scholarly journals Structural changes during HCN channel gating defined by high affinity metal bridges

2012 ◽  
Vol 140 (3) ◽  
pp. 279-291 ◽  
Author(s):  
Daniel C.H. Kwan ◽  
David L. Prole ◽  
Gary Yellen
Keyword(s):  
Structural Changes ◽  
Structural Model ◽  
Channel Gating ◽  
Structural Basis ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Membrane Hyperpolarization ◽  
Linker Region ◽  
High Affinity ◽  
Gating Mechanism

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.

Microscale Structural Model of Alzheimer's Aβ(1-40) Amyloid Fibril: Comparative Study of 2- and 3-fold Morphologies

2009 ◽  
Vol 1240 ◽  
Author(s):  
Raffaella Paparcone ◽  
Markus J Buehler
Keyword(s):  
Structural Changes ◽  
Amyloid Fibrils ◽  
Structural Model ◽  
Amyloid Fibril ◽  
Physical Models ◽  
Structural Basis ◽  
Stable Configuration ◽  
Pathological Feature ◽  
Amyloid Fibers ◽  
Degenerative Disorders

AbstractAmyloid fibrils aggregation is a key pathological feature of many severe degenerative disorders including Alzheimer’s disease and clinical dementia. Moreover, amyloids have been classified as intriguing molecules due to their exceptional strength, sturdiness and elasticity. However, physical models that explain the structural basis of these properties remain largely elusive, preventing the description of the link between their hierarchical structure and physical properties. Here we present an atomistic-based multiscale analysis based on computational materiomics, utilized to predict the structure of the two known polymorphous Alzheimer’s Aβ(1–40) amyloid fibers. We report an analysis of the energies, structural changes and H-bonding for varying amyloid fibril lengths, elucidating their size dependent properties. We also propose an explanation for the different stability of the two morphologies. A structural model of amyloid fibers with lengths of hundreds of nanometers at atomistic resolution is obtained. It predicts the formation of twisted amyloid microfibers in close agreement with experimental results. The approach used here provides a link between the fibril geometry, the chemical interactions and the most stable configuration, and resolves the issue of missing atomistic structures for long amyloid fibers.

scholarly journals HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

2020 ◽  
Author(s):  
Ze-Jun Wang ◽  
Ismary Blanco ◽  
Sebastien Hayoz ◽  
Tinatin I. Brelidze
Keyword(s):  
Cyclic Nucleotide ◽  
Transmembrane Domain ◽  
Rhythmic Activity ◽  
Surface Expression ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Structural Element ◽  
Functional Link ◽  
Membrane Hyperpolarization ◽  
Membrane Spanning

ABSTRACTHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane domain via the C-linker. Previous functional analysis of HCN channels suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of the coupling were unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed HCN domain (HCND), forms a direct structural link between the VSD and C-linker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We then identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the surface expression of HCN channels, and provides a functional link between the voltage- and cAMP-dependent mechanisms of HCN channel gating.

scholarly journals The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

2019 ◽  
Author(s):  
Gucan Dai ◽  
Teresa K. Aman ◽  
Frank DiMaio ◽  
William N. Zagotta
Keyword(s):  
Ion Channels ◽  
Metal Ion ◽  
Voltage Sensor ◽  
The Body ◽  
Resting Membrane Potential ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Transmembrane Voltage

Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body1. They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells1. In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration2. VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore3–6. These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD)3 and within the membrane’s electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization7. HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization8–10. The mechanism underlying this “reverse gating” is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels11, no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.

scholarly journals Altered channel gating mechanism for CFTR inhibition by a high-affinity thiazolidinone blocker

FEBS Letters ◽  
2004 ◽  
Vol 558 (1-3) ◽  
pp. 52-56 ◽  
Author(s):  
Alessandro Taddei ◽  
Chiara Folli ◽  
Olga Zegarra-Moran ◽  
Pascale Fanen ◽  
A.S Verkman ◽  
...  
Keyword(s):  
Channel Gating ◽  
High Affinity ◽  
Gating Mechanism

scholarly journals Structural basis of BAK activation in mitochondrial apoptosis initiation

