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 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 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 Allostery between two binding sites in the ion channel subunit TRIP8b confers binding specificity to HCN channels

2017 ◽  
Vol 292 (43) ◽  
pp. 17718-17730 ◽  
Author(s):  
Kyle A. Lyman ◽  
Ye Han ◽  
Robert J. Heuermann ◽  
Xiangying Cheng ◽  
Jonathan E. Kurz ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cyclic Nucleotide ◽  
Tetratricopeptide Repeat ◽  
Hcn Channels ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein–protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal motifs. A homolog of PEX5, tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide (HCN)–gated channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide–binding domain and the C terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN cyclic nucleotide–binding domain through a 37-residue domain and the HCN C terminus through the TPR domains. Using a combination of fluorescence polarization– and co-immunoprecipitation–based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to type 1 peroxisomal targeting signal substrates. These results raise the possibility that other TPR domains may be similarly influenced by allosteric mechanisms as a general feature of protein–protein interactions.

scholarly journals A conserved interaction between a C-terminal motif in Norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation

2015 ◽  
Author(s):  
Eoin N Leen ◽  
Frédéric Sorgeloos ◽  
Samantha Correia ◽  
Yasmin Chaudhry ◽  
Fabien Cannac ◽  
...  
Keyword(s):  
Binding Site ◽  
Translation Initiation ◽  
Specific Binding ◽  
Helical Conformation ◽  
Titration Calorimetry ◽  
Murine Norovirus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Binding Interface ◽  
C Terminus

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5 ́ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5 ́-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C-terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C- terminal peptide of MNV VPg to inhibit translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.

scholarly journals Insights into the Function of theN-Acetyltransferase SatA That Detoxifies Streptothricin inBacillus subtilisandBacillus anthracis

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Rachel M. Burckhardt ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Binding Site ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Side Chains ◽  
Missense Mutations ◽  
Content Type ◽  
C Terminus ◽  
Exclusion Chromatography ◽  
Domains Of Life

ABSTRACTAcylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed thatBacillus subtilisandBacillus anthracisencode the GCN5-relatedN-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues ofB. subtilisSatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of theB. anthracisAmes SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations intosatA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 ofBaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCEThis work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

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 Cyclic nucleotide-regulated channels (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Elvir Becirovic ◽  
Martin Biel ◽  
Verena Hammelmann ◽  
Franz Hofmann ◽  
U. Benjamin Kaupp
Keyword(s):  
Cyclic Gmp ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Cardiac Cells ◽  
Cation Channels ◽  
Sensory Cells ◽  
Hcn Channels ◽  
Excitable Cells ◽  
C Terminus ◽  
Voltage Gated Ion Channels

Cyclic nucleotide-gated (CNG) channels are responsible for signalling in the primary sensory cells of the vertebrate visual and olfactory systems.CNG channels are voltage-independent cation channels formed as tetramers. Each subunit has 6TM, with the pore-forming domain between TM5 and TM6. CNG channels were first found in rod photoreceptors [69, 98], where light signals through rhodopsin and transducin to stimulate phosphodiesterase and reduce intracellular cyclic GMP level. This results in a closure of CNG channels and a reduced ‘dark current’. Similar channels were found in the cilia of olfactory neurons [153] and the pineal gland [60]. The cyclic nucleotides bind to a domain in the C terminus of the subunit protein: other channels directly binding cyclic nucleotides include HCN, eag and certain plant potassium channels.Hyperpolarisation-activated, cyclic nucleotide-gated (HCN)The hyperpolarisation-activated, cyclic nucleotide-gated (HCN) channels are cation channels that are activated by hyperpolarisation at voltages negative to ~-50 mV. The cyclic nucleotides cyclic AMP and cyclic GMP directly activate the channels and shift the activation curves of HCN channels to more positive voltages, thereby enhancing channel activity. HCN channels underlie pacemaker currents found in many excitable cells including cardiac cells and neurons [56, 164]. In native cells, these currents have a variety of names, such as Ih, Iq and If. The four known HCN channels have six transmembrane domains and form tetramers. It is believed that the channels can form heteromers with each other, as has been shown for HCN1 and HCN4 [2]. High resolution structural studies of CNG and HCN channels has provided insight into the the gating processes of these channels [117, 121]. A standardised nomenclature for CNG and HCN channels has been proposed by the NC-IUPHAR subcommittee on voltage-gated ion channels [88].

