Conformational Changes in Orotidine 5′-Monophosphate Decarboxylase: “Remote” Residues That Stabilize the Active Conformation

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca2+alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca2+activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.

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Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes

Nucleic Acids Research ◽

10.1093/nar/gkaa935 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11664-11674 ◽

Cited By ~ 1

Author(s):

Ivana Duic ◽

Hisashi Tadakuma ◽

Yoshie Harada ◽

Ryo Yamaue ◽

Katashi Deguchi ◽

...

Keyword(s):

Conformational Changes ◽

Mammalian Cells ◽

Atp Hydrolysis ◽

Antiviral Response ◽

Fiber Formation ◽

Viral Rna ◽

Active Conformation ◽

Viral Dsrna ◽

Fiber Assembly ◽

Signaling Domain

Abstract Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-β induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.

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Hairpin and hammerhead ribozymes: how different are they?

Biochemical Society Transactions ◽

10.1042/bst0301116 ◽

2002 ◽

Vol 30 (6) ◽

pp. 1116-1119 ◽

Cited By ~ 6

Author(s):

J. M. Burke

Keyword(s):

Metal Ions ◽

Conformational Changes ◽

Experimental Work ◽

Hammerhead Ribozyme ◽

Active Conformation ◽

Hairpin Ribozyme ◽

Hammerhead Ribozymes ◽

Active Structure ◽

Catalytic Function ◽

Recent Experimental Work

Recent experimental work on the hairpin and hammerhead ribozymes suggests that they have more similarities than previously suspected. Notably, each is now known to function as a true RNA catalyst, not requiring metal ions for folding or catalytic function. The active conformation of the hairpin ribozyme has been established by crystallography, and is well supported by biochemical and biophysical evidence that has identified conformational changes and key nucleotides required for catalysis. Analogous work is under way to establish the active structure of the hammerhead ribozyme.

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Self-association configures the NAD+-binding site of plant NLR TIR domains

10.1101/2021.10.02.462850 ◽

2021 ◽

Author(s):

Hayden Burdett ◽

Xiahao Hu ◽

Maxwell X Rank ◽

Natsumi Maruta ◽

Bostjan Kobe

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Active Site ◽

Binding Sites ◽

Molecular Mechanisms ◽

Structural Features ◽

Active Conformation ◽

Effector Triggered Immunity ◽

Self Association ◽

Tir Domains

TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the ″AE interface″ (mediated by helices A and E), the ″BB-loop″ (connecting β-strand B and helix B in the structure), and the ″BE interface″ (mediated by the BB-loop from one TIR and the ″DE surface″ of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact ″DE surface″ is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

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Comparison of the Thermostability Properties of Three Acid Phosphatases from Molds: Aspergillus fumigatusPhytase, A. niger Phytase, and A. nigerpH 2.5 Acid Phosphatase

Applied and Environmental Microbiology ◽

10.1128/aem.64.11.4446-4451.1998 ◽

1998 ◽

Vol 64 (11) ◽

pp. 4446-4451 ◽

Cited By ~ 93

Author(s):

Markus Wyss ◽

Luis Pasamontes ◽

Roland Rémy ◽

Josiane Kohler ◽

Eric Kusznir ◽

...

Keyword(s):

Acid Phosphatase ◽

Aspergillus Niger ◽

Enzymatic Activity ◽

Conformational Changes ◽

Animal Feed ◽

Heat Denaturation ◽

Acid Phosphatases ◽

Active Conformation ◽

Feed Supplements ◽

Optimum Ph

ABSTRACT Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase,Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases ofA. fumigatus and A. niger were both denatured at temperatures between 50 and 70°C. After heat denaturation at temperatures up to 90°C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases,A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of ≥80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of enzymatic activity was considerably higher for A. fumigatusphytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80°C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.

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Molecular dynamics of the immune checkpoint programmed cell death protein I, PD-1: conformational changes of the BC-loop upon binding of the ligand PD-L1 and the monoclonal antibody nivolumab

BMC Bioinformatics ◽

10.1186/s12859-020-03904-9 ◽

2020 ◽

Vol 21 (S17) ◽

Cited By ~ 1

Author(s):

Bernhard Roither ◽

Chris Oostenbrink ◽

Wolfgang Schreiner

Keyword(s):

