Structure of dystrophia myotonica protein kinase

Abstract

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled-coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM-8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled-coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully-ordered and correctly positioned alphaC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C-terminal extension of the kinase domain is bound to the N-terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C-terminal extension compared to the closely related Rho-associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM-8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.

Figure 1

Domain arrangement of DMPK. The BIM-8 inhibitor in the active site is shown as a ball-and-stick representation. The N-terminal kinase lobe is mostly above the BIM-8 inhibitor and the C-terminal kinase lobe mostly below. The C-terminal section of the protein is colored purple, the activation loop is colored red, the αEF/αF loop is colored yellow, and the glycine rich loop colored blue. The N-terminal helices involved in the dimerisation interface are colored orange.

Figure 2

Comparison of the binding of the C-termini of DMPK and ROCK1 to the N-terminal lobe. (A) The PXXP motif, the ROCK1 C-terminus is in yellow, and DMPK is colored as in Figure 1. (B) The turn motif. (C) Sequence alignment of DMPK and the Rho kinases over the region covering the PXXP, active-site tether and turn motifs. The VSGGG insertion present in isoforms 1 and 2 of DMPK is indicated with red lettering.

Figure 3

DMPK dimer. (A) The DMPK biological dimer, with each molecule colored blue to red from N- to C-terminus. (B) The dimerisation interface of DMPK. The surface is shown for one DMPK monomer, colored according to hydrophobicity, with blue more hydrophobic and red less hydrophobic. The other DMPK monomer is shown as a ribbon, with residues that are conserved between ROCK1 and DMPK illustrated. The DMPK ribbon is colored as in Figure 1. (C) Partial sequence alignment of the N-terminal regions of DMPK and ROCK1 involved in the dimer interface. (D) Partial sequence alignment of the C-terminal regions of DMPK and ROCK1 involved in the dimer interface.

Figure 4

Self association of DMPK studied by analytical ultracentrifugation. (A) Sedimentation velocity experiment, the concentration of DMPK was 25 μM. At that concentration the protein was mainly populated as a dimer as indicated by the peak at 4.8 Swedberg units. The molecular weights determined from the velocity data were ∼48 and ∼100 kDa, respectively, consistent with the expected molecular weight of a DMPK monomer and dimer. (B) Sedimentation equilibrium experiment of DMPK. The upper panel shows residuals to a nonlinear least-squares fit to a monomer–dimer model, shown as a solid line. The determined association constant was in the range 1–5 μM.

Figure 5

(A) Activation segment of DMPK, coloured as in Figure 1. (B) Partial sequence alignment of the activation loop and αEF/αF loop regions of DMPK against the Rho kinases 1 and 2, which are colored as in (A). The residues equivalent to those phosphorylated in MRCK are indicated with green stars. A full sequence alignment of the DMPK subfamily can be seen as Supplementary Figure 1.

Figure 6

The active site of DMPK with inhibitor BIM-8 bound.