The structure of the MAP2K MEK6 reveals an autoinhibitory dimer

Abstract

MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/DeltaN/DD) has been solved at 2.3 angstroms resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/DeltaN/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare "arginine stack" between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.

Figure 1. The structure of MEK6 /ΔN/DD

A: Left panel shows the two dimers in the asymmetric unit; and the center panel shows one of the crystallographic dimers. The two monomers are in yellow and pink. The disordered activation loop is shown in dotted line. B: A Monomer is depicted with the side-chains of residues Glu68, Lys82, Asp99, Asp197, Phe198 and Tyr203 represented in ball and stick. TAO2 structure in the same orientation is shown here for comparison. The red box highlights the Ω-loop in MEK6/ΔN/DD and compares it with β1-strand, and β2-strand enclosed in red box in TAO2. C: Close-up view of the C-terminus, helices J and K. Residues in the “DVD” motif are shown as yellow spheres, His317, Thr322, Val324, Phe327, Val328, and Ile331. We mutated the residues in the dimer interface, Asp124, Tyr64, Arg83, Arg61, Leu59 and Tyr203 (shown as orange spheres) to alanine. Leu332 (green sphere) is in the DVD motif and was also mutated. Note that the second subunit of the dimer is nearby (in yellow). D: Close-up view of the activation loop and PYMAPER loop in the same color scheme as in A. Arg178, Asp179, Lys181, Pro218, Tyr219, Leu229 and Arg224. Drawn in Pymol (DeLano, 2002).

Figure 2. The dimer interface

A: Stereo view of the two Ω-loops bound to the opposite subunit active sites, and contacts with C-terminus. Two subunits shown, and rendered in cartoon. Side chains of Leu59, Arg61, Tyr64, Leu332, backbone of Gly60 and Arg61 rendered for both monomers. Dotted lines denote hydrogen bonds (2.8-3.0 Å) and B. View 180° rotation about Y from panel A showing the Arginine Stack. Arg83 and Ala86 Asp124 and Trp126 are rendered in ball and stick. Electron density contoured at 1σ covers selected side-chains.

Figure 3. Low angle x-ray scattering of MEK6/ΔN/DD

A. Buffer-subtracted scattering profile (black) of the MEK6/ΔN/DD is consistent with an extended conformation. The fit to the experimental data of one ab initio DAMMIN model is shown in red. B. The electron pair distribution function of the experimental data shows a general bell-shaped curve, but with an asymmetric biased toward the longer distances, indicating an extended structure. Dmax was 140 Å. C. Data in the Guinier region is linear, indicating monodispersity of the sample in solution. D. Averaged and filtered ab initio shape prediction for MEK6/ΔN/DD in solution was calculated by the program DAMMIN and aligned using the DAMAVER program suite. The overall volume is consistent with two molecules of MEK6/ΔN/DD and the crystallographically observed dimer.

Figure 4

Comparison of elution volumes from analytical gel-filtration (Superdex 200 10/300) of MEK6/ΔN/DD in the presence(red) and absence (green) of ATP in the column buffer and a double mutant D124A/W126S (blue). A shift toward lower molecular weight in the presence of ATP and for the double mutant indicates a change in dimer-monomer equilibrium.

Figure 5

A. Sequence alignments of MAP2Ks in regions forming the dimer interfaces in MEK6 and MEK1/2. B. The MEK6 dimer, with conserved interface residues rendered as balls. C. the MEK1/2 dimer from (Ohren et al) (PDB file 1S9J). A cartoon representation shows the differences in packing of the dimers in the two MAP2Ks.