Molecular Dynamics ◽

Signal Transduction ◽

T Cells ◽

Cell Death ◽

Programmed Cell Death ◽

Conformational Changes ◽

Immune Checkpoint ◽

Active Conformation ◽

Dynamics Simulations ◽

Cell Death Protein

Abstract Background The immune checkpoint receptor programmed cell death protein I (PD-1) has been identified as a key target in immunotherapy. PD-1 reduces the risk of autoimmunity by inducing apoptosis in antigen-specific T cells upon interaction with programmed cell death protein ligand I (PD-L1). Various cancer types overexpress PD-L1 to evade the immune system by inducing apoptosis in tumor-specific CD8+ T cells. The clinically used blocking antibody nivolumab binds to PD-1 and inhibits the immunosuppressive interaction with PD-L1. Even though PD-1 is already used as a drug target, the exact mechanism of the receptor is still a matter of debate. For instance, it is hypothesized that the signal transduction is based on an active conformation of PD-1. Results Here we present the results of the first molecular dynamics simulations of PD-1 with a complete extracellular domain with a focus on the role of the BC-loop of PD-1 upon binding PD-L1 or nivolumab. We could demonstrate that the BC-loop can form three conformations. Nivolumab binds to the BC-loop according to the conformational selection model whereas PD-L1 induces allosterically a conformational change of the BC-loop. Conclusion Due to the structural differences of the BC-loop, a signal transduction based on active conformation cannot be ruled out. These findings will have an impact on drug design and will help to refine immunotherapy blocking antibodies.

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Agonist Binding and G Protein Coupling in Histamine H2 Receptor: A Molecular Dynamics Study

International Journal of Molecular Sciences ◽

10.3390/ijms21186693 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6693

Author(s):

Marcus Conrad ◽

Christian A. Söldner ◽

Yinglong Miao ◽

Heinrich Sticht

Keyword(s):

Molecular Dynamics ◽

G Protein ◽

Conformational Changes ◽

Structural Information ◽

Conformational Stability ◽

Site Directed Mutagenesis ◽

Active Conformation ◽

Histamine H2 Receptor ◽

H2 Receptor ◽

Ulcer Disease

The histamine H2 receptor (H2R) plays an important role in the regulation of gastric acid secretion. Therefore, it is a main drug target for the treatment of gastroesophageal reflux or peptic ulcer disease. However, there is as of yet no 3D-structural information available hampering a mechanistic understanding of H2R. Therefore, we created a model of the histamine-H2R-Gs complex based on the structure of the ternary complex of the β2-adrenoceptor and investigated the conformational stability of this active GPCR conformation. Since the physiologically relevant motions with respect to ligand binding and conformational changes of GPCRs can only partly be assessed on the timescale of conventional MD (cMD) simulations, we also applied metadynamics and Gaussian accelerated molecular dynamics (GaMD) simulations. A multiple walker metadynamics simulation in combination with cMD was applied for the determination of the histamine binding mode. The preferential binding pose detected is in good agreement with previous data from site directed mutagenesis and provides a basis for rational ligand design. Inspection of the H2R-Gs interface reveals a network of polar interactions that may contribute to H2R coupling selectivity. The cMD and GaMD simulations demonstrate that the active conformation is retained on a μs-timescale in the ternary histamine-H2R-Gs complex and in a truncated complex that contains only Gs helix α5 instead of the entire G protein. In contrast, histamine alone is unable to stabilize the active conformation, which is in line with previous studies of other GPCRs.

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Conformational Changes in Orotidine 5′-Monophosphate Decarboxylase: A Structure-Based Explanation for How the 5′-Phosphate Group Activates the Enzyme

Biochemistry ◽

10.1021/bi301188k ◽

2012 ◽

Vol 51 (43) ◽

pp. 8665-8678 ◽

Cited By ~ 13

Author(s):

Bijoy J. Desai ◽

B. McKay Wood ◽

Alexander A. Fedorov ◽

Elena V. Fedorov ◽

Bogdana Goryanova ◽

...

Keyword(s):

Conformational Changes ◽

Phosphate Group ◽

Monophosphate Decarboxylase

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Enzyme Architecture: Erection of Active Orotidine 5′-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes

Journal of the American Chemical Society ◽

10.1021/jacs.7b08897 ◽

2017 ◽

Vol 139 (45) ◽

pp. 16048-16051 ◽

Cited By ~ 8

Author(s):

Archie C. Reyes ◽

Tina L. Amyes ◽

John P. Richard

Keyword(s):

Conformational Changes ◽

Monophosphate Decarboxylase

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Microsecond Timescale Simulations at the Transition State of PmHMGR Predict Remote Allosteric Residues

10.26434/chemrxiv.9999545.v1 ◽

2019 ◽

Author(s):

Taylor Quinn ◽

Calvin N. Steussy ◽

Brandon E. Haines ◽

Jinping Lei ◽

Wei Wang ◽

...

Keyword(s):

Transition State ◽

Molecular Mechanics ◽

Conformational Changes ◽

Enzymatic Catalysis ◽

Dynamic Effect ◽

Md Simulations ◽

Site Directed Mutagenesis ◽

Pseudomonas Mevalonii ◽

Remote Residues ◽

Coa Reductase

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. QM/MM methods have been extensively used to study these reactions, but the difficulties arising from the hybrid treatment of the system are well documented. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to react the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale. Application of this approach the second hydride transfer transition state of HMG-CoA reductase from Pseudomonas mevalonii (PmHMGR) identified three remote residues, R396 E399 and L407, (15-27 Å away from the active site) that have a remote dynamic effect on enzyme activity. The predictions were subsequently validated experimentally via site-directed mutagenesis. These results show that microsecond timescale MD simulations of transition states are possible and can predict rather than just rationalize remote allosteric residues.

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