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

2020 ◽  
Author(s):  
Marco Lolicato ◽  
Andrew M. Natale ◽  
Fayal Abderemane-Ali ◽  
David Crottès ◽  
Sara Capponi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Anomalous Scattering ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Ion Binding Sites ◽  
Physical And Chemical

K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action of their selectivity filter (C-type) gate3-6. Although structural studies show K2P conformations that impact activity7-13, no selectivity filter conformational changes have been observed. Here, combining K2P2.1 (TREK-1) X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and functional studies, we uncover the unprecedented, asymmetric, potassium-dependent conformational changes underlying K2P C-type gating. Low potassium concentrations evoke conformational changes in selectivity filter strand 1 (SF1), selectivity filter strand 2 (SF2), and the SF2-transmembrane helix 4 loop (SF2-M4 loop) that destroy the S1 and S2 ion binding sites and are suppressed by C-type gate activator ML335. Shortening the uniquely long SF2-M4 loop to match the canonical length found in other potassium channels or disrupting the conserved Glu234 hydrogen bond network supporting this loop blunts C-type gate response to various physical and chemical stimuli. Glu234 network destabilization also compromises ion selectivity, but can be reversed by channel activation, indicating that the ion binding site loss reduces selectivity similar to other channels14. Together, our data establish that C-type gating occurs through potassium-dependent order-disorder transitions in the selectivity filter and adjacent loops that respond to gating cues relayed through the SF2-M4 loop. These findings underscore the potential for targeting the SF2-M4 loop for the development of new, selective K2P channel modulators.

scholarly journals Identifying dynamic, partially occupied residues using anomalous scattering

2019 ◽  
Author(s):  
Serena Rocchio ◽  
Ramona Duman ◽  
Kamel El Omari ◽  
Vitaliy Mykhaylyk ◽  
Zhen Yan ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Structural Changes ◽  
Structural Data ◽  
Anomalous Scattering ◽  
Long Wavelength ◽  
X Ray Crystallography ◽  
Structural Ensembles ◽  
Analysis Strategy ◽  
Chain Conformations

AbstractX-ray crystallography is generally used to take single snapshots of a protein’s conformation. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes Residual Anomalous and Electron Density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data collection and analysis strategy for partially occupied iodine anomalous signals. Using the long wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with as low as ~12% occupancy is demonstrated. The number and position of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly dynamic sections of crystal structures.SynopsisStructural studies on partially occupied, dynamic protein systems by crystallography are difficult. We present methods here for detecting these states in crystals.

scholarly journals Identifying dynamic, partially occupied residues using anomalous scattering

2019 ◽  
Vol 75 (12) ◽  
pp. 1084-1095 ◽  
Author(s):  
Serena Rocchio ◽  
Ramona Duman ◽  
Kamel El Omari ◽  
Vitaliy Mykhaylyk ◽  
Christian Orr ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Structural Changes ◽  
Structural Data ◽  
Anomalous Scattering ◽  
Long Wavelength ◽  
X Ray Crystallography ◽  
Structural Ensembles ◽  
Analysis Strategy ◽  
Chain Conformations

Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes residual electron and anomalous density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data-collection and analysis strategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly disordered sections of crystal structures.

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 Two distinct mechanisms of transcriptional regulation by the redox sensor YodB

2016 ◽  
Vol 113 (35) ◽  
pp. E5202-E5211 ◽  
Author(s):  
Sang Jae Lee ◽  
In-Gyun Lee ◽  
Ki-Young Lee ◽  
Dong-Gyun Kim ◽  
Hyun-Jong Eun ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Disulfide Bonds ◽  
Structural Changes ◽  
High Specificity ◽  
Redox Balance ◽  
Structural Basis ◽  
X Ray Crystallography ◽  
Redox Sensor ◽  
Regulatory Systems ◽  
Sense And Respond

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis. This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch.

scholarly journals Structural basis for C-type inactivation in a Shaker family voltage gated K+ channel

2021 ◽  
Author(s):  
Ravikumar Reddi ◽  
Kimberly Matulef ◽  
Erika A. Riederer ◽  
Matthew R. Whorton ◽  
Francis I. Valiyaveetil
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Structural Changes ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Ion Binding ◽  
Channel Pore ◽  
Voltage Gated ◽  
Ion Binding Sites

AbstractC-type inactivation is a process by which ion flux through a voltage-gated K+ (Kv) channel is regulated at the selectivity filter. While prior studies have indicated that C-type inactivation involves structural changes at the selectivity filter, the nature of the changes have not been resolved. Here we report the crystal structure of the Kv1.2 channel in a C-type inactivated state. The structure shows that C-type inactivation involves changes in the selectivity filter that disrupt the outer two ion binding sites in the filter. The changes at the selectivity filter propagate to the extracellular mouth and the turret regions of the channel pore. The structural changes observed are consistent with the functional hallmarks of C-type inactivation. This study highlights the intricate interplay between K+ occupancy at the ion binding sites and the interactions of the selectivity filter in determining the balance between the conductive and the inactivated conformations of the filter.

scholarly journals ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Mark F. Mabanglo ◽  
Elisa Leung ◽  
Siavash Vahidi ◽  
Thiago V. Seraphim ◽  
Bryan T. Eger ◽  
...  
Keyword(s):  
Small Molecule ◽  
Conformational Changes ◽  
Electrostatic Interaction ◽  
Structural Changes ◽  
Structural Heterogeneity ◽  
Interaction Networks ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Bacterial Cell Death

Abstract Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

Conformational Changes and Gating at the Selectivity Filter of Potassium Channels

2008 ◽  
Vol 130 (29) ◽  
pp. 9474-9480 ◽  
Author(s):  
Carmen Domene ◽  
Michael L. Klein ◽  
Davide Branduardi ◽  
Francesco L. Gervasio ◽  
Michele Parrinello
Keyword(s):  
Potassium Channels ◽  
Conformational Changes ◽  
Selectivity Filter

scholarly journals Selectivity filter ion binding affinity determines inactivation in a potassium channel

2020 ◽  
Vol 117 (47) ◽  
pp. 29968-29978
Author(s):  
Céline Boiteux ◽  
David J. Posson ◽  
Toby W. Allen ◽  
Crina M. Nimigean
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Bk Channels ◽  
Selectivity Filter ◽  
Ion Binding ◽  
X Ray Crystallography ◽  
Extracellular Side ◽  
Opening And Closing ◽  
Low K ◽  
Conductive State

Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca2+-activated K+channel fromMethanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K+concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF’s four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K+affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K+, similar to KcsA, but that even a single K+binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K+concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK’s S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

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