Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK.
Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK.
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DOI：
10.1021/acs.biochem.5b00593
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发表时间：
2015-08-18
期刊：
影响因子：
2.9
通讯作者：
Cobb, Melanie H.
中科院分区：
文献类型：
--
作者：
Taylor, Clinton A.;Juang, Yu-Chi;Earnest, Svetlana;Sengupta, Samarpita;Goldsmith, Elizabeth J.;Cobb, Melanie H.
相关的蛋白激酶SPAK和OSR 1部分通过磷酸化阳离子协同转运蛋白家族成员来调节离子稳态。OSR 1的激酶结构域的结构以非磷酸化的非活性形式被解析，并且像其他一些Ste 20激酶一样，表现出结构域交换的激活环。为了进一步探索SPAK/OSR 1中结构域交换的作用，我们在3.1 nm和2.5 nm分辨率下测定了SPAK 63-403和SPAK 63-390 T243 D的晶体结构。这些结构包括激酶结构域和C-末端尾的不同部分，较长的没有，较短的具有激活点突变T243 D。T243 D蛋白的结构揭示了相对于未磷酸化的SPAK和OSR 1的显著构象差异，但也具有失活激酶的一些特征。两种结构都是结构域交换的二聚体。鉴定并突变参与结构域交换的序列以产生具有激酶活性的SPAK单体突变体，表明单体形式是活性的。单体突变体被WNK 1激活，但对其底物NKCC 2的活性降低，表明结构域交换的调节作用。部分活性SPAK T243 D的结构与多阶段活化过程一致，其中磷酸化诱导SPAK构象，其需要进一步重塑以构建活性结构。
The related protein kinases SPAK and OSR1 regulate ion homeostasis in part by phosphorylating cation cotransporter family members. The structure of the kinase domain of OSR1 was solved in the unphosphorylated inactive form, and like some other Ste20 kinases, exhibited a domain-swapped activation loop. To further probe the role of domain swapping in SPAK/OSR1, we have determined the crystal structures of SPAK 63–403 at 3.1 Å and SPAK 63–390 T243D at 2.5 Å resolutions. These structures encompass the kinase domain and different portions of the C-terminal tail, the longer without, and the shorter with an activating point mutation T243D. The structure of the T243D protein reveals significant conformational differences relative to unphosphorylated SPAK and OSR1, but also has some features of an inactive kinase. Both structures are domain-swapped dimers. Sequences involved in domain swapping were identified and mutated to create a SPAK monomeric mutant with kinase activity, indicating that monomeric forms are active. The monomeric mutant is activated by WNK1, but has reduced activity toward its substrate NKCC2, suggesting regulatory roles for domain swapping. The structure of the partially active SPAK T243D is consistent with a multi-stage activation process in which phosphorylation induces a SPAK conformation that requires further remodeling to build the active structure.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
16.2
作者：
Leiserson, WM;Harkins, EW;Keshishian, H
通讯作者：
Keshishian, H
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
4.6
作者：
Delpire, Eric;Gagnon, Kenneth B. E.
通讯作者：
Gagnon, Kenneth B. E.
影响因子：
16
作者：
Chang, CI;Xu, BE;Goldsmith, EJ
通讯作者：
Goldsmith, EJ