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Geetika Singh ◽  
Cristina D. Guibao ◽  
Jayaraman Seetharaman ◽  
Anup Aggarwal ◽  
Christy R. Grace ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Basis ◽  
Ligand Affinity ◽  
High Affinity ◽  
Direct Activation ◽  
In Trans ◽  
Hit And Run ◽  
Membrane Poration ◽  
Apoptosis Initiation

AbstractBCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

scholarly journals The HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms

2020 ◽  
Vol 295 (24) ◽  
pp. 8164-8173
Author(s):  
Ze-Jun Wang ◽  
Ismary Blanco ◽  
Sebastien Hayoz ◽  
Tinatin I. Brelidze
Keyword(s):  
Cell Surface ◽  
Cyclic Nucleotide ◽  
Rhythmic Activity ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Structural Element ◽  
Functional Link ◽  
Membrane Hyperpolarization

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD), and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane segment via the C-linker. Previous functional analysis of HCN channels has suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of this coupling remain unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed the HCN domain (HCND), forms a direct structural link between the VSD and C-linker–CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified Arg237 and Gly239 residues on the S2 of the VSD that form direct interactions with Ile135 on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker–CNBD (Glu478, Gln482, and His559) that form direct interactions with residues Arg154 and Ser158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the cell-surface expression of HCN channels and provides a functional link between voltage- and cAMP-dependent mechanisms of HCN channel gating.

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins IIbβ3 and Vβ3

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2559-2568 ◽  
Author(s):  
Hirokazu Kashiwagi ◽  
Yoshiaki Tomiyama ◽  
Seiji Tadokoro ◽  
Shigenori Honda ◽  
Masamichi Shiraga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Relevant Variable ◽  
High Affinity ◽  
293 Cells ◽  
Inside Out

Abstract Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins IIbβ3 and Vβ3

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2559-2568 ◽  
Author(s):  
Hirokazu Kashiwagi ◽  
Yoshiaki Tomiyama ◽  
Seiji Tadokoro ◽  
Shigenori Honda ◽  
Masamichi Shiraga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Relevant Variable ◽  
High Affinity ◽  
293 Cells ◽  
Inside Out

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

scholarly journals Structural Basis for the C-Terminal Domain of Mycobacterium tuberculosis Ribosome Maturation Factor RimM to Bind Ribosomal Protein S19

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 597
Author(s):  
Haoran Zhang ◽  
Qiuxiang Zhou ◽  
Chenyun Guo ◽  
Liubin Feng ◽  
Huilin Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ribosomal Protein ◽  
Solution Structure ◽  
Molecular Mechanisms ◽  
Structural Model ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Hydrophobic Core ◽  
Terminal Domain ◽  
Ribosomal Protein S19

Multidrug-resistant tuberculosis (TB) is a serious threat to public health, calling for the development of new anti-TB drugs. Chaperon protein RimM, involved in the assembly of ribosomal protein S19 into 30S ribosomal subunit during ribosome maturation, is a potential drug target for TB treatment. The C-terminal domain (CTD) of RimM is primarily responsible for binding S19. However, both the CTD structure of RimM from Mycobacterium tuberculosis (MtbRimMCTD) and the molecular mechanisms underlying MtbRimMCTD binding S19 remain elusive. Here, we report the solution structure, dynamics features of MtbRimMCTD, and its interaction with S19. MtbRimMCTD has a rigid hydrophobic core comprised of a relatively conservative six-strand β-barrel, tailed with a short α-helix and interspersed with flexible loops. Using several biophysical techniques including surface plasmon resonance (SPR) affinity assays, nuclear magnetic resonance (NMR) assays, and molecular docking, we established a structural model of the MtbRimMCTD–S19 complex and indicated that the β4-β5 loop and two nonconserved key residues (D105 and H129) significantly contributed to the unique pattern of MtbRimMCTD binding S19, which might be implicated in a form of orthogonality for species-dependent RimM–S19 interaction. Our study provides the structural basis for MtbRimMCTD binding S19 and is beneficial to the further exploration of MtbRimM as a potential target for the development of new anti-TB drugs.

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