Bringing Dynamic Molecular Machines into Focus by Methyl-TROSY NMR

2014 ◽  
Vol 83 (1) ◽  
pp. 291-315 ◽  
Author(s):  
Rina Rosenzweig ◽  
Lewis E. Kay
Keyword(s):  
Molecular Machines ◽  
Methyl Trosy ◽  
Methyl Trosy Nmr

Investigating the Dynamics of Destabilized Nucleosomes Using Methyl-TROSY NMR

2018 ◽  
Vol 140 (14) ◽  
pp. 4774-4777 ◽  
Author(s):  
Julianne L. Kitevski-LeBlanc ◽  
Tairan Yuwen ◽  
Pamela N. Dyer ◽  
Johannes Rudolph ◽  
Karolin Luger ◽  
...  
Keyword(s):  
Methyl Trosy ◽  
Methyl Trosy Nmr

scholarly journals Exploring long-range cooperativity in the 20S proteasome core particle from Thermoplasma acidophilum using methyl-TROSY–based NMR

2020 ◽  
Vol 117 (10) ◽  
pp. 5298-5309
Author(s):  
Enrico Rennella ◽  
Rui Huang ◽  
Zanlin Yu ◽  
Lewis E. Kay
Keyword(s):  
Molecular Machines ◽  
Molecular Assembly ◽  
20S Proteasome ◽  
Core Particle ◽  
Thermoplasma Acidophilum ◽  
Transverse Relaxation ◽  
Catalytic Sites ◽  
Methyl Trosy ◽  
Transverse Relaxation Optimized Spectroscopy ◽  
Allosteric Pathway

The 20S core particle (CP) proteasome is a molecular assembly catalyzing the degradation of misfolded proteins or proteins no longer required for function. It is composed of four stacked heptameric rings that form a barrel-like structure, sequestering proteolytic sites inside its lumen. Proteasome function is regulated by gates derived from the termini of α-rings and through binding of regulatory particles (RPs) to one or both ends of the barrel. The CP is dynamic, with an extensive allosteric pathway extending from one end of the molecule to catalytic sites in its center. Here, using methyl-transverse relaxation optimized spectroscopy (TROSY)-based NMR optimized for studies of high–molecular-weight complexes, we evaluate whether the pathway extends over the entire 150-Å length of the molecule. By exploiting a number of different labeling schemes, the two halves of the molecule can be distinguished, so that the effects of 11S RP binding, or the introduction of gate or allosteric pathway mutations at one end of the barrel can be evaluated at the distal end. Our results establish that while 11S binding and the introduction of key mutations affect each half of the CP allosterically, they do not further couple opposite ends of the molecule. This may have implications for the function of so-called “hybrid” proteasomes where each end of the CP is bound with a different regulator, allowing the CP to be responsive to both RPs simultaneously. The methodology presented introduces a general NMR strategy for dissecting pathways of communication in homo-oligomeric molecular machines.

scholarly journals Oligomeric assembly regulating mitochondrial HtrA2 function as examined by methyl-TROSY NMR

2021 ◽  
Vol 118 (11) ◽  
pp. e2025022118
Author(s):  
Yuki Toyama ◽  
Robert W. Harkness ◽  
Tim Y. T. Lee ◽  
Jason T. Maynes ◽  
Lewis E. Kay
Keyword(s):  
Active Sites ◽  
Transverse Relaxation ◽  
Catalytic Center ◽  
Protective Enzyme ◽  
Biochemical Assays ◽  
Methyl Trosy ◽  
Oligomeric Assembly ◽  
Domain Reorientation ◽  
Methyl Trosy Nmr ◽  
Trimeric Structure

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.

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

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

scholarly journals Conformational dynamics of the HIV Vif protein complex

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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