An NMR comparison of the changes produced by different guanosine 5'-triphosphate analogs in wild-type and oncogenic mutant p21ras.
An NMR comparison of the changes produced by different guanosine 5'-triphosphate analogs in wild-type and oncogenic mutant p21ras.
复制标题
野生型和致癌突变体 p21ras 中不同鸟苷 5-三磷酸类似物产生的变化的 NMR 比较。
DOI：
10.1021/bi00080a006
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Redfield,AG
中科院分区：
文献类型：
--
作者：
Miller,AF;Halkides,CJ;Redfield,AG
摘要：我们使用核磁共振波谱比较了野生型p21 ras和致癌突变型（G12 D）p21 ras中GTP类似物鸟苷5 '-G-（3-硫代三磷酸）（GTP 7S）和鸟苷酰（β，7-亚氨基）二磷酸（GMPPNP）取代结合GDP所产生的构象变化。我们已经使用同位素编辑的核磁共振光谱观察选择性[15 N]甘氨酸和[15 N]异亮氨酸标记的p21 ras-核苷酸复合物的酰胺共振。我们发现，对GTP 7S和GMPPNP结合反应强烈的9个共振中有8个是相同的，但作用的性质似乎不同。在GTP 7S中，七个新的共振取代了与GDP-p21 ras特异性相关的八个共振，但在GMPPNP-p21 ras中，只有两个共振取代了丢失的GDP特异性共振。除了甘氨酸10、12、13、15和75以及异亮氨酸36、21和另一个被米勒等人发现对GTP 7S应答之外，Gly 60的共振清楚地显示对GMPPNP置换GDP应答。[米勒，A. F.、帕帕斯塔夫罗斯湾Z.，& Redfield，A. G.（1992）Biochemistry 31，10208-10216）。观察到的两个GMPPNP特异性共振出现在与GTP 7S特异性共振相似的位置，GTP 7S特异性共振虽然没有完全消失，但比它们所取代的GDP特异性共振弱。因此，这两个GTP类似物对p21 ras的光谱具有相似的作用，表明这种作用是由于两个类似物共有的特征。我们建议，活性位点的共振强度是特别衰减时，GTP类似物的绑定，因为与7-磷酸的GTP类似物的相互作用耦合的灵活的环2和4的刚性环1的活性位点。环2和环4的构象异质性和动力学将受到环1的约束，但也传递到它。在一个共同的中间时间尺度上的耦合构象交换可以解释同时失去的共振从所有三个环的活性位点。在我们对野生型和（G12 D）GDP-p21 ras的比较中，我们发现在（G12 D）p21 ras中看不到lie 36的共振。在（G12 D）p21 ras中，GDP被GTP 7S取代导致甘氨酸10、13、15、60和75以及异亮氨酸21和其他四个氨基酸的共振从它们的GDP特异性位置移动。GTP 7S特异性共振观察到所有，但其中两个。所有的共振峰都对应于活性位点上的残基或与其相连的残基，但只有四个对应的共振峰是GMPPNP-（G12 d）p21 ras特异性的。因此，替换甘氨酸12天冬氨酸只有轻微改变的基态p21 ras的核苷酸替换的响应性。此外，所观察到的（G12 D）p21 ras的GTP 7S特异性共振接近于野生型p21 ras的GTP 7S特异性共振，即使几种GDP特异性（G12 D）p21 ras共振与野生型p21 ras的那些显著不同。1990年）。它发生在三个非常相似的变体中，称为N-，H-和K-p21 ras，当GTP 1结合时具有生理活性，当GDP结合时无活性。它在其他蛋白质的控制下在这两种状态之间循环（Bourne et al.，1990年）。GTP酶激活蛋白（GAP; Trahey & McCormick，1987）加速GTP-p21 ras的hy-
Revised Manuscript Received May 6, 1993 abstract: We have used nuclear magnetic resonance spectroscopy to compare the conformational changes produced by replacement of bound GDP by the GTP analogs guanosine 5'-G-(3-thiotriphosphate)(GTP7S) and guanylyl (/3, 7-imido) diphosphate (GMPPNP) in wild-type p21ras as well as the oncogenic mutant (G12D) p21ras. We have used isotope-edited nuclear magnetic resonance spectroscopy to observe the amide resonances of selectively [I5N] glycine and [15N] isoleucine labeled p21ras-nucleotide complexes. We find that eight of the nine resonances that respond strongly toGTP7S and GMPPNP binding are the same but that the nature of the effect appears different. With GTP7S, seven new resonances replace the eight resonances specifically associated with GDP-p21ras, but in GMPPNP-p21ras only two resonances replace the GDP-specific resonances that are lost. The resonance of Gly 60 is clearly shown to be responsive to replacement of GDP by GMPPNP, in addition to glycines 10, 12, 13, 15, and 75 and isoleucines 36, 21, and one other, that were found to respond to GTP7S by Miller et al.[Miller, A.-F., Papastavros, M. Z., & Redfield, A. G.(1992) Biochemistry 31, 10208-10216). The two GMPPNP-specific resonances observed appear in positions similar to GTP7S-specific resonances, andthe GTP7S-specific resonances, although not lost altogether, are weaker than the GDP-specific resonances they replace. Thus, the two GTP analogs have similar effects on the spectrum of p21ras, suggesting that the effects are due to features common to both analogs. We propose that active site resonance intensities are specifically attenuated when GTP analogs are bound because interactions with the 7-phosphate of GTP analogs couple the flexible loops 2 and 4 to the rigid loop 1 of the active site. The conformational heterogeneity and dynamics of loops 2 and 4 would be constrainedby loop 1 but also transmitted to it. Coupled conformational exchange on a common intermediate time scale could explain the simultaneous loss of resonances from all three loops in the active site. In our comparison of wild-type and (G12D) GDP-p21ras, we find that the resonance of lie 36 is not visible in (G12D) p21ras. In (G12D) p21ras, replacement of GDP by GTP7S causes the resonances of glycines 10, 13, 15, 60, and 75 and isoleucine 21 and four others toshift from their GDP-specific positions. GTP7S-specific resonances are observed for all but two of these. The assigned responsive resonances all correspond to residues in the active site or connected to it. Largely the same resonances respond to GMPPNP binding, but only four corresponding resonances specific to GMPPNP-(G12d) p21ras are observed. Thus, replacement of glycine 12 by aspartate only slightly alters the responsiveness of the ground state of p21ras to nucleotide replacement. Furthermore, the observed GTP7S-specific resonances of (G12D) p21ras are close to GTP7S-specific resonances of wild-type p21ras, even though several of the GDP-specific (G12D) p21ras resonances differ significantly from those of wild-type p21ras.Human p21ras is a guanine-nucleotide binding protein implicated in regulation of cell differentiation and proliferation (Bourne et al., 1990). It occurs in three very similar variants called N-, H-, and K-p21rasand is physiologically active when GTP1 is bound and inactive when GDP is bound. It cycles between these two states under the control of other proteins (Bourne et al., 1990). GTPase activating protein (GAP; Trahey & McCormick, 1987) accelerates GTP-p21ras’s hy-