scholarly journals AMPylation targets the rate-limiting step of BiP’s ATPase cycle for its functional inactivation

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Steffen Preissler ◽  
Lukas Rohland ◽  
Yahui Yan ◽  
Ruming Chen ◽  
Randy J Read ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Inverse Correlation ◽  
Rate Limiting Step ◽  
Hsp70 Chaperone ◽  
Tightly Coupled ◽  
Client Proteins ◽  
Functional Inactivation ◽  
Atp Binding And Hydrolysis ◽  
Rate Limiting ◽  
J Protein

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP’s activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.

Anion transport as related to hemoglobin A1c in erythrocytes of diabetic children.

1988 ◽  
Vol 34 (7) ◽  
pp. 1414-1416
Author(s):  
J Brahm ◽  
H B Mortensen
Keyword(s):  
Erythrocyte Membrane ◽  
Hemoglobin A1c ◽  
Bicarbonate Transport ◽  
Physiological Conditions ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Diabetic Children ◽  
Tightly Coupled ◽  
Integral Transport ◽  
Rate Limiting

Abstract We determined chloride and bicarbonate transport [Jcl and Jbic, nmol/(cm2.s)] under physiological conditions (Cl- 110 and HCO3- 25 mmol per liter, respectively, pH 7.4, 38 degrees C) across the erythrocyte membrane in blood samples from 12 diabetic (Jcl 26.1, SD +/- 3.7, n = 24; Jbic 7.6, SD +/- 0.9, n = 19) and 10 non-diabetic children (Jcl 30.6, SD +/- 4.6, n = 16; Jbic 7.3, SD +/- 1.0, n = 20) with mean hemoglobin A1c values of 11.08% (SD +/- 2.45%) and 5.36% (SD +/- 0.25%), respectively. The concentration of HbA1c, which also reflects the degree of glycation of the membrane proteins, differed significantly (P greater than 0.001) between the two groups, whereas there was no significant variation (P greater than 0.1) in Jcl and Jbic. We conclude that glycation of the integral transport protein in the erythrocyte membrane, capnophorin (also called "band 3"), which mediates a tightly coupled anion exchange, does not change the capacity of the transport system under physiological conditions. Thus the rate-limiting step of the exploitation of the CO2 transport capacity of the blood is not impaired in diabetics and consequently does not endanger the compensatory hyperventilation after ketoacidosis.

scholarly journals Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

2021 ◽  
Author(s):  
Mrinal Shekhar ◽  
Chitrak Gupta ◽  
Kano Suzuki ◽  
Abhishek Singharoy ◽  
Takeshi Murata
Keyword(s):  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Hydrolysis Product ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Dynamics Simulations ◽  
Rate Limiting

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or 'dwell' is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transfor-mations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain level interactions transcend into larger scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

scholarly journals Author response: AMPylation targets the rate-limiting step of BiP’s ATPase cycle for its functional inactivation

2017 ◽  
Author(s):  
Steffen Preissler ◽  
Lukas Rohland ◽  
Yahui Yan ◽  
Ruming Chen ◽  
Randy J Read ◽  
...  
Keyword(s):  
Author Response ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Functional Inactivation ◽  
Rate Limiting

scholarly journals Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1

2020 ◽  
Author(s):  
Yanxin Liu ◽  
Ming Sun ◽  
Alexander G. Myasnikov ◽  
Daniel Elnatan ◽  
Nicolas Delaeter ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Activation Mechanism ◽  
Client Protein ◽  
Protein Maturation ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Client Proteins ◽  
Rate Limiting

AbstractHsp90 is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of cellular “client” proteins. The ATP-driven client maturation process is regulated by a large number of co-chaperones. Among them, Aha1 is the most potent activator of Hsp90 ATPase activity and thus dramatically affects Hsp90’s client proteins. To understand the Aha1 activation mechanism, we determined full-length Hsp90:Aha1 structures in six different states by cryo-electron microscopy, including nucleotide-free semi-closed, nucleotide-bound pre-hydrolysis, and semi-hydrolyzed states. Our structures demonstrate that the two Aha1 domains can each interact with Hsp90 in two different modes, uncovering a complex multistep activation mechanism. The results show that Aha1 accelerates the chemical step of ATP hydrolysis like a conventional enzyme, but most unusually, catalyzes the rate-limiting large-scale conformational changes of Hsp90 fundamentally required for ATP hydrolysis. Our work provides a structural framework to guide small molecule development targeting this critical modulator of client protein maturation.

scholarly journals Effects of phospholipid fatty acyl chain length on phosphorylation and dephosphorylation of the Ca2+-ATPase

1995 ◽  
Vol 310 (3) ◽  
pp. 875-879 ◽  
Author(s):  
A P Starling ◽  
J M East ◽  
A G Lee
Keyword(s):  
Atp Hydrolysis ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
Phosphoryl Transfer ◽  
Acyl Chain Length ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Kinetics Of

The kinetics of the Ca(2+)-ATPase purified from sarcoplasmic reticulum have been studied after reconstitution into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC], dioleoylphosphatidylcholine[di(C18:1)PC] and dinervonylphosphatidylcholine [di(C24:1)PC]. In di(C24:1)PC the rate of phosphorylation of the ATPase by ATP was comparable with that in di(C18:1)PC (about 70 s-1), but in di(C14:1)PC the rate was much lower (21 s-1). Fluorescence responses of the ATPase suggest changes in the phosphoryl-transfer step rather than in the preceding conformational change E1Ca2ATP<-->E1′Ca2ATP. The rate of dephosphorylation of the phosphorylated ATPase was found to decrease in the order di(C24:1)PC < di(C14:1)PC < di(C18:1)PC. For the ATPase in di(C24:1)PC the rate of dephosphorylation (3.3 s-1) was slow enough to be the rate-limiting step for ATP hydrolysis; in di(C14:1)PC, it is suggested that both phosphorylation and dephosphorylation contribute to rate limitation. Phosphorylation of the ATPase in di(C24:1)PC by Pi was normal, but no phosphoenzyme could be detected in di(C14:1)PC. The rate of the Ca(2+)-transport step was normal in di(C24:1)PC, suggesting that the single Ca2+ ion bound to the ATPase in di(C24:1)PC could be transported.

scholarly journals Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

2009 ◽  
Vol 20 (19) ◽  
pp. 4256-4266 ◽  
Author(s):  
Fu-Cheng Liang ◽  
Umesh K. Bageshwar ◽  
Siegfried M. Musser
Keyword(s):  
Protein Transport ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Precursor Proteins ◽  
Inverted Membrane Vesicles ◽  
Rate Limiting

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.

scholarly journals Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ling Wang ◽  
Zachary Lee Johnson ◽  
Michael R Wasserman ◽  
Jesper Levring ◽  
Jue Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Chemotherapeutic Agents ◽  
Multidrug Resistance Protein 1 ◽  
Rate Limiting Step ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Resistance Protein ◽  
Rate Limiting

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.

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