Propogation of the Allosteric Signal in Bacillus Stearothermophilus Phosphofructokinase Examined by Methyl-TROSY NMR

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.

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Bringing Dynamic Molecular Machines into Focus by Methyl-TROSY NMR

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-060713-035829 ◽

2014 ◽

Vol 83 (1) ◽

pp. 291-315 ◽

Cited By ~ 134

Author(s):

Rina Rosenzweig ◽

Lewis E. Kay

Keyword(s):

Molecular Machines ◽

Methyl Trosy ◽

Methyl Trosy Nmr

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Ligand modulation of sidechain dynamics in a wild-type human GPCR

eLife ◽

10.7554/elife.28505 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 41

Author(s):

Lindsay D Clark ◽

Igor Dikiy ◽

Karen Chapman ◽

Karin EJ Rödström ◽

James Aramini ◽

...

Keyword(s):

Human Physiology ◽

Structural Changes ◽

Inverse Agonist ◽

Methyl Groups ◽

Signaling Proteins ◽

Wild Type ◽

Biophysical Studies ◽

Methyl Trosy ◽

Conformational Regulation ◽

Methyl Trosy Nmr

GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (A2AR) that are deuterated apart from 1H/13C NMR probes at isoleucine δ1 methyl groups, which facilitated 1H/13C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na+] is required to allow large agonist-induced structural changes in A2AR, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics.

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Measurement of Active Site Ionization Equilibria in the 670 kDa Proteasome Core Particle Using Methyl-TROSY NMR

Journal of the American Chemical Society ◽

10.1021/ja403091c ◽

2013 ◽

Vol 135 (25) ◽

pp. 9259-9262 ◽

Cited By ~ 21

Author(s):

Algirdas Velyvis ◽

Lewis E. Kay

Keyword(s):

Active Site ◽

Core Particle ◽

Methyl Trosy ◽

Ionization Equilibria ◽

Methyl Trosy Nmr

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Conformational dynamics of the HIV Vif protein complex

10.1101/483099 ◽

2018 ◽

Author(s):

K. A. Ball ◽

L. M. Chan ◽

D. J. Stanley ◽

E. Tierney ◽

S. Thapa ◽

...

Keyword(s):

Crystal Structure ◽

Protein Complex ◽

Conformational Dynamics ◽

Intrinsically Disordered Protein ◽

Md Simulations ◽

Global Dynamics ◽

Intrinsically Disordered ◽

Ring E3 Ligase ◽

Methyl Trosy ◽

Methyl Trosy Nmr

AbstractHIV-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 antiviral proteins. Vif folds when it binds the Cullin-RING E3 ligase CRL5 and the transcription cofactor CBF-β. A five-protein complex containing the substrate receptor (Vif, CBF-β, Elongin-B, Elongin-C) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use Molecular Dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and APOBEC3 bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, while VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation optimized spectroscopy (methyl-TROSY) NMR data, which indicates that the entire VCBC complex without CUL5 is dynamic on the μs-ms timescale. Vif binds APOBEC3 to recruit it to the complex, and methyl-TROSY NMR shows that the VCBC complex is more conformationally restricted when bound to APOBEC3F, consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjuction with the global dynamics of the complex. Like other ubiquitin substrate receptors, VCBC can exist alone or in complex with CUL5 in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

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Biphasic activation of β-arrestin 1 upon interaction with a GPCR revealed by methyl-TROSY NMR

Nature Communications ◽

10.1038/s41467-021-27482-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yutaro Shiraishi ◽

Yutaka Kofuku ◽

Takumi Ueda ◽

Shubhi Pandey ◽

Hemlata Dwivedi-Agnihotri ◽

...

Keyword(s):

Nuclear Magnetic Resonance ◽

Nmr Spectroscopy ◽

Magnetic Resonance ◽

Conformational Changes ◽

G Protein Coupled Receptors ◽

Binding Modes ◽

Partial Activation ◽

Methyl Trosy ◽

G Protein Coupled ◽

Methyl Trosy Nmr

Abstractβ-arrestins (βarrs) play multifaceted roles in the function of G protein-coupled receptors (GPCRs). βarrs typically interact with phosphorylated C-terminal tail (C tail) and transmembrane core (TM core) of GPCRs. However, the effects of the C tail- and TM core-mediated interactions on the conformational activation of βarrs have remained elusive. Here, we show the conformational changes for βarr activation upon the C tail- and TM core-mediated interactions with a prototypical GPCR by nuclear magnetic resonance (NMR) spectroscopy. Our NMR analyses demonstrated that while the C tail-mediated interaction alone induces partial activation, in which βarr exists in equilibrium between basal and activated conformations, the TM core- and the C tail-mediated interactions together completely shift the equilibrium toward the activated conformation. The conformation-selective antibody, Fab30, promotes partially activated βarr into the activated-like conformation. This plasticity of βarr conformation in complex with GPCRs engaged in different binding modes may explain the multifunctionality of βarrs.

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