Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation

AbstractPupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.

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Unravelling the role of active-site isolation in reactivity and reaction pathway control for acetylene hydrogenation

Chemical Communications ◽

10.1039/d0cc01889a ◽

2020 ◽

Vol 56 (47) ◽

pp. 6372-6375

Author(s):

Siyang Liu ◽

Yiming Niu ◽

Yongzhao Wang ◽

Junnan Chen ◽

Xueping Quan ◽

...

Keyword(s):

Active Site ◽

Bimetallic Catalysts ◽

Reaction Pathway ◽

Isolation Effect ◽

Site Isolation ◽

Acetylene Hydrogenation ◽

Supported Pd

Supported PdxCuy bimetallic catalysts were prepared and characterized to illustrate the active-site isolation effect on controlling the reactivity and reaction pathway of acetylene hydrogenation.

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The role of post-translational modification in the photoregulation of Fe-type nitrile hydrataseElectronic supplementary information (ESI) available: Selected structural parameters of the experimentally observed NHase active site and DFT-optimised models 1 and 2, and graphical MO representations of the calculated INDO/S spectroscopic transitions for the models discussed. See http://www.rsc.org/suppdata/cc/b2/b207027h/

Chemical Communications ◽

10.1039/b207027h ◽

2002 ◽

pp. 2386-2387 ◽

Cited By ~ 6

Author(s):

Shannon N. Greene ◽

Christopher H. Chang ◽

Nigel G. J. Richards

Keyword(s):

Active Site ◽

Structural Parameters ◽

Supplementary Information ◽

Post Translational Modification

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Temporary Intermediates of L-Trp Along the Reaction Pathway of Human Indoleamine 2,3-Dioxygenase 1 and Identification of an Exo Site

International Journal of Tryptophan Research ◽

10.1177/11786469211052964 ◽

2021 ◽

Vol 14 ◽

pp. 117864692110529

Author(s):

Manon Mirgaux ◽

Laurence Leherte ◽

Johan Wouters

Keyword(s):

Molecular Dynamics ◽

Reaction Mechanism ◽

Crystal Structures ◽

Protein Dynamics ◽

Active Site ◽

Reaction Pathway ◽

Small Unit ◽

Indoleamine 2,3 Dioxygenase ◽

Dynamics Simulations

Protein dynamics governs most of the fundamental processes in the human body. Particularly, the dynamics of loops located near an active site can be involved in the positioning of the substrate and the reaction mechanism. The understanding of the functioning of dynamic loops is therefore a challenge, and often requires the use of a multi-disciplinary approach mixing, for example, crystallographic experiments and molecular dynamics simulations. In the present work, the dynamic behavior of the JK-loop of the human indoleamine 2,3-dioxygenase 1 hemoprotein, a target for immunotherapy, is investigated. To overcome the lack of knowledge on this dynamism, the study reported here is based on 3 crystal structures presenting different conformations of the loop, completed with molecular dynamics trajectories and MM-GBSA analyses, in order to trace the reaction pathway of the enzyme. In addition, the crystal structures identify an exo site in the small unit of the enzyme, that is populated redundantly by the substrate or the product of the reaction. The role of this newer reported exo site still needs to be investigated.

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Atomic view into Plasmodium actin polymerization, ATP hydrolysis, and phosphate release

10.1101/467423 ◽

2018 ◽

Author(s):

Esa-Pekka Kumpula ◽

Andrea J. López ◽

Leila Tajedin ◽

Huijong Han ◽

Inari Kursula

Keyword(s):

