Crystallographic and mass spectrometric characterisation of elF4E with N7-alkylated cap derivatives
Crystallographic and mass spectrometric characterisation of elF4E with N7-alkylated cap derivatives
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DOI：
10.1016/j.jmb.2007.06.033
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发表时间：
2007-09-07
影响因子：
5.6
通讯作者：
Walkinshaw, Malcolm D.
中科院分区：
文献类型：
--
作者：
Brown, Christopher J.;McNae, Iain;Walkinshaw, Malcolm D.
真核生物翻译起始因子4 E（eIF 4 E）与一系列N-7-烷基化鸟苷衍生物mRNA帽类似物结构的结构复合物已被表征。使用质谱法测定eIF 4 E与7-甲基-GTP（m（7）GTP）、GTP和GMP的表观气相平衡解离常数（K-d）值分别为0.15 μ M、13.6 μ M和55.7 μ M。为了与eIF 4 E单核苷酸肽结合位点紧密和特异性结合，似乎明确要求鸟苷衍生物具有N-7-甲基化鸟嘌呤系统的离域正电荷和至少一个磷酸基团。我们发现，N-7-苄基化单磷酸7-苄基-GMP（Bn（7）GMP）和7-（对氟苄基）-GMP（FBn（7）GMP）与eIF 4 E的结合比非N-7-烷基化鸟苷衍生物（Kd值分别为7.0 μ M和2.0 μ M）更紧密。具有Bn（7）GMP和FBn（7）GMP的eIF 4 E复合物晶体结构表明，苄基与eIF 4 E的额外有利接触贡献了结合能，其补偿了1和γ-磷酸的损失。N-7-苄基填充到两个色氨酸侧链后面的疏水口袋中，所述色氨酸侧链参与帽和eIF 4 E单核苷酸肽结合位点之间的阳离子-π堆积相互作用。这个口袋是由诱导的配合形成的，其中涉及帽结合的色氨酸残基之一相对于具有N-7-甲基化帽衍生物的结构翻转180度。在此进行的这一观察和其他观察将有助于设计新的eIF 4 E抑制剂家族，其可能在癌症中具有潜在的治疗应用。(c)2007爱思唯尔有限公司保留所有权利。
Structural complexes of the eukaryotic translation initiation factor 4E (eIF4E) with a series of N-7-alkylated guanosine derivative mRNA cap analogue structures have been characterised. Mass spectrometry was used to determine apparent gas-phase equilibrium dissociation constants (K-d) values of 0.15 mu M, 13.6 mu M, and 55.7 mu M for eIF4E with 7-methyl-GTP (m(7)GTP), GTP, and GMP, respectively. For tight and specific binding to the eIF4E mononucleotide binding site, there seems to be a clear requirement for guanosine derivatives to possess both the delocalised positive charge of the N-7-methlated guanine system and at least one phosphate group. We show that the N-7-benzylated monophosphates 7-benzyl-GMP (Bn(7)GMP) and 7-(p-fluorobenzyl)-GMP (FBn(7)GMP) bind eIF4E substantially more tightly than non-N-7-alkylated guanosine derivatives (K-d values of 7.0 mu M and 2.0 mu M, respectively). The eIF4E complex crystal structures with Bn(7)GMP and FBn(7)GMP show that additional favourable contacts of the benzyl groups with eIF4E contribute binding energy that compensates for loss of the 1 and gamma-phosphates. The N-7-benzyl groups pack into a hydrophobic pocket behind the two tryptophan side-chains that are involved in the cation-pi stacking interaction between the cap and the eIF4E mononucleotide binding site. This pocket is formed by an induced fit in which one of the tryptophan residues involved in cap binding flips through 180 degrees relative to structures with N-7-methylated cap derivatives. This and other observations made here will be useful in the design of new families of eIF4E inhibitors, which may have potential therapeutic applications in cancer. (c) 2007 Elsevier Ltd. All rights reserved.