scholarly journals Molecular mechanism of depolarization-dependent inactivation in W366F mutant of Kv1.2

2018 ◽  
Author(s):  
H. X. Kondo ◽  
N. Yoshida ◽  
M. Shirota ◽  
K. Kinoshita
Keyword(s):  
Membrane Potential ◽  
Potassium Channels ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Structural Changes ◽  
Membrane Depolarization ◽  
Potassium Ions ◽  
Selective Filter ◽  
Dependent Inactivation ◽  
Dynamics Simulations

ABSTRACTVoltage-gated potassium channels play crucial roles in regulating membrane potential. They are activated by membrane depolarization, allowing the selective permeation of potassium ions across the plasma membrane, and enter a nonconducting state after lasting depolarization of membrane potential, a process known as inactivation. Inactivation in voltage-activated potassium channels occurs through two distinct mechanisms, N-type inactivation and C-type inactivation. C-type inactivation is caused by conformational changes in the extracellular mouth of the channel, while N-type inactivation is elicited by changes in the cytoplasmic mouth of the protein. The W434F-mutated Shaker channel is known as a nonconducting mutant and is in a C-type inactivation state at a depolarizing membrane potential. To clarify the structural properties of C-type inactivated protein, we performed molecular dynamics simulations of the wild-type and W366F (corresponding to W434F in Shaker) mutant of the Kv1.2-2.1 chimera channel. The W366F mutant was in a nearly nonconducting state with a depolarizing voltage and recovered from inactivation with a reverse voltage. Our simulations and 3D-RISM analysis suggested that structural changes in the selective filter upon membrane depolarization trap potassium ions around the entrance of the selectivity filter and prevent ion permeation. This pore restriction is involved in the molecular mechanism of C-type inactivation.

scholarly journals The conduction pathway of potassium channels is water free under physiological conditions

2019 ◽  
Vol 5 (7) ◽  
pp. eaaw6756 ◽  
Author(s):  
Carl Öster ◽  
Kitty Hendriks ◽  
Wojciech Kopec ◽  
Veniamin Chevelkov ◽  
Chaowei Shi ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Direct Contact ◽  
Selectivity Filter ◽  
Water Molecules ◽  
Potassium Ions ◽  
Ion Conduction ◽  
Physiological Conditions ◽  
Conduction Pathway ◽  
Fundamental Process ◽  
Dynamics Simulations

Ion conduction through potassium channels is a fundamental process of life. On the basis of crystallographic data, it was originally proposed that potassium ions and water molecules are transported through the selectivity filter in an alternating arrangement, suggesting a “water-mediated” knock-on mechanism. Later on, this view was challenged by results from molecular dynamics simulations that revealed a “direct” knock-on mechanism where ions are in direct contact. Using solid-state nuclear magnetic resonance techniques tailored to characterize the interaction between water molecules and the ion channel, we show here that the selectivity filter of a potassium channel is free of water under physiological conditions. Our results are fully consistent with the direct knock-on mechanism of ion conduction but contradict the previously proposed water-mediated knock-on mechanism.

scholarly journals Structural insights into the synthesis of FMN in prokaryotic organisms

2015 ◽  
Vol 71 (12) ◽  
pp. 2526-2542 ◽  
Author(s):  
Beatriz Herguedas ◽  
Isaias Lans ◽  
María Sebastián ◽  
Juan A. Hermoso ◽  
Marta Martínez-Júlvez ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Substrate Inhibition ◽  
Bifunctional Enzyme ◽  
Computational Simulations ◽  
Binding Studies ◽  
Oligomeric Protein ◽  
Catalytic Sites ◽  
Dynamics Simulations ◽  
Structural Insights

Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyltransferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure ofCorynebacterium ammoniagenesFADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module ofCaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-lengthCaFADS protomer and the quaternary organization.

scholarly journals Differences in Thermal Structural Changes and Melting Between Mesophilic and Thermophilic Dihydrofolate Reductase Enzymes

2020 ◽  
Author(s):  
Irene Maffucci ◽  
Damien Laage ◽  
Guillaume Stirnemann ◽  
Fabio Sterpone
Keyword(s):  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Thermotoga Maritima ◽  
Conformational Sampling ◽  
Detailed Understanding ◽  
Wide Range ◽  
The Difference ◽  
Dynamics Simulations

