Molecular model of a soluble guanylyl cyclase fragment determined by small-angle X-ray scattering and chemical cross-linking.
Molecular model of a soluble guanylyl cyclase fragment determined by small-angle X-ray scattering and chemical cross-linking.
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DOI：
10.1021/bi301570m
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发表时间：
2013-03-05
期刊：
影响因子：
2.9
通讯作者：
Montfort WR
中科院分区：
文献类型：
--
作者：
Fritz BG;Roberts SA;Ahmed A;Breci L;Li W;Weichsel A;Brailey JL;Wysocki VH;Tama F;Montfort WR
可溶性鸟苷酸/鸟苷酸环化酶（sGC）在结合一氧化氮后将GTP转化为cGMP，导致平滑肌松弛和血管舒张。受损的sGC活性在心血管疾病中是常见的，并且sGC刺激化合物被大量寻找。sGC是具有两个H-NOX结构域（一个具有血红素，一个不具有血红素）、两个PAS结构域、卷曲螺旋结构域和两个环化酶结构域的150 kDa异二聚体蛋白。NO与sGC血红素的结合导致近端组氨酸释放和催化活性的刺激。为了开始理解结合如何导致激活，我们检查了来自烟草天蛾（Manduca sexta）的截短sGC蛋白，其结合NO、CO和刺激性化合物YC-1，但缺乏环化酶结构域。我们使用分析超离心和小角X射线散射（SAXS）确定了截短的Ms sGC的整体形状，揭示了115 μ m × 90 μ m × 75 μ m的细长分子。NO、CO或YC-1的结合对形状影响不大。使用化学交联和串联质谱，我们确定了20个分子间的联系，使我们能够适应SAXS衍生的分子包膜的各个域的同源模型。所得到的模型显示了一个中心平行卷曲螺旋平台，H-NOX和PAS域组装在该平台上。β1 H-NOX和α1 PAS结构域接触并形成核心信号传导复合物，而α1 H-NOX结构域可以被去除而对配体结合或整体形状没有显著影响。从C-末端去除21个残基产生一种蛋白质，其在NO结合时具有显著增加的近端组氨酸释放速率。
Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease and sGC stimulatory compounds are greatly sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin understanding how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO and stimulatory compound YC-1, but lack the cyclase domains. We determined the overall shape of truncated Ms sGC using analytical ultracentrifugation and small angle X-ray scattering (SAXS), revealing an elongated molecule 115 Å by 90 Å by 75 Å. Binding of NO, CO or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The β1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.
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影响因子：
3
作者：
Ahmed, Aqeel;Whitford, Paul C.;Sanbonmatsu, Karissa Y.;Tama, Florence
通讯作者：
Tama, Florence
影响因子：
11.4
作者：
Ma, Xiaolei;Sayed, Nazish;van den Akker, Focco
通讯作者：
van den Akker, Focco
影响因子：
6.1
作者：
Konarev, PV;Volkov, VV;Svergun, DI
通讯作者：
Svergun, DI
影响因子：
4.8
作者：
Koglin, M;Behrends, S
通讯作者：
Behrends, S
DOI：
10.1006/bbrc.1997.7470
发表时间：
1997-10-09
影响因子：
3.1
作者：
Kharitonov, VG;Russwurm, M;Koesling, D
通讯作者：
Koesling, D