X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr
X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr
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DOI：
10.1073/pnas.192368699
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发表时间：
2002-10-15
影响因子：
11.1
通讯作者：
Nessler, S
中科院分区：
文献类型：
--
作者：
Fieulaine, S;Morera, S;Nessler, S
HPr激酶/磷酸化酶（HprK/P）控制磷酸载体蛋白HPr的磷酸化状态，调控革兰氏阳性菌对碳源的利用。它既能催化HPr的Ser-46的atp依赖性磷酸化，也能催化其通过磷酸化解去磷酸化。后一反应以无机磷酸盐为底物，产生焦磷酸盐。我们在这里展示了干酪乳杆菌HprK/P与枯草芽孢杆菌HPr催化结构域复合物的两个晶体结构，都是在2.8埃的分辨率下。其中一种结构是在过量焦磷酸盐存在下获得的，逆转了磷酸化反应，含有丝氨酸磷酸化的HPr。该复合物有6个HPr分子与六聚体激酶结合。两个相邻的酶亚基与每个HPr分子接触，一个通过其活性位点，另一个通过其c端螺旋。在丝氨酸磷酸化的HPr复合物中，一个磷酸离子处于对丝氨酸进行亲核攻击的位置。虽然磷酸化反应的机制类似于真核蛋白激酶，但无机磷酸盐的去磷酸化是HprK/P家族激酶所特有的。本研究提供了蛋白激酶及其蛋白底物复合物的结构，从而深入了解了两个方向上磷酸转移反应的化学性质。
HPr kinase/phosphorylase (HprK/P) controls the phosphorylation state of the phosphocarrier protein HPr and regulates the utilization of carbon sources by Gram-positive bacteria. It catalyzes both the ATP-dependent phosphorylation of Ser-46 of HPr and its dephosphorylation by phosphorolysis. The latter reaction uses inorganic phosphate as substrate and produces pyrophosphate. We present here two crystal structures of a complex of the catalytic domain of Lactobacillus casei HprK/P with Bacillus subtilis HPr, both at 2.8-Angstrom resolution. One of the structures was obtained in the presence of excess pyrophosphate, reversing the phosphorolysis reaction and contains serine-phosphorylated HPr. The complex has six HPr molecules bound to the hexameric kinase. Two adjacent enzyme subunits are in contact with each HPr molecule, one through its active site and the other through its C-terminal helix. In the complex with serine-phosphorylated HPr, a phosphate ion is in a position to perform a nucleophilic attack on the phosphoserine. Although the mechanism of the phosphorylation reaction resembles that of eukaryotic protein kinases, the dephosphorylation by inorganic phosphate is unique to the HprK/P family of kinases. This study provides the structure of a protein kinase in complex with its protein substrate, giving insights into the chemistry of the phospho-transfer reactions in both directions.