A key aspect of life's evolution on Earth is the adaptation of proteins to be stable and work in a very wide range of temperature conditions. A detailed understanding of the associated molecular mechanisms would also help to design enzymes optimized for biotechnological processes. Despite important advances, a comprehensive picture of how thermophilic enzymes succeed in functioning under extreme temperatures remains incomplete. Here, we examine the temperature dependence of stability and of flexibility in the mesophilic monomeric Escherichia coli (Ec) and thermophilic dimeric Thermotoga maritima (Tm) homologs of the paradigm dihydrofolate reductase (DHFR) enzyme. We use all-atom molecular dynamics simulations and a replica-exchange scheme that allows to enhance the conformational sampling while providing at the same time a detailed understanding of the enzymes' behavior at increasing temperatures. We show that this approach reproduces the stability shift between the two homologs, and provides a molecular description of the denaturation mechanism by identifying the sequence of secondary structure elements melting as temperature increases, which is not straightforwardly obtained in the experiments. By repeating our approach on the hypothetical TmDHFR monomer, we further determine the respective effects of sequence and oligomerization in the exceptional stability of TmDFHR. We show that the intuitive expectation that protein flexibility and thermal stability are correlated is not verified. Finally, our simulations reveal that significant conformational fluctuations already take place much below the melting temperature. While the difference between the TmDHFR and EcDHFR catalytic activities is often interpreted via a simplified two-state picture involving the open and closed conformations of the key M20 loop, our simulations suggest that the two homologs' markedly different activity temperature dependences are caused by changes in the ligand-cofactor distance distributions in response to these conformational changes.

scholarly journals Molecular mechanism of mitochondrial phosphatidate transfer by Ups1/Mdm35

2019 ◽  
Author(s):  
Jiuwei Lu ◽  
Kevin Chan ◽  
Leiye Yu ◽  
Jun Fan ◽  
Yujia Zhai ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Conformational Changes ◽  
Transfer Rate ◽  
Mitochondrial Membrane ◽  
Physiological Function ◽  
Bound State ◽  
Outer Mitochondrial Membrane ◽  
Structural Elements ◽  
Inner Mitochondrial Membrane ◽  
Dynamics Simulations

ABSTRACTCardiolipin plays many important roles for mitochondrial physiological function and is synthesized from phosphatidic acid (PA) at inner mitochondrial membrane (IMM). PA synthesized from endoplasmic reticulum needs to transfer to IMM via outer mitochondrial membrane (OMM). The transfer of PA between IMM and OMM is mediated by Ups1/Mdm35 protein family. Although there are many structures of this family available, the detailed molecular mechanism of how PA is transferred between membranes is yet unknown. Here, we report another crystal structures of Ups1/Mdm35 in the authentic monomeric apo state and the DHPA bound state. By combining subsequent all-atom molecular dynamics simulations, extensive structural comparisons and biophysical assays, we discovered the conformational changes of Ups1/Mdm35, identified key structural elements and residues during membrane binding and PA entry. We found the monomeric Ups1 on membrane is an important transit for the success of PA transfer, and the equilibrium between monomeric Ups1 and Ups1/Mdm35 complex on membrane affects the PA transfer rate and can be regulated by many factors including environmental pH.

scholarly journals The RCK1 domain of the human BKCa channel transduces Ca2+ binding into structural rearrangements

2010 ◽  
Vol 136 (2) ◽  
pp. 189-202 ◽  
Author(s):  
Taleh Yusifov ◽  
Anoosh D. Javaherian ◽  
Antonios Pantazis ◽  
Chris S. Gandhi ◽  
Riccardo Olcese
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Bkca Channel ◽  
Structural Rearrangements ◽  
Bkca Channels ◽  
Time Resolved ◽  
High Affinity ◽  
Cytosolic Domains

Large-conductance voltage- and Ca2+-activated K+ (BKCa) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca2+ homeostasis. The molecular mechanism of Ca2+-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BKCa RCK1 domain adopts an α/β fold, binds Ca2+, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca2+-induced conformational changes in physiologically relevant [Ca2+]. The neutralization of residues known to be involved in high-affinity Ca2+ sensing (D362 and D367) prevented Ca2+-induced structural transitions in RCK1 but did not abolish Ca2+ binding. We provide evidence that the RCK1 domain is a high-affinity Ca2+ sensor that transduces Ca2+ binding into structural rearrangements, likely representing elementary steps in the Ca2+-dependent activation of human BKCa channels.

scholarly journals Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions

2019 ◽  
Vol 5 (7) ◽  
pp. eaaw1531 ◽  
Author(s):  
Oskar Berntsson ◽  
Ryan Rodriguez ◽  
Léocadie Henry ◽  
Matthijs R. Panman ◽  
Ashley J. Hughes ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Conformational Changes ◽  
Structural Changes ◽  
Structural Response ◽  
Biochemical Activity ◽  
Time Resolved ◽  
X Ray ◽  
Molecular Events ◽  
Carboxyl Terminal ◽  
Dynamics Simulations

