NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir
NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir
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异种鼠白血病病毒相关病毒蛋白酶与安普那韦复合物的核磁共振研究
DOI：
10.1016/j.bbrc.2012.07.083
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Masato Katahira
中科院分区：
文献类型：
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作者：
Ayako Furukawa;Hideyasu Okamura;Ryo Morishita;Satoko Matsunaga;Naohiro Kobayashi;Takahisa Ikegami;Tsutomu Kodaki;Akifumi Takaori-Kondo;Ryo Akihide;Takashi Nagata;Masato Katahira
异源性鼠白血病病毒相关病毒（XMRV）是两种鼠白血病原病毒在人前列腺癌细胞在无胸腺裸鼠体内传代过程中在人工条件下重组产生的病毒。 XMRV 的同二聚蛋白酶 (PR) 在功能性病毒蛋白的产生中发挥着关键作用，是病毒复制的先决条件。我们使用小麦胚芽无细胞表达系统合成了 XMRV PR，并通过 NMR 对 XMRV PR 与抑制剂安普那韦 (APV) 的复合物进行了结构分析。制备了五个不同的组合 15N 标记样品，并通过应用 Otting 方法进行主链共振分配，使用五个样品自动识别 [1H,15N]HSQC 共振的氨基酸类型（Wu 等，2006）[14]。涉及 APV 的滴定实验表明，一个 APV 分子与一个 XMRV PR 二聚体结合。对于许多残基，观察到两个不同的共振，这被认为是由于 APV:XMRV PR=1:2 复合物中两个原聚体之间的结构异质性。基于化学位移扰动和通过过滤 NOE 实验鉴定分子间 NOE，已鉴定出与 APV 界面处的 PR 残基。有趣的是，XMRV PR 的两个原体之间的化学位移异质性不仅在与 APV 的界面处观察到，而且在界面以外的区域也观察到。这表明APV与XMRV PR二聚体结合的不对称性引起的结构异质性被传递到遥远的区域。这与 APV:HIV-1 PR 复合物的情况相反，其中结构异质性仅位于界面处。 XMRV PR 复合物的结构变化的长距离传输可用于发现新型药物。
Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially15N-labeled samples were prepared and backbone resonance assignments were made by applying Otting’s method, with which the amino acid types of the [1H,15N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) [14]. A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR=1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions. This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug.