2HV9

Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase in complex with sinefungin

2HV9 の概要

• エントリーDOI: 10.2210/pdb2hv9/pdb
• 関連するPDBエントリー: 1ri1 1ri2 1ri3 1ri4 1ri5 1z3c
• 分子名称: mRNA cap guanine-N7 methyltransferase, SINEFUNGIN (3 entities in total)
• 機能のキーワード: mrna, cap, methyltransferase, inhibitor, transferase
• 由来する生物種: Encephalitozoon cuniculi
• 細胞内の位置: Nucleus (By similarity): Q8SR66
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 35205.07

構造登録者

Lima, C.D. (登録日: 2006-07-27, 公開日: 2006-09-19, 最終更新日: 2023-08-30)

主引用文献

Zheng, S.,Hausmann, S.,Liu, Q.,Ghosh, A.,Schwer, B.,Lima, C.D.,Shuman, S.
Mutational Analysis of Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase, Structure of the Enzyme Bound to Sinefungin, and Evidence That Cap Methyltransferase Is the Target of Sinefungin's Antifungal Activity
J.Biol.Chem., 281:35904-35913, 2006

PubMed Abstract: Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.

PubMed: 16971388
DOI: 10.1074/jbc.M607292200

実験手法

X-RAY DIFFRACTION (2.6 Å)