A mobile loop near the active site acts as a switch between the dual activities of a viral protease/deubiquitinase

PLoS Pathogens

Lucile Jomat¹  Isabelle Jupin¹  Stéphane Bressanelli²  Sonia Fieulaine²  Maya Ayach² 

[1] Institut Jacques Monod, CNRS—Univ Paris-Diderot, Paris, France;Institute for Integrative Biology of the Cell, CEA—CNRS—Univ Paris-Saclay, Gif sur Yvette, France

关键词: Viral replication;    Crystal structure;    Arabidopsis thaliana;    Leaves;    Microbial mutation;    Molecular dynamics;    Crystals;    Molecular structure;   

DOI  :  10.1371/journal.ppat.1006714

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

The positive-strand RNA virus Turnip yellow mosaic virus (TYMV) encodes an ovarian tumor (OTU)-like protease/deubiquitinase (PRO/DUB) protein domain involved both in proteolytic processing of the viral polyprotein through its PRO activity, and in removal of ubiquitin chains from ubiquitylated substrates through its DUB activity. Here, the crystal structures of TYMV PRO/DUB mutants and molecular dynamics simulations reveal that an idiosyncratic mobile loop participates in reversibly constricting its unusual catalytic site by adopting "open", "intermediate" or "closed" conformations. The two cis-prolines of the loop form a rigid flap that in the most closed conformation zips up against the other side of the catalytic cleft. The intermediate and closed conformations also correlate with a reordering of the TYMV PRO/DUB catalytic dyad, that then assumes a classical, yet still unusually mobile, OTU DUB alignment. Further structure-based mutants designed to interfere with the loop's mobility were assessed for enzymatic activity in vitro and in vivo, and were shown to display reduced DUB activity while retaining PRO activity. This indicates that control of the switching between the dual PRO/DUB activities resides prominently within this loop next to the active site. Introduction of mutations into the viral genome revealed that the DUB activity contributes to the extent of viral RNA accumulation both in single cells and in whole plants. In addition, the conformation of the mobile flap was also found to influence symptoms severity in planta. Such mutants now provide powerful tools with which to study the specific roles of reversible ubiquitylation in viral infection.

【 授权许可】

CC BY