4ZMF
Phosphorylated Aspartate in the Crystal Structure of the Alpha-kinase domain of Myosin-II Heavy Chain Kinase A
Summary for 4ZMF
| Entry DOI | 10.2210/pdb4zmf/pdb |
| Related | 3LKM 3LLA 3LMH 3LMI 3PDT |
| Descriptor | Myosin heavy chain kinase A, ADENOSINE MONOPHOSPHATE, PHOSPHATE ION, ... (5 entities in total) |
| Functional Keywords | aspartyl phosphate intermediate, transferase |
| Biological source | Dictyostelium discoideum (Slime mold) |
| Total number of polymer chains | 2 |
| Total formula weight | 70228.32 |
| Authors | |
| Primary citation | Yang, Y.,Ye, Q.,Jia, Z.,Cote, G.P. Characterization of the Catalytic and Nucleotide Binding Properties of the alpha-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A. J.Biol.Chem., 290:23935-23946, 2015 Cited by PubMed Abstract: The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min(-1), respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2'/3'-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μM, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg(2+) ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3-6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site. PubMed: 26260792DOI: 10.1074/jbc.M115.672410 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (2.39 Å) |
Structure validation
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