scholarly journals Decreased conformational stability in the oncogenic N92I mutant of Ras-related C3 botulinum toxin substrate 1

2019 ◽  
Vol 5 (8) ◽  
pp. eaax1595
Author(s):  
Yuki Toyama ◽  
Kenji Kontani ◽  
Toshiaki Katada ◽  
Ichio Shimada
Keyword(s):  
Botulinum Toxin ◽  
Conformational Stability ◽  
Bound State ◽  
Activation Mechanism ◽  
Resonance Spectroscopy ◽  
Activation Process ◽  
Dissociation Reaction ◽  
Static Structure ◽  
Guanosine Diphosphate ◽  
Solution Nuclear Magnetic Resonance

Ras-related C3 botulinum toxin substrate 1 (Rac1) functions as a molecular switch by cycling between an inactive guanosine diphosphate (GDP)–bound state and an active guanosine triphosphate (GTP)–bound state. An oncogenic mutant of Rac1, an N92I mutant, strongly promotes cell proliferation and subsequent oncogenic activities by facilitating the intrinsic GDP dissociation in the inactive GDP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the N92I mutant. We found that the static structure of the GDP binding site is not markedly perturbed by the mutation, but the overall conformational stability decreases in the N92I mutant, which then facilitates GDP dissociation by lowering the activation energy for the dissociation reaction. On the basis of these results, we proposed the activation mechanism of the N92I mutant, in which the decreased conformational stability plays important roles in its activation process.

scholarly journals Conformational landscape alternations promote oncogenic activities of Ras-related C3 botulinum toxin substrate 1 as revealed by NMR

2019 ◽  
Vol 5 (3) ◽  
pp. eaav8945 ◽  
Author(s):  
Yuki Toyama ◽  
Kenji Kontani ◽  
Toshiaki Katada ◽  
Ichio Shimada
Keyword(s):  
Botulinum Toxin ◽  
Bound States ◽  
Conformational Equilibrium ◽  
Bound State ◽  
Activation Mechanism ◽  
Resonance Spectroscopy ◽  
Guanosine Triphosphate ◽  
Guanosine Diphosphate ◽  
Conformational Landscape ◽  
Solution Nuclear Magnetic Resonance

Ras-related C3 botulinum toxin substrate 1 (Rac1) plays critical roles in the maintenance of cell morphology by cycling between inactive guanosine diphosphate (GDP)–bound and active guanosine triphosphate (GTP)–bound states. Rac1 P29S mutant is known to strongly promote oncogenesis by facilitating its intrinsic GDP dissociation and thereby increasing the level of the GTP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the oncogenic P29S mutant. We demonstrate that the conformational landscape is markedly altered in the mutant, and the preexisting equilibrium is shifted toward the conformation with reduced affinity for Mg2+, a cofactor that is critical for maintaining stable GDP binding. Our results suggest that the alternation of the preexisting conformational equilibrium of proteins is one of the fundamental mechanisms underlying their oncogenic activities.

scholarly journals Butyl Isocyanide as a Probe of the Activation Mechanism of Soluble Guanylate Cyclase

2007 ◽  
Vol 282 (49) ◽  
pp. 35741-35748 ◽  
Author(s):  
Emily R. Derbyshire ◽  
Michael A. Marletta
Keyword(s):  
Nitric Oxide ◽  
Binding Site ◽  
Guanylate Cyclase ◽  
Electronic Absorption ◽  
Soluble Guanylate Cyclase ◽  
Activation Mechanism ◽  
Activation Process ◽  
Absorbance Maximum ◽  
No Activation ◽  
Allosteric Activator

Nitric oxide (NO) is a physiologically relevant activator of the hemoprotein soluble guanylate cyclase (sGC). In the presence of NO, sGC is activated several hundredfold above the basal level by a mechanism that remains to be elucidated. The heme ligand n-butyl isocyanide (BIC) was used to probe the mechanism of NO activation of sGC. Electronic absorption spectroscopy was used to show that BIC binds to the sGC heme, forming a 6-coordinate complex with an absorbance maximum at 429 nm. BIC activates sGC 2-5-fold, and synergizes with the allosteric activator YC-1, to activate the enzyme 15-25-fold. YC-1 activates the sGC-BIC complex, and leads to an increase in both the Vmax and Km. BIC was also used to probe the mechanism of NO activation. The activity of the sGC-BIC complex increases 15-fold in the presence of NO, without displacing BIC at the heme, which is consistent with previous reports that proposed the involvement of a non-heme NO binding site in the activation process.

scholarly journals The allosteric activation mechanism of a phospholipase A2-like toxin from Bothrops jararacussu venom: a dynamic description

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Antoniel A. S. Gomes ◽  
Fabio F. Cardoso ◽  
Maximilia F. Souza ◽  
Cristiano L. P. Oliveira ◽  
David Perahia ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Activation Mechanism ◽  
Activation Process ◽  
Structural Determinants ◽  
Allosteric Activation ◽  
Bothrops Jararacussu ◽  
Inactive State ◽  
Dynamic Description ◽  
Comprehensive Study