High Resolution ◽

Active Site ◽

Actin Polymerization ◽

Atp Hydrolysis ◽

Potassium Ion ◽

Structural Factors ◽

Comprehensive Model ◽

Phosphate Release ◽

Magnesium Binding

AbstractPlasmodium actins form very short filaments and have a non-canonical link between ATP hydrolysis and polymerization. Long filaments are detrimental to the parasites, but the structural factors constraining Plasmodium microfilament lengths are currently unknown. Using high-resolution crystallography, we show that magnesium binding activates the Plasmodium actin I monomer before polymerization by a slight flattening, which is reversed upon phosphate release. A coordinated potassium ion resides in the active site during hydrolysis and leaves together with the phosphate, a process governed by the position of the Arg178/Asp180-containing A-loop. Asp180 interacts with either Lys270 or His74, depending on protonation, while Arg178 links the inner and outer domains. Hence, the A-loop is a switch between stable and non-stable filament conformations. Our data provide a comprehensive model for polymerization, phosphate release, and the inherent instability of parasite microfilaments.

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Trp–His covalent adduct in bilirubin oxidase is crucial for effective bilirubin binding but has a minor role in electron transfer

Scientific Reports ◽

10.1038/s41598-019-50105-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Tomáš Kovaľ ◽

Leona Švecová ◽

Lars H. Østergaard ◽

Tereza Skalova ◽

Jarmila Dušková ◽

...

Keyword(s):

Electron Transfer ◽

Active Site ◽

Substrate Binding ◽

Minor Role ◽

Post Translational Modification ◽

Unique Type ◽

Bilirubin Oxidase ◽

Bilirubin Binding ◽

A Minor

Abstract Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396–His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp–His adduct mainly for binding and to a smaller extent for electron transfer.

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High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

eLife ◽

10.7554/elife.73724 ◽

2021 ◽

Vol 10 ◽

Author(s):

Sabrina Pospich ◽

H Lee Sweeney ◽

Anne Houdusse ◽

Stefan Raunser

Keyword(s):

High Resolution ◽

Bound States ◽

Atp Hydrolysis ◽

Molecular Motor ◽

Structural Data ◽

Underlying Mechanism ◽

Actin Binding ◽

Structural Transitions ◽

Myosin V ◽

High Flexibility

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin.

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Supplemental Material for High Resolution Examination of the Role of Sleep Disturbance in Predicting Functioning and Psychotic Symptoms in Schizophrenia: A Novel Experience Sampling Study

Journal of Abnormal Psychology ◽

10.1037/abn0000180.supp ◽

2016 ◽

Keyword(s):

High Resolution ◽

Sleep Disturbance ◽

Experience Sampling ◽

Psychotic Symptoms

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Quantitative accuracy of serotonergic neurotrans-mission imaging with high-resolution 123I SPECT

Nuklearmedizin ◽

10.1055/s-0038-1623914 ◽

2004 ◽

Vol 43 (06) ◽

pp. 185-189 ◽

Cited By ~ 4

Author(s):

J. T. Kuikka

Keyword(s):

High Resolution ◽

Scatter Correction ◽

Region Of Interest ◽

Binding Potential ◽

Partial Volume ◽

Therapeutic Drugs ◽

Quantitative Accuracy ◽

Critical Error ◽

Statistical Noise

Summary Aim: Serotonin transporter (SERT) imaging can be used to study the role of regional abnormalities of neurotransmitter release in various mental disorders and to study the mechanism of action of therapeutic drugs or drugs’ abuse. We examine the quantitative accuracy and reproducibility that can be achieved with high-resolution SPECT of serotonergic neurotransmission. Method: Binding potential (BP) of 123I labeled tracer specific for midbrain SERT was assessed in 20 healthy persons. The effects of scatter, attenuation, partial volume, mis-registration and statistical noise were estimated using phantom and human studies. Results: Without any correction, BP was underestimated by 73%. The partial volume error was the major component in this underestimation whereas the most critical error for the reproducibility was misplacement of region of interest (ROI). Conclusion: The proper ROI registration, the use of the multiple head gamma camera with transmission based scatter correction introduce more relevant results. However, due to the small dimensions of the midbrain SERT structures and poor spatial resolution of SPECT, the improvement without the partial volume correction is not great enough to restore the estimate of BP to that of the true one.

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