Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

被引：151

作者：

Auld, Douglas S. ^{[1
]}

Lovell, Scott ^{[2
]}

Thorne, Natasha ^{[1
]}

Lea, Wendy A. ^{[1
]}

Maloney, David J. ^{[1
]}

Shen, Min ^{[1
]}

Rai, Ganesha ^{[1
]}

Battaile, Kevin P. ^{[3
]}

Thomas, Craig J. ^{[1
]}

Simeonov, Anton ^{[1
]}

Hanzlik, Robert P. ^{[2
]}

Inglese, James ^{[1
]}

机构：

[1] NIH, Chem Genom Ctr, Bethesda, MD 20892 USA

[2] Univ Kansas, Struct Biol Ctr, Lawrence, KS 66047 USA

[3] Argonne Natl Lab, Adv Photon Source, Ind Macromol Crystallog Assoc Collaborat Access T, Argonne, IL 60439 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2010年 / 107卷 / 11期

基金：

美国国家卫生研究院;

关键词：

Ataluren; multisubstrate adduct inhibitor; protein stability; reporter gene assay; X-ray crystallography; MULTISUBSTRATE ADDUCT INHIBITOR; CRYSTAL-STRUCTURE; BIOLUMINESCENCE; MECHANISM; REPORTER; ENZYME; ASSAYS; TRANSFORMYLASE; IDENTIFICATION; SPECIFICITY;

D O I：

10.1073/pnas.0909141107

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl] benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 angstrom cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K(D) = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the "off-target" effect of a small molecule is mediated by an MAI mechanism.

引用

页码：4878 / 4883

页数：6

共 31 条

[1] A specific mechanism for nonspecific activation in reporter-gene assays [J].