Molecular dynamics simulations and in vitro analysis of the CRMP2 thiol switch

TL;DR

This study combines molecular dynamics simulations and in vitro experiments to elucidate how redox regulation via disulfide bonds influences CRMP2 structure and function, revealing insights into its phosphorylation and oxidation interplay.

Contribution

It provides the first detailed structural analysis of CRMP2's redox switch and its impact on protein stability and regulation, integrating computational and experimental approaches.

Findings

CRMP2 aggregates below 200 mM bivalent ion concentration.

Redox state alters solvent accessibility of Ser518.

Disulfide bond reduction causes significant structural relaxation.

Abstract

Collapsin response mediator protein CRMP2 (gene: DPYSL2) is crucial for neuronal development. The homotetrameric CRMP2 complex is regulated via two mechanisms, first by phosphorylation at, and second by reduction and oxidation of the Cys504 residues of two adjacent subunits. Here, we analyzed the effects of this redox switch on the protein in vitro combined with force field molecular dynamics (MD). Earlier X-ray data contain the structure of the rigid body of the molecule but lack the flexible C-terminus with the important sites for phosphorylation and redox regulation. An in silico model for this part was established by replica exchange simulations and homology modelling, which is consistent with results gained from CD spectroscopy with recombinant protein. Thermofluor data indicated that the protein aggregates at bivalent ion concentrations below 200 mM. In simulations the protein surface was covered at these conditions by large amounts of ions, which most likely prevent aggregation. A tryptophan residue (Trp295) in close proximity to the forming disulfide allowed the measurement of the structural relaxation of the rigid body upon reduction by fluorescent quenching. We were also able to determine the second order rate constant of CRMP2 oxidation by H2O2. The simulated solvent accessible surface of the hydroxyl group of Ser518 significantly increased upon reduction of the disulfide bond. Our results give first detailed insight in the profound structural changes of the tetrameric CRMP2 due to oxidation and indicate a tightly connected regulation by phosphorylation and redox modification.