Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K

Abstract The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a t...

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Autores principales:	Manoj K. Rout, Brian L. Lee, Aiyang Lin, Wei Xiao, Leo Spyracopoulos
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/847413dcf7314bdbbe3d72508e66203f
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spelling	oai:doaj.org-article:847413dcf7314bdbbe3d72508e66203f2021-12-02T15:09:04ZActive Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K10.1038/s41598-018-25476-82045-2322https://doaj.org/article/847413dcf7314bdbbe3d72508e66203f2018-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-25476-8https://doaj.org/toc/2045-2322Abstract The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.Manoj K. RoutBrian L. LeeAiyang LinWei XiaoLeo SpyracopoulosNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-15 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Manoj K. Rout Brian L. Lee Aiyang Lin Wei Xiao Leo Spyracopoulos Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K
description	Abstract The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.
format	article
author	Manoj K. Rout Brian L. Lee Aiyang Lin Wei Xiao Leo Spyracopoulos
author_facet	Manoj K. Rout Brian L. Lee Aiyang Lin Wei Xiao Leo Spyracopoulos
author_sort	Manoj K. Rout
title	Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K
title_short	Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K
title_full	Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K
title_fullStr	Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K
title_full_unstemmed	Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K
title_sort	active site gate dynamics modulate the catalytic activity of the ubiquitination enzyme e2-25k
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/847413dcf7314bdbbe3d72508e66203f
work_keys_str_mv	AT manojkrout activesitegatedynamicsmodulatethecatalyticactivityoftheubiquitinationenzymee225k AT brianllee activesitegatedynamicsmodulatethecatalyticactivityoftheubiquitinationenzymee225k AT aiyanglin activesitegatedynamicsmodulatethecatalyticactivityoftheubiquitinationenzymee225k AT weixiao activesitegatedynamicsmodulatethecatalyticactivityoftheubiquitinationenzymee225k AT leospyracopoulos activesitegatedynamicsmodulatethecatalyticactivityoftheubiquitinationenzymee225k
_version_	1718387950885011456

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K

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