A Novel Signal Transduction Protein PII Variant from Synechococcus elongatus PCC 7942 Indicates a Two-Step Process for NAGK-PII Complex Formation
A Novel Signal Transduction Protein PII Variant from Synechococcus elongatus PCC 7942 Indicates a Two-Step Process for NAGK-PII Complex Formation
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DOI：
10.1016/j.jmb.2010.04.018
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发表时间：
2010-06-11
影响因子：
5.6
通讯作者：
Forchhammer, Karl
中科院分区：
文献类型：
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作者：
Fokina, Oleksandra;Chellamuthu, Vasuki-Ranjani;Forchhammer, Karl
P-II信号转导蛋白在细菌、古生菌和植物中高度保守，并且通过整合来自细胞的碳、氮和能量状态的信号而在协调中心代谢中具有关键功能。在蓝细菌细长聚球藻PCC 7942中，P-II以协同方式结合ATP和2-酮戊二酸（2-OG），ATP结合位点也接受ADP。根据其效应分子结合状态，P-II（来自该蓝藻和其他产氧光养生物）复合并调节环鸟氨酸途径的精氨酸控制酶N-乙酰基-L-谷氨酸激酶（NAGK），以控制精氨酸生物合成。为了更深入地了解P-II与NAGK结合的过程，我们搜索了具有改变的结合特征的P-II变体，并发现P-II变体I86 N和I86 T能够结合先前显示不能结合野生型P-II蛋白的NAGK变体（R233 A）。对这些P-II变体与野生型NAGK以及与NAGK R233 A变体之间的相互作用的分析表明，P-II I86 N变体是超活性NAGK结合剂。为了揭示这种性质的结构基础，我们以原子分辨率解析了P-II I86 N变体的晶体结构。在P-II蛋白的大多数受体相互作用中普遍存在的大T环以紧密弯曲的构象存在，其模拟S的T环。elongatusP-II在已经闩锁到NAGK上之后。此外，两个P-II 186变体在2-OG结合中显示特定缺陷，这意味着残基186在2-OG结合中的作用。我们提出了一个两步模型来解释P-II-NAGK复合物的形成机制：在起始步骤中，NAGK的R233和P-II的E85之间的接触引发了P-II的延伸T环的弯曲，然后是第二步，弯曲的T环深深插入NAGK裂缝中形成紧密的复合物。(C)2010爱思唯尔有限公司版权所有。
P-II signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, P-II binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, P-II (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-L-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of P-II binding to NAGK, we searched for P-II variants with altered binding characteristics and found P-II variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type P-II protein. Analysis of interactions between these P-II variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the P-II I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the P-II I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of P-II proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus P-II after having latched onto NAGK. Moreover, both P-II 186 variants display a specific defect in 2-OG binding, implying a role of residue 186 in 2-OG binding. We propose a two-step model for the mechanism of P-II-NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of P-II initiates the bending of the extended T-loop of P-II, followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex. (C) 2010 Elsevier Ltd. All rights reserved.