Combining NMR and Computer Simulations to Evaluate Cdc25B Protein Flexibility

Abstract

Listen

Translate article

Cdc25B is a phosphatase involved in cell cycle checkpoints and has become an important drug target because it is overexpressed in several types of cancer. Crystal structures indicate that the C-terminus of the Cdc25B catalytic domain is in close contact with the active site. On the other hand, computer simulations and bioinformatic predictors suggest a large conformational flexibility in this region. In order to obtain detailed insight on the dynamics of the Cdc25B C-terminal segment in solution, we produced isotopically enriched protein samples for nuclear magnetic resonance (NMR) data acquisition. Agreement between backbone dihedral angles obtained from the crystal structure and predicted from NMR chemical shifts (CS) suggest consistency between the crystal and the solution structure of Cdc25B. Comparisons between experimental backbone CS and values back-calculated from long molecular dynamics (MD) simulations (total aggregate MD time of 10 us) suggest significant conformational flexibility in several Cdc25B regions. In particular, the greater amplitude of CS fluctuations for the terminal residues in comparison with the non-terminal ones is consistent with higher flexibility at the terminal regions. For instance, Ser373 located at the N-terminus populates two different states characterized by a 13Calpha CS of 58 and 59.5 ppm. These fluctuations are mainly associated with changes in psi and chi1 dihedral angles observed along the MD trajactory. Our results illustrate the power of the combination of NMR and MD simulations in order to obtain a faithful description of the conformational distribution of proteins in solution.