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. 2016 May 23:6:26634.
doi: 10.1038/srep26634.

Structure of the Dictyostelium Myosin-II Heavy Chain Kinase A (MHCK-A) α-kinase domain apoenzyme reveals a novel autoinhibited conformation

Qilu Ye  1 , Yidai Yang  1 , Laura van Staalduinen  1 , Scott William Crawley  1 , Linda Liu  1 , Stephanie Brennan  1 , Graham P Côté  1 , Zongchao Jia  1
Affiliations

Structure of the Dictyostelium Myosin-II Heavy Chain Kinase A (MHCK-A) α-kinase domain apoenzyme reveals a novel autoinhibited conformation

Qilu Ye et al. Sci Rep. .

Abstract

The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain.

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Figures

Figure 1
Figure 1. Schematic diagram showing the domain organization of MHCK-A.
MHCK-A consists of an N-terminal coiled-coil domain (left), a central α-kinase domain and a C-terminal WD repeat domain (right). The Thr825 site of autophosphorylation required for activity is indicated by a P.
Figure 2
Figure 2. Organization of molecules in the Apo-A-CAT asymmetric unit.
(a) Surface view of the Apo-A-CAT asymmetric unit with the eight monomers labeled A-H. (b) Cartoon representation of the A-D tetramer in the same orientation and coloring as in panel A. The phosphothreonine (P-Thr612) in B that binds to the Pi pocket in A is shown in stick representation. (c) Cartoon representation showing the front and back of molecule A. Residues involved in interfaces with B, C, D and G are shown as sticks and colored as indicated in the legend. Labels indicate the N- and C-termini (N and C), α-helices, β strands, N/D-loop, P-loop and P-Thr-612.
Figure 3
Figure 3. Binding of P-Thr612 to the Pi-pocket.
(a) The Pi-pocket region in molecule B is shown as a surface representation and is colored according to electrostatic potential (red-negative, blue-positive). Molecule A is colored grey and displayed in ribbon representation with P-Thr612, Thr613, Thr614 and Ile620 shown as sticks. (b) Stereo view showing P-Thr612 from molecule A (grey) bound to the Pi-pocket of molecule B (cyan). Residues involved in interactions are shown as sticks and are labeled black for molecule A and blue for molecule B. Interactions are indicated by dashed lines. (c) The kinase activity of A-CAT-Δ809 was assayed in the presence of peptides with the sequences QQG(p)TMVMPD (closed circles) or a QQG(p)TMSMPD (open circles). Hyperbolic curves fit to the data yielded a Kd of 38 ± 12 μM and a Vmax of 61 ± 4% for the QQG(p)TMVMPD peptide and a Kd of 98 ± 12 μM and a Vmax of 51 ± 3% for the QQG(p)TMSMPD peptide. Kinase activities are reported as a percentage of wild-type A-CAT activity.
Figure 4
Figure 4. Rotation of the N-lobe opens up the catalytic cleft of Apo-A-CAT.
(a) Surface representations of Apo-A-CAT, A-CAT·AMP, A-CAT-D663A and A-CAT·AMP·P-D766 showing the catalytic cleft. In the Apo-A-CAT structure P-Asp663 is shown as spheres and Arg592, Tyr647 and Asp766, which form a barrier dividing the cleft, are colored green. In the other structures AMP is shown as spheres. (b) Superimposition of the structures of Apo-A-CAT (white) and A-CAT-AMP (blue). Broken arrows with numbers show the direction and displacement (in Å) for the α-carbons of the indicated residues. The red dot indicates the pivot point for the N-lobe rotation (Trp692). (c) Superimposition of the eight molecules in the Apo-A-CAT asymmetric unit shows variability in the position of the P-loop and Arg592 (shown as sticks). Molecules are colored as in Fig. 2a.
Figure 5
Figure 5. Phosphorylation of Asp663 alters the conformation of the A-CAT active site.
(a) Stereo view showing the P-loop and N/D-loop of Apo-A-CAT (grey) and A-CAT·AMP (blue). In Apo-A-CAT the side chain of Arg592 occupies the same position as the α-phosphoryl group of AMP (shown as semi-transparent sticks) in A-CAT·AMP. Distances are given in Å for residues connected by red dashed lines. Black dashed lines indicate the interaction between Arg592 and Asp766. (b) Hydrophobic residues (Leu591, Tyr647, Leu659, Tyr660 and Leu779; shown as red spheres) assemble in A-CAT·AMP (left panel) to organize the active site but are disassembled in Apo-A-CAT (right panel). (c) Asp663 is phosphorylated in Apo-A-CAT. The purple mesh shows the 2Fo−Fc electron density map contoured at the 2σ level. Interactions supporting Arg592 are indicated by black dashed lines. (d) The structure of A-CAT·AMP·PD766 indicates that Asp663 is hydrogen bonded to the phosphoryl group of P-Asp766. The purple mesh shows the 2Fo−Fc electron density map contoured at the 2σ level. Tyr647 in Apo-A-CAT (white) is superimposed to illustrate the displacement of this residue (red dashed line) induced by Asp663 phosphorylation.
Figure 6
Figure 6. Effects of Asp663 and Tyr647 mutations on the conformation of the catalytic cleft and catalytic activity.
The structures of A-CAT-D663A (salmon) and (a) A-CAT-AMP (blue) and (b) A-CAT·AMP·P-D766 (wheat) are superimposed. The D663A mutation and phosphorylation of Asp766 induce similar conformational changes within the right-hand side of the catalytic cleft.
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