Muegge I, Schweins T, Langen R, Warshel A
Department of Chemistry, University of Southern California, Los Angeles 90089-1062, USA.
Structure. 1996 Apr 15;4(4):475-89. doi: 10.1016/s0969-2126(96)00052-4.
p21ras is one of the GTP-binding proteins that act as intercellular molecular switches. The GTP-bound form of p21ras sends a growth-promoting signal that is terminated once the protein is cycled back into its GDP-bound form. The interaction of guanine-nucleotide-exchange factors (GEFs) with p21ras leads to activation of the protein by promoting GDP --> GTP exchange. Oncogenic mutations of p21ras trap the protein in its biological active GTP-bound form. Other mutations interfere with the activity of GEF. Thus, it is important to explore the structural basis for the action of different mutations.
The crystal structures of p21ras are correlated with the binding affinities of GTP and GDP by calculating the relevant electrostatic energies. It is demonstrated that such calculations can provide a road map to the location of 'hot' residues whose mutations are likely to change functional properties of the protein. Furthermore, calculations of the effect of specific mutations on GTP and GDP binding are consistent with those observed. This helps to analyze and locate functionally important parts of the protein.
Our calculations indicate that the protein main chain provides a major contribution to the binding energies of nucleotides and probably plays a key role in relaying the effect of GEF action. Analysis of p21ras mutations in residues that are important for the proper function of GEFs suggests that the region comprising residues 62-67 in p21ras is the major GEF-binding site. This analysis and our computer simulations indicate that the effect of GEF is probably propagated to the P-loop (residues 10-17) through interaction between Gly60 and Gly12. This then reduces the interaction between the main-chain dipoles of the P-loop and the nucleotide. Finally, the results also suggest a possible relationship between the GTP --> GDP structural transition and the catalytic effect of the GTPase-activating protein.
p21ras是一种作为细胞间分子开关的GTP结合蛋白。p21ras的GTP结合形式发出促进生长的信号，一旦该蛋白循环回到其GDP结合形式，该信号就会终止。鸟嘌呤核苷酸交换因子（GEF）与p21ras的相互作用通过促进GDP→GTP交换导致该蛋白激活。p21ras的致癌突变使该蛋白被困在其生物活性GTP结合形式中。其他突变会干扰GEF的活性。因此，探索不同突变作用的结构基础很重要。
通过计算相关静电能，将p21ras的晶体结构与GTP和GDP的结合亲和力相关联。结果表明，这种计算可以为“热点”残基的位置提供路线图，其突变可能会改变蛋白质的功能特性。此外，特定突变对GTP和GDP结合影响的计算结果与观察结果一致。这有助于分析和定位蛋白质功能上重要的部分。
我们的计算表明，蛋白质主链对核苷酸的结合能有主要贡献，并且可能在传递GEF作用的效果中起关键作用。对p21ras中对GEF正常功能重要的残基突变的分析表明，p21ras中包含62 - 67位残基的区域是主要的GEF结合位点。该分析和我们的计算机模拟表明，GEF的作用可能通过Gly60和Gly12之间的相互作用传递到P环（10 - 17位残基）。这进而减少了P环主链偶极与核苷酸之间的相互作用。最后，结果还表明了GTP→GDP结构转变与GTP酶激活蛋白催化作用之间可能存在的关系。
