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トップダウン紫外線光解離質量スペクトロメトリーによるディヒドロフォラートリドゥクタゼ複合体の構造的特徴づけ

Michael B Cammarata¹, Ross Thyer¹, Jake Rosenberg¹

¹†Department of Chemistry and ‡Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, United States.

Journal of the American Chemical Society

|July 1, 2015

まとめ

193 nmのUVPD質量スペクトロメトリーは,二水酸化葉酸還元酵素 (DHFR) の構成変化を追跡します. この技術は,NADPHとメトトレキサート (MTX) のリガンド結合部位をマッピングし,その触媒サイクル中の酵素の柔軟性を明らかにします.

科学分野:

バイオケミストリーバイオケミストリー
構造生物学構造生物学とは
マススペクトロメトリーによる質量スペクトロメトリーです.

背景:

ディヒドロフォラート還元酵素 (DHFR) は,ディヒドロフォラートをテトラヒドロフォラートに還元する過程で,重要な構造変化を経験します.
これらのダイナミックな変化を理解することは,酵素の機能を理解し,標的型阻害剤の設計に不可欠です.

研究の目的:

193nm UVPDを用いてコファクターNADPHと阻害剤メトトレキサート (MTX) の存在下におけるDHFRのコンフォーマーションランドスケープを特徴づける.
DHFR内のNADPHとMTXの結合部位をマッピングし,断片化パターンを構造的再配置と相関させる.

主な方法:

193nm紫外線光解離 (UVPD) 質量スペクトロメトリーは,二次 (DHFR·NADPH,DHFR·MTX) と三次 (DHFR·NADPH·MTX) 複合体を分析するために使用されました.
広範な配列のカバーが達成され,リガンド相互作用と構成状態を示す断片イオンが得られました.

主要な成果:

UVPDは高いシーケンスカバー率 (>80%) を提供し,リガンド結合した断片を明らかにし,NADPHをアデノシン結合ドメインとMTXをループドメインにマッピングしました.
断片化パターンは,骨幹の裂け方傾向の有意な変動を示し,リガンド結合時にサブドメインの回転やループの動きなどの構成変化を反映した.
特定の領域は,強化された裂け方 (柔軟性の増加) または抑制された裂け方 (リガンドシールド/分子内相互作用) を示した.

結論:

193nm UVPDは,触媒サイクル中の酵素構成動態を監視するための多用途かつ敏感な技術です.
この研究では,UVPDの断片化とDHFRの既知の構造的,機能的側面との相関関係が成功し,この方法の有用性を検証しました.
発見は,三元複合体の移行状態と酵素の柔軟性についての洞察を提供します.

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