4ZMF

Phosphorylated Aspartate in the Crystal Structure of the Alpha-kinase domain of Myosin-II Heavy Chain Kinase A

4ZMF の概要

• エントリーDOI: 10.2210/pdb4zmf/pdb
• 関連するPDBエントリー: 3LKM 3LLA 3LMH 3LMI 3PDT
• 分子名称: Myosin heavy chain kinase A, ADENOSINE MONOPHOSPHATE, PHOSPHATE ION, ... (5 entities in total)
• 機能のキーワード: aspartyl phosphate intermediate, transferase
• 由来する生物種: Dictyostelium discoideum (Slime mold)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 70228.32

構造登録者

Ye, Q.,Jia, Z. (登録日: 2015-05-04, 公開日: 2015-08-19, 最終更新日: 2023-09-27)

主引用文献

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

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: 26260792
DOI: 10.1074/jbc.M115.672410

実験手法

X-RAY DIFFRACTION (2.39 Å)