A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the β-phosphate to destabilize GDP on ARF1
A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the β-phosphate to destabilize GDP on ARF1
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DOI：
10.1093/emboj/17.13.3651
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发表时间：
1998-07-01
期刊：
影响因子：
11.4
通讯作者：
Antonny, B
中科院分区：
文献类型：
--
作者：
Béraud-Dufour, S;Robineau, S;Antonny, B
鸟嘌呤核苷酸交换因子ARNO （ARNO-Sec7）的Sec7结构域负责gtp结合蛋白ARF1的交换活性。ARNO-Sec7与无核苷酸形式的[Delta 17]ARF1 （ARF1的可溶截断形式）形成稳定的配合物。ARNO-Sec7的晶体结构最近得到了解决，一种位点定向诱变方法确定了一个疏水槽和一个邻近的亲水环作为arf1的结合位点。我们发现，ARNO-Sec7亲水环中的Glu156参与了Mg2+和GDP在ARF1中的失稳。保守突变E156D和电荷反转突变E156K使ARNO-Sec7的交换活性降低了几个数量级。此外，[E156K]ARNO-Sec7与无Mg2+形式的[Delta 17]ARF1-GDP形成配合物，而不诱导GDP的释放。ARNO-Sec7和[Delta 17]ARF1的其他突变表明，ARF1开关I区突出的疏水残基插入到Sec7结构域的凹槽中，ARF1开关II区的Lys73与ARNO-Sec7的Asp183形成离子对。
The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta 17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta 17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta 17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the snitch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.