Cryptochromes are blue-light photoreceptor proteins, which provide input to circadian clocks. The cryptochrome from Drosophila melanogaster (DmCry) modulates the degradation of Timeless and itself. It is unclear how light absorption by the chromophore and the subsequent redox reactions trigger these events. Here, we use nano- to millisecond time-resolved x-ray solution scattering to reveal the light-activated conformational changes in DmCry and the related (6-4) photolyase. DmCry undergoes a series of structural changes, culminating in the release of the carboxyl-terminal tail (CTT). The photolyase has a simpler structural response. We find that the CTT release in DmCry depends on pH. Mutation of a conserved histidine, important for the biochemical activity of DmCry, does not affect transduction of the structural signal to the CTT. Instead, molecular dynamics simulations suggest that it stabilizes the CTT in the resting-state conformation. Our structural photocycle unravels the first molecular events of signal transduction in an animal cryptochrome.

scholarly journals K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

2020 ◽  
Vol 6 (44) ◽  
pp. eabc9174
Author(s):  
Marco Lolicato ◽  
Andrew M. Natale ◽  
Fayal Abderemane-Ali ◽  
David Crottès ◽  
Sara Capponi ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Conformational Changes ◽  
Structural Changes ◽  
Selectivity Filter ◽  
Anomalous Scattering ◽  
X Ray Crystallography ◽  
Gating Mechanisms ◽  
Cellular Excitability ◽  
Ion Binding Sites ◽  
Channel Modulator

K2P potassium channels regulate cellular excitability using their selectivity filter (C-type) gate. C-type gating mechanisms, best characterized in homotetrameric potassium channels, remain controversial and are attributed to selectivity filter pinching, dilation, or subtle structural changes. The extent to which such mechanisms control C-type gating of innately heterodimeric K2Ps is unknown. Here, combining K2P2.1 (TREK-1) x-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and electrophysiology, we uncover unprecedented, asymmetric, potassium-dependent conformational changes that underlie K2P C-type gating. These asymmetric order-disorder transitions, enabled by the K2P heterodimeric architecture, encompass pinching and dilation, disrupt the S1 and S2 ion binding sites, require the uniquely long K2P SF2-M4 loop and conserved “M3 glutamate network,” and are suppressed by the K2P C-type gate activator ML335. These findings demonstrate that two distinct C-type gating mechanisms can operate in one channel and underscore the SF2-M4 loop as a target for K2P channel modulator development.

scholarly journals Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel

2018 ◽  
Vol 115 (45) ◽  
pp. E10556-E10565 ◽  
Author(s):  
Kaiqian Wang ◽  
Christian Holt ◽  
Jocelyn Lu ◽  
Malene Brohus ◽  
Kamilla Taunsig Larsen ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Conformational Changes ◽  
Structural Changes ◽  
Voltage Gated ◽  
Cardiac Ion Channels ◽  
Disease Mutations ◽  
Dependent Inactivation ◽  
Ef Hand ◽  
Voltage Gated Calcium Channel ◽  
Structural Insights

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in theCALMgenes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (CaV1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the CaV1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca2+by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca2+-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca2+-dependent inactivation of the voltage-gated calcium channel.

scholarly journals Structural Changes of Sarco/Endoplasmic Reticulum Ca2+-ATPase Induced by Rutin Arachidonate: A Molecular Dynamics Study

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 214
Author(s):  
Yoel Rodríguez ◽  
Magdaléna Májeková
Keyword(s):  
Molecular Dynamics ◽  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Structural Changes ◽  
Acid Group ◽  
Bilayer Membrane ◽  
Salt Bridges ◽  
Drug Candidates ◽  
Dynamics Simulations

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) maintains the level of calcium concentration in cells by pumping calcium ions from the cytoplasm to the lumen while undergoing substantial conformational changes, which can be stabilized or prevented by various compounds. Here we attempted to clarify the molecular mechanism of action of new inhibitor rutin arachidonate, one of the series of the acylated rutin derivatives. We performed molecular dynamics simulations of SERCA1a protein bound to rutin arachidonate positioned in a pure dipalmitoylphosphatidylcholine bilayer membrane. Our study predicted the molecular basis for the binding of rutin arachidonate towards SERCA1a in the vicinity of the binding site of calcium ions and near the location of the well-known inhibitor thapsigargin. The stable hydrogen bond between Glu771 and rutin arachidonate plays a key role in the binding. SERCA1a is kept in the E2 conformation preventing the formation of important salt bridges between the side chains of several residues, primarily Glu90 and Lys297. All in all, the structural changes induced by the binding of rutin arachidonate to SERCA1a may shift proton balance near the titrable residues Glu771 and Glu309 into neutral species, hence preventing the binding of calcium ions to the transmembrane binding sites and thus affecting calcium homeostasis. Our results could lead towards the design of new types of inhibitors, potential drug candidates for cancer treatment, which could be anchored to the transmembrane region of SERCA1a by a lipophilic fatty acid group.

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