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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
La base para un interruptor de tioester de proteína similar a la ubiquitina que cambia la afinidad E1-E2.
Danny T Huang1, Harold W Hunt, Min Zhuang
1Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Nature
|January 16, 2007
Resumen
El estudio revela cómo las enzimas de conjugación de proteínas similares a la ubiquitina (UBL) cambian afinidades de unión a través de un mecanismo de tioester. Este cambio conformacional impulsa pasos secuenciales en las vías de modificación de UBL.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Biología Estructural Biología estructural.
- Biología celular molecular Biología celular molecular
Sus antecedentes:
- Las proteínas similares a la ubiquitina (UBL) se modifican a través de cascadas dinámicas de enzimas E1-E2-E3.
- Las enzimas E1 activan las UBL a través de la adenilación, formando intermedios tioesters cruciales para los siguientes pasos de conjugación.
Objetivo del estudio:
- Para aclarar el mecanismo estructural de la activación y transferencia de UBL dentro de la vía NEDD8 humana.
- Para comprender cómo las interacciones enzima-sustrato y los cambios conformacionales impulsan las cascadas de conjugación UBL.
Principales métodos:
- Análisis estructural reportado de un complejo de activación UBL atrapado.
- Utilizó un complejo que contiene la enzima NEDD8 E1 (APPBP1-UBA3), dos moléculas NEDD8, una enzima E2 inactiva (Ubc12) y MgATP.
Principales resultados:
- Se identificó un mecanismo de "cambio de tioester" que modula las afinidades E1-E2.
- Se revelaron dos sitios de unión E2 dependientes del enlace tioester de NEDD8 con E1, uno desenmascarado por el cambio conformacional de E1.
- Se demostró que la transferencia de NEDD8 a E2 induce la reversión conformacional de E1, facilitando la liberación del producto.
Conclusiones:
- La transferencia de enlaces tioéstricos entre las enzimas de conjugación impulsa cambios conformacionales y altera las redes de interacción.
- Este mecanismo es esencial para impulsar pasos consecutivos en cascadas de conjugación UBL.
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