Figure 2. S-Nitrosylation of MEF2 at Cys³⁹ in the MADS-Box DNA Binding Domain.

(A) Recombinant MEF2C protein was infused into a mass spectrometer (LTQ-Orbitrap-XL with ETD) to obtain molecular ion spectra in the presence or absence of NO donor, 50 µM SNOC (n = 2 independent experiments).

(B) HEK293T cells transfected with WT myc-tagged MEF2C (WT), MEF2C(C39A) or MEF2C(C41A) were incubated with SNOC. SNO-MEF2C was determined by biotin switch. SNO proteins were precipitated and SNO-MEF2C detected with anti-myc antibody (n = 3 independent experiments; quantification shown in Figure S2A).

(C) HEK293 cells stably expressing nNOS were transfected with V5-tagged MEF2C (WT) or V5-tagged MEF2C(C39A), and nNOS activated with Ca²⁺ ionophore A23187. V5-tagged MEF2C proteins were immunoprecipitated with anti-V5 antibody and subjected to DAN assay. Values are mean + SEM (n = 3 independent experiments, *p < 0.05 by t test).

(D) Sequence alignment of MADS-box DNA binding domains of MEF2 (human isoforms MEF2A-D; Drosophila D-MEF2). α-Helix (bars) and β-strands (arrows). Cys³⁹ S-nitrosylation site (yellow). Residues involved in pocket formation (red).

(E) Cys³⁹ (yellow) is symmetrically located at the hinge between α1-helix and β1-strands in the MEF2 (monomer A in blue, monomer B in green)-DNA (pink) complex (PDB ID: 1EGW). Right: viewed at 90°.

(F) Electrostatic view of MEF2 (negative charge in red, positive charge in blue). Cys³⁹ is present at the bottom of the pocket created by Glu¹⁴, Arg¹⁷, Ser⁵⁰ Ser⁵¹, Asn⁴⁹, Phe²¹, and Gln¹⁸.

(G) Cys³⁹ of MEF2 monomer A (blue) is surrounded by residues (Glu¹⁴ Arg¹⁷, Ser⁵⁰, Ser⁵¹, Asn⁴⁹, Phe²¹, and Gln¹⁸; red) of monomer B (green). Right: complex viewed at 90°.

See also Figure S2.