  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.