Abstract The activation process of phospholipase A2-like (PLA2-like) toxins is a key step in their molecular mechanism, which involves oligomeric changes leading to the exposure of specific sites. Few studies have focused on the characterization of allosteric activators and the features that distinguish them from inhibitors. Herein, a comprehensive study with the BthTX-I toxin from Bothrops jararacussu venom bound or unbound to α-tocopherol (αT) was carried out. The oligomerization state of BthTX-I bound or unbound to αT in solution was studied and indicated that the toxin is predominantly monomeric but tends to oligomerize when complexed with αT. In silico molecular simulations showed the toxin presents higher conformational changes in the absence of αT, which suggests that it is important to stabilize the structure of the toxin. The transition between the two states (active/inactive) was also studied, showing that only the unbound BthTX-I system could migrate to the inactive state. In contrast, the presence of αT induces the toxin to leave the inactive state, guiding it towards the active state, with more regions exposed to the solvent, particularly its active site. Finally, the structural determinants necessary for a molecule to be an inhibitor or activator were analyzed in light of the obtained results.

Evaluation of Reactions of a Dilute Cross-Linker Using Solution Nuclear Magnetic Resonance Spectroscopy with Isotopically Labeled Cross-Linkers

2004 ◽  
Vol 37 (10) ◽  
pp. 3699-3706 ◽  
Author(s):  
Lon J. Mathias ◽  
Rick D. Davis ◽  
Scott J. Steadman ◽  
William L. Jarrett ◽  
Richard D. Redfearn ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Nuclear Magnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Cross Linker ◽  
Solution Nuclear Magnetic Resonance ◽  
Nuclear Magnetic

scholarly journals Intramolecular hydrogen bonding and conformational preferences on 2-fluoro-phenylaminocyclohexanol

2018 ◽  
Author(s):  
Cassia Chiari ◽  
Claudio Francisco Tormena ◽  
Kahlil Schwanka Salome ◽  
Laiza Bruzadelle Loureiro ◽  
Renan Vidal Viesser
Keyword(s):  
Hydrogen Bonding ◽  
Driving Force ◽  
Conformational Stability ◽  
Intramolecular Hydrogen Bonding ◽  
Quantum Mechanical ◽  
Resonance Spectroscopy ◽  
Quantum Mechanical Calculations ◽  
Conformational Preferences ◽  
Intramolecular Hydrogen ◽  
Theoretical Results

The aim of this study is to evaluate the influence and strength of possible intramolecular hydrogen bonding (IntraHB) involving N-H--O, O-H--N, O-H--F and N-H--F molecular moieties as a driving force on the conformational preferences of 2-fluoro-phenylaminocyclohexanol. To achieve our purpose we synthesized the compound and performed it's characterization using Nuclear Magnetic Resonance Spectroscopy (NMR). Quantum mechanical calculations were carried out to evaluate the effect of IntraHB on the conformational stability. Experimental and theoretical results showed that N-H--F and O-H--N IntraHB have a greater influence on the conformacional preferably adopted by the molecule.

scholarly journals Elucidating the Molecular Basis of pH Activation of an Engineered Mechanosensitive Channel

2019 ◽  
Author(s):  
Kalyan Immadisetty ◽  
Adithya Polasa ◽  
Reid Shelton ◽  
Mahmoud Moradi
Keyword(s):  
Mechanical Energy ◽  
Salt Bridge ◽  
Md Simulations ◽  
Pressure Profile ◽  
Chemical Signals ◽  
Activation Mechanism ◽  
Activation Process ◽  
Ph Change ◽  
Non Equilibrium Molecular Dynamics ◽  
First Time

AbstractMechanosensitive (MS) channels detect and respond to changes in the pressure profile of cellular membranes and transduce the mechanical energy into electrical and/or chemical signals. By re-engineering, however, the activation of some MS channels can be triggered by chemical signals such as pH change. Here, for the first time, we have elucidated, at an atomic level, the activation mechanism of an engineered MscL channel in response to the pH changes of the environment through a combination of equilibrium and non-equilibrium molecular dynamics (MD) simulations. The key highlights of our proposed activation mechanism are that: (1) periplasmic loops play a key role in activation, (2) loss of various hydrogen bonding and salt bridge interactions in the engineered MscL channel causes the opening of the channel, and (3) the most significant interactions lost during the activation process are those between the transmembrane (TM) helices 1 and 2 (TM1 and TM2). The orientation-based method in this work for generating and optimizing an open model of engineered MscL is a promising method for generating unknown states of proteins and for studying the activation processes in ion channels. This work facilitates the studies aimed at designing pH-triggered drug delivery liposomes (DDL), which embed MscL as a nanovalve.

scholarly journals Structure and activation mechanism of the hexameric plasma membrane H+-ATPase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Zhao ◽  
Chaoran Zhao ◽  
Dandan Chen ◽  
Caihong Yun ◽  
Huilin Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Conformational Changes ◽  
Liquid Crystalline ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Proton Pumping ◽  
Antifungal Drugs ◽  
Activation Mechanism ◽  
Activation Process ◽  
Α Helix

AbstractThe S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.

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