Evidence for differing roles for each lobe of the calmodulin-like domain in a calcium-dependent protein kinase
Evidence for differing roles for each lobe of the calmodulin-like domain in a calcium-dependent protein kinase
复制标题
DOI：
10.1074/jbc.m401297200
复制
发表时间：
2004-07-09
影响因子：
4.8
通讯作者：
Chazin, WJ
中科院分区：
文献类型：
--
作者：
Christodoulou, J;Malmendal, A;Chazin, WJ
钙依赖蛋白激酶(CDPKs)是在植物和某些原生动物中发现的结构独特的丝氨酸/苏氨酸蛋白激酶。它们的区别在于有一个钙调素样调节装置(钙调素样结构域(CaM-LD))，它通过连接(J)区域连接到激酶催化域的C末端。与CaM一样，CaM-LD由两个球状的EF结构域(N叶和C叶)组成，每个结构域都包含一对钙结合位点。光谱分析表明，CaM-LD是由螺旋元素组成的，但在没有Ca~(2+)的情况下，孤立的CaM-LD不形成构象均一的三级结构。在含有J和CaM-LD(JC)的结构中，添加亚化学计量的钙离子足以稳定C-末端叶，但不只是在CaM-LD中。此外，当J滴定到钙离子饱和的CaM-LD中时，与CaM-LD的C-叶比N-叶的相互作用更强。对JC的钙亲和力测定表明，C叶有两个协同作用的高亲和力结合部位(K-d，20 mM KCl时K-d=5.6 nM)，N叶有两个较弱的结合部位(20 mM KCl时K-d，K-Mean=110 nM)。在分离的CaM-LD中，相应的钙结合常数降低了2个数量级以上，这表明J区在稳定CDPK调控装置的结构方面具有重要作用。这两个结构域之间的巨大差异亲和力加上之前对疟原虫CDPK的研究(赵，Y.，Pokutta，S.，Maurer，P.，Lindt，M.，Franklin，R.M.和Kappes，B.(1994)BioChemical 33,3714-3721)提出了一个模型，即即使在正常低的细胞内钙离子水平，C-叶与连接相互作用，但该激酶保持在自抑制状态。当钙离子水平上升，填满N-叶中两个较弱的亲和力结合部位时，激活就会发生，从而触发构象变化，导致自抑制区的释放。
Calcium-dependent protein kinases (CDPKs) are structurally unique Ser/Thr kinases found in plants and certain protozoa. They are distinguished by a calmodulin-like regulatory apparatus (calmodulin-like domain (CaM-LD)) that is joined via a junction (J) region to the C-terminal end of the kinase catalytic domain. Like CaM, the CaM-LD is composed of two globular EF structural domains (N-lobe,C-lobe), each containing a pair of Ca2+ binding sites. Spectroscopic analysis shows that the CaM-LD is comprised of helical elements, but the isolated CaM-LD does not form a conformationally homogeneous tertiary structure in the absence of Ca2+. The addition of substoichiometric amounts of Ca2+ is sufficient to stabilize the C-terminal lobe in a construct containing J and CaM-LD (JC) but not in the CaM-LD alone. Moreover, as J is titrated into Ca2+-saturated CaM-LD, interactions are stronger with the C-lobe than the N-lobe of the CaM-LD. Measurements of Ca2+ affinity for JC reveal two cooperatively interacting high affinity binding sites (K-d,(mean) = 5.6 nM at 20 mM KCl) in the C-lobe and two weaker sites in the N-lobe (K-d,K-mean = 110 nM at 20 mM KCl). The corresponding Ca2+ binding constants in the isolated CaM-LD are lower by more than 2 orders of magnitude, which indicates that the J region has an essential role in stabilizing the structure of the CDPK regulatory apparatus. The large differential affinity between the two domains together with previous studies on a plasmodium CDPK (Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R. M., and Kappes, B. (1994) Biochemistry 33, 3714-3721) suggests a model whereby even at normally low cytosolic levels of Ca2+, the C-lobe interacts with the junction, but the kinase remains in an autoinhibited state. Activation then occurs when Ca2+ levels rise to fill the two weaker affinity binding sites in the N-lobe, thereby triggering a conformational change that leads to release of the autoinhibitory region.