scholarly journals Salt Bridges and Gating in the COOH-terminal Region of HCN2 and CNGA1 Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 663-677 ◽  
Author(s):  
Kimberley B. Craven ◽  
William N. Zagotta
Keyword(s):  
Crystal Structure ◽  
Cyclic Nucleotide ◽  
Sequence Similarity ◽  
Terminal Region ◽  
Hcn Channels ◽  
Salt Bridges ◽  
Linker Region ◽  
Channel Opening ◽  
Intersubunit Interactions ◽  
Cnga1 Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels and cyclic nucleotide-gated (CNG) channels are activated by the direct binding of cyclic nucleotides. The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels. This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. Recently, the structure of the HCN2 COOH-terminal region was solved and shown to contain intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. We show that breaking these salt bridges in both HCN2 and CNGA1 channels through mutation causes an increase in the favorability of channel opening. The wild-type behavior of both HCN2 and CNGA1 channels is rescued by switching the position of the positive and negative residues, thus restoring the salt bridges. These results suggest that the salt bridges seen in the HCN2 COOH-terminal crystal structure are also present in the intact HCN2 channel. Furthermore, the similar effects of the mutations on HCN2 and CNGA1 channels suggest that these salt bridge interactions are also present in the intact CNGA1 channel. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 COOH-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.

scholarly journals CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

2017 ◽  
Vol 114 (17) ◽  
pp. 4430-4435 ◽  
Author(s):  
Zachary M. James ◽  
Andrew J. Borst ◽  
Yoni Haitin ◽  
Brandon Frenz ◽  
Frank DiMaio ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
Sequence Similarity ◽  
Nucleotide Binding ◽  
Cardiac Pace ◽  
Hcn Channels ◽  
Cyclic Nucleotide Binding ◽  
Channel Opening ◽  
Cng Channel ◽  
Linker Domain

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae—which shares sequence similarity to eukaryotic CNG and HCN channels—in the presence of a saturating concentration of cAMP. A short S4–S5 linker connects nearby voltage-sensing and pore domains to produce a non–domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.

scholarly journals Crystal Structure of d-Hydantoinase from Burkholderia pickettii at a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability

2003 ◽  
Vol 185 (14) ◽  
pp. 4038-4049 ◽  
Author(s):  
Zhen Xu ◽  
Yunqing Liu ◽  
Yunliu Yang ◽  
Weihong Jiang ◽  
Eddy Arnold ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Active Site ◽  
Molecular Basis ◽  
Bacillus Stearothermophilus ◽  
Sequence Similarity ◽  
Amino Acid Residues ◽  
Salt Bridges ◽  
Aromatic Residues ◽  
Catalytic Active Site

ABSTRACT d-Hydantoinase (d-HYD) is an industrial enzyme that is widely used in the production of d-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of d-hydantoinase from Burkholderia pickettii (HYDBp) at a 2.7-Å resolution. The structure of HYDBp consists of a core (α/β)8 triose phosphate isomerase barrel fold and a β-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYDBp shares only moderate sequence similarity with d-HYDs from Thermus sp. (HYDTsp) and Bacillus stearothermophilus (HYDBst), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of d-HYDs. The more thermostable HYDTsp contains more aromatic residues in the interior of the structure than HYDBp and HYDBst. Changes of large aromatic residues in HYDTsp to smaller residues in HYDBp or HYDBst decrease the hydrophobicity and create cavities inside the structure. HYDTsp has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYDBp and HYDBst. Besides, HYDTsp also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.

scholarly journals Kinetic Relationship between the Voltage Sensor and the Activation Gate in spHCN Channels

2007 ◽  
Vol 130 (1) ◽  
pp. 71-81 ◽  
Author(s):  
Andrew Bruening-Wright ◽  
Fredrik Elinder ◽  
H. Peter Larsson
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Ionic Current ◽  
Current Data ◽  
Terminal Region ◽  
Hcn Channels ◽  
Mode Shift ◽  
Channel Opening ◽  
Rate Limiting ◽  
Voltage Activation