scholarly journals Cyclic nucleotide-regulated channels (CNG) in GtoPdb v.2021.3

2021 ◽  
Vol 2021 (3) ◽  
Author(s):  
Elvir Becirovic ◽  
Martin Biel ◽  
Stefanie Fenske ◽  
Verena Hammelmann ◽  
Franz Hofmann ◽  
...  
Keyword(s):  
Cyclic Gmp ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Cardiac Cells ◽  
Cation Channels ◽  
Sensory Cells ◽  
Hcn Channels ◽  
C Terminus ◽  
Olfactory Systems ◽  
Voltage Gated Ion Channels

Cyclic nucleotide-gated (CNG) channels are responsible for signalling in the primary sensory cells of the vertebrate visual and olfactory systems. CNG channels are voltage-independent cation channels formed as tetramers. Each subunit has 6TM, with the pore-forming domain between TM5 and TM6. CNG channels were first found in rod photoreceptors [83, 120], where light signals through rhodopsin and transducin to stimulate phosphodiesterase and reduce intracellular cyclic GMP level. This results in a closure of CNG channels and a reduced ‘dark current’. Similar channels were found in the cilia of olfactory neurons [181] and the pineal gland [71]. The cyclic nucleotides bind to a domain in the C terminus of the subunit protein: other channels directly binding cyclic nucleotides include hyperolarisation-activated, cyclic nucleotide-gated channels (HCN), ether-a-go-go and certain plant potassium channels.The HCN channels are cation channels that are activated by hyperpolarisation at voltages negative to ~-50 mV. The cyclic nucleotides cyclic AMP and cyclic GMP directly bind to the cyclic nucleotide-binding domain of HCN channels and shift their activation curves to more positive voltages, thereby enhancing channel activity. HCN channels underlie pacemaker currents found in many excitable cells including cardiac cells and neurons [64, 192]. In native cells, these currents have a variety of names, such as Ih, Iq and If. The four known HCN channels have six transmembrane domains and form tetramers. It is believed that the channels can form heteromers with each other, as has been shown for HCN1 and HCN4 [2]. High resolution structural studies of CNG and HCN channels has provided insight into the the gating processes of these channels [139, 146, 140]. A standardised nomenclature for CNG and HCN channels has been proposed by the NC-IUPHAR Subcommittee on voltage-gated ion channels [108].

scholarly journals Cellular context and multiple channel domains determine cAMP sensitivity of HCN4 channels: Ligand-independent relief of autoinhibition in HCN4

2012 ◽  
Vol 140 (5) ◽  
pp. 557-566 ◽  
Author(s):  
Zhandi Liao ◽  
Dean Lockhead ◽  
Joshua R. St. Clair ◽  
Eric D. Larson ◽  
Courtney E. Wilson ◽  
...  
Keyword(s):  
Cho Cells ◽  
Cyclic Nucleotide ◽  
Voltage Dependence ◽  
Chinese Hamster ◽  
Hcn Channels ◽  
Dependent Manner ◽  
Multiple Channel ◽  
Hek Cells ◽  
C Terminus ◽  
Cellular Context

Hyperpolarization-activated, cyclic nucleotide–sensitive (HCN) channels produce the If and Ih currents, which are critical for cardiac pacemaking and neuronal excitability, respectively. HCN channels are modulated by cyclic AMP (cAMP), which binds to a conserved cyclic nucleotide–binding domain (CNBD) in the C terminus. The unliganded CNBD has been shown to inhibit voltage-dependent gating of HCNs, and cAMP binding relieves this “autoinhibition,” causing a depolarizing shift in the voltage dependence of activation. Here we report that relief of autoinhibition can occur in the absence of cAMP in a cellular context- and isoform-dependent manner: when the HCN4 isoform was expressed in Chinese hamster ovary (CHO) cells, the basal voltage dependence was already shifted to more depolarized potentials and cAMP had no further effect on channel activation. This “pre-relief” of autoinhibition was specific both to HCN4 and to CHO cells; cAMP shifted the voltage dependence of HCN2 in CHO cells and of HCN4 in human embryonic kidney (HEK) cells. The pre-relief phenotype did not result from different concentrations of soluble intracellular factors in CHO and HEK cells, as it persisted in excised cell-free patches. Likewise, it did not arise from a failure of cAMP to bind to the CNBD of HCN4 in CHOs, as indicated by cAMP-dependent slowing of deactivation. Instead, a unique ∼300–amino acid region of the distal C terminus of HCN4 (residues 719–1012, downstream of the CNBD) was found to be necessary, but not sufficient, for the depolarized basal voltage dependence and cAMP insensitivity of HCN4 in CHO cells. Collectively, these data suggest a model in which multiple HCN4 channel domains conspire with membrane-associated intracellular factors in CHO cells to relieve autoinhibition in HCN4 channels in the absence of cAMP. These findings raise the possibility that such ligand-independent regulation could tune the activity of HCN channels and other CNBD-containing proteins in many physiological systems.

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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