cdc25B ligands would benefit from detailed knowledge of its tertiary structure. The conformational flexibility of the C-terminal region of the Cdc25B catalytic domain has been debated recently and suggested to play an important structural role. Here, a combination of experimental NMR measurements and molecular dynamics simulations for the complete catalytic domain of the Cdc25B phosphatase is presented. The stability of the C-terminal α-helix is confirmed, but the last 20 residues in the complete catalytic domain are very flexible, partially occlude the active site and may establish transient contacts with the protein core. This flexibility in the C-terminal tail may modulate the molecular recognition of natural substrates and competitive inhibitors by Cdc25B. Proteins 2016; 84:1567-1575. © 2016 Wiley Periodicals, Inc.

Combining free energy simulations and NMR chemical-shift perturbation to identify transient cation-π contacts in proteins

“…Side-chains of R488 and R492 make strong electrostatic interactions and remain buried within the secondary phosphate binding pocket. 15,21 Side-chains of R485, R490 and R562 are fully exposed to solvent but do not perform a stable cation-π interaction with W550, as shown by the dissociative free energy profiles estimated here (Figs. 3 and S1).…”

“…Combining free energy simulations and NMR chemical-shift perturbation to identify transient cation-π contacts in proteins 1.884 Note the ratio between 1 H and 15 N scaling factors (j α /k α ) is similar to 0.15, the standard value used in CSP studies, 21,38 for all N-H systems except for Trp Nǫ1-H. S1 Table S2: Side-chain distance (in nm) to W550 in the crystallographic x-ray structure 14 and minimum distance (in nm) observed along 6 µs of MD simulation 21 for all Arg residues of the complete Cdc25B C-terminal domain. Table S3: Side-chain distance (in nm) to W550 in the crystallographic x-ray structure, 14 minimum distance (in nm) observed along 6 µs of MD simulation, 21 Figure S1: Free energy profiles for the cation-π interaction between W550 side-chain and Arg 490 (panel A), 554 (B) and 556 (C) side-chains. Shadows around lines in all panels show the statistical uncertainty.…”

“…Profiles were obtained after 100 ns of total simulation time per US window and the first 30ns were discarded from analysis. These simulations were not carried out longer because the dissociative profiles shown and the low conservation of these Arg residues 21 suggested these are not important interactions for the Cdc25B conformational ensemble. Figure S2: NMR spectra.…”

Combining Free Energy Simulations and NMR Chemical-Shift Perturbation To Identify Transient Cation−π Contacts in Proteins

“…Sixteen residues (A551-Q566) lacking from the native C-terminal structure 14 and seven residues in the N-terminal part of the experimental con-struction (see below) were manually added in an extended conformation. 21 Crystallographic water, ions and β-mercaptoethanol (BMER) were removed. Hydrogen atoms were added and a standard protonation state at pH=7 was assumed for side-chains.…”

“…Human Cdc25B catalytic domain contains 17 Arg, 13 Lys and only one Trp residue. We first analyzed the distance of each cationic residue to W550 in the Cdc25B x-ray structure 14 and in a previous long MD simulation (total time of 6 µs, Table S2) 21,23 to check which cation-π interactions could be formed. We discarded Trp-Lys interactions and concentrated in Trp-Arg pairs, which interactions were shown above to be well described by the CHARMM36 force-field.…”