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarizations that cause an inward movement of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. The mechanism of how the motion of S4 charges triggers channel opening is unknown. Here, we used voltage clamp fluorometry (VCF) to detect S4 conformational changes and to correlate these to the different activation steps in spHCN channels. We show that S4 undergoes two distinct conformational changes during voltage activation. Analysis of the fluorescence signals suggests that the N-terminal region of S4 undergoes conformational changes during a previously characterized mode shift in HCN channel voltage dependence, while a more C-terminal region undergoes an additional conformational change during gating charge movements. We fit our fluorescence and ionic current data to a previously proposed 10-state allosteric model for HCN channels. Our results are not compatible with a fast S4 motion and rate-limiting channel opening. Instead, our data and modeling suggest that spHCN channels open after only two S4s have moved and that S4 motion is rate limiting during voltage activation of spHCN channels.

scholarly journals Structural Insights into a Bifunctional Peptide Methionine Sulfoxide Reductase MsrA/B Fusion Protein from Helicobacter pylori

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 389
Author(s):  
Sulhee Kim ◽  
Kitaik Lee ◽  
Sun-Ha Park ◽  
Geun-Hee Kwak ◽  
Min Seok Kim ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Helicobacter Pylori ◽  
Fusion Protein ◽  
Pathogenic Bacteria ◽  
Catalytic Efficiency ◽  
Salt Bridge ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Salt Bridges ◽  
Linker Region

Methionine sulfoxide reductase (Msr) is a family of enzymes that reduces oxidized methionine and plays an important role in the survival of bacteria under oxidative stress conditions. MsrA and MsrB exist in a fusion protein form (MsrAB) in some pathogenic bacteria, such as Helicobacter pylori (Hp), Streptococcus pneumoniae, and Treponema denticola. To understand the fused form instead of the separated enzyme at the molecular level, we determined the crystal structure of HpMsrABC44S/C318S at 2.2 Å, which showed that a linker region (Hpiloop, 193–205) between two domains interacted with each HpMsrA or HpMsrB domain via three salt bridges (E193-K107, D197-R103, and K200-D339). Two acetate molecules in the active site pocket showed an sp2 planar electron density map in the crystal structure, which interacted with the conserved residues in fusion MsrABs from the pathogen. Biochemical and kinetic analyses revealed that Hpiloop is required to increase the catalytic efficiency of HpMsrAB. Two salt bridge mutants (D193A and E199A) were located at the entrance or tailgate of Hpiloop. Therefore, the linker region of the MsrAB fusion enzyme plays a key role in the structural stability and catalytic efficiency and provides a better understanding of why MsrAB exists in a fused form.

A second S4 movement opens hyperpolarization-activated HCN channels

2021 ◽  
Vol 118 (37) ◽  
pp. e2102036118
Author(s):  
Xiaoan Wu ◽  
Rosamary Ramentol ◽  
Marta E. Perez ◽  
Sergei Yu Noskov ◽  
H. Peter Larsson
Keyword(s):  
Sea Urchin ◽  
Cyclic Nucleotide ◽  
Voltage Dependence ◽  
Rhythmic Activity ◽  
Hcn Channels ◽  
Molecular Models ◽  
Transmembrane Segment ◽  
Pacemaker Cells ◽  
Gate Opening ◽  
Channel Opening

Rhythmic activity in pacemaker cells, as in the sino-atrial node in the heart, depends on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. As in depolarization-activated K+ channels, the fourth transmembrane segment S4 functions as the voltage sensor in hyperpolarization-activated HCN channels. But how the inward movement of S4 in HCN channels at hyperpolarized voltages couples to channel opening is not understood. Using voltage clamp fluorometry, we found here that S4 in HCN channels moves in two steps in response to hyperpolarizations and that the second S4 step correlates with gate opening. We found a mutation in sea urchin HCN channels that separate the two S4 steps in voltage dependence. The E356A mutation in S4 shifts the main S4 movement to positive voltages, but channel opening remains at negative voltages. In addition, E356A reveals a second S4 movement at negative voltages that correlates with gate opening. Cysteine accessibility and molecular models suggest that the second S4 movement opens up an intracellular crevice between S4 and S5 that would allow radial movement of the intracellular ends of S5 and S6 to open HCN channels.

scholarly journals Structure of the dodecamer of the aminopeptidase APDkam598 from the archaeonDesulfurococcus kamchatkensis

2015 ◽  
Vol 71 (3) ◽  
pp. 277-285 ◽  
Author(s):  
T. E. Petrova ◽  
E. S. Slutskaya ◽  
K. M. Boyko ◽  
O. S. Sokolova ◽  
T. V. Rakitina ◽  
...  
Keyword(s):  
Crystal Structure ◽  
External Surface ◽  
Sequence Similarity ◽  
Salt Bridges ◽  
High Sequence Similarity ◽  
Pyrococcus Horikoshii ◽  
Sequence Identity ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Spatial Locations

The crystal structure of the aminopeptidase APDkam589 from the thermophilic crenarchaeonDesulfurococcus kamchatkensiswas determined at a resolution of 3.0 Å. In the crystal, the monomer of APDkam589 and its symmetry-related monomers are densely packed to form a 12-subunit complex. Single-particle electron-microscopy analysis confirms that APDkam589 is present as a compact dodecamer in solution. The APDkam589 molecule is built similarly to the molecules of the PhTET peptidases, which have the highest sequence identity to APDkam589 among known structures and were isolated from the more thermostable archaeonPyrococcus horikoshii. A comparison of the interactions of the subunits in APDkam589 with those in PhTET1, PhTET2 and PhTET3 reveals that APDkam589 has a much lower total number of salt bridges, which correlates with the lower thermostability of APDkam589. The monomer of APDkam589 has six Trp residues, five of which are on the external surface of the dodecamer. A superposition of the structure of APDkam589 with those having a high sequence similarity to APDkam589 reveals that, although the positions of Trp45, Trp252 and Trp358 are not conserved in the sequences, the spatial locations of the Trp residues in these models are similar.

scholarly journals Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide–gated ion channel

2019 ◽  
Vol 151 (10) ◽  
pp. 1190-1212 ◽  
Author(s):  
Leo C.T. Ng ◽  
Meiying Zhuang ◽  
Filip Van Petegem ◽  
Yue Xian Li ◽  
Eric A. Accili
Keyword(s):  
Binding Site ◽  
Cyclic Nucleotide ◽  
Single Point ◽  
Titration Calorimetry ◽  
Hcn Channels ◽  
Negative Cooperativity ◽  
Response Data ◽  
C Terminus ◽  
Channel Opening ◽  
Ligand Sensitivity

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open more easily when cAMP or cGMP bind to a domain in the intracellular C-terminus in each of four identical subunits. How sensitivity of the channels to these ligands is determined is not well understood. Here, we apply a mathematical model, which incorporates negative cooperativity, to gating and mutagenesis data available in the literature and combine the results with binding data collected using isothermal titration calorimetry. This model recapitulates the concentration–response data for the effects of cAMP and cGMP on wild-type HCN2 channel opening and, remarkably, predicts the concentration–response data for a subset of mutants with single-point amino acid substitutions in the binding site. Our results suggest that ligand sensitivity is determined by negative cooperativity and asymmetric effects on structure and channel opening, which are tuned by ligand-specific interactions and residues within the binding site.

scholarly journals The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alessandro Porro ◽  
Andrea Saponaro ◽  
Federica Gasparri ◽  
Daniel Bauer ◽  
Christine Gross ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Channel Gating ◽  
Hcn Channels ◽  
Structural Mechanism ◽  
Cyclic Nucleotide Gated Channels ◽  
Channel Opening ◽  
Mode Of Operation ◽  
Voltage Dependent ◽  
Upward Displacement ◽  
Voltage Sensing Domain

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms.

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