Eukaryotic Signal Transduction Research Articles

Genome-Wide Identification of Mitogen-activated Protein Kinase Cascade Genes and Transcriptional Profiling Analysis during Organ Development in Eucommia ulmoides

The mitogen-activated protein kinase (MAPK) cascades, which play crucial roles in plant development processes, are universal modules of signal transduction in eukaryotes and consist of a core module of three sequentially phosphorylated kinases: MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). This is the first report on the identification and analysis of MAPK cascades in Eucommia ulmoides. We conducted a genome-wide screening and identified 13 EuMAPKs, five EuMAPKKs, and 57 EuMAPKKKs. The construction of phylogenetic trees revealed that EuMAPKs and EuMAPKKs were divided into four groups (A, B, C, and D), and EuMAPKKKs were divided into three subfamilies (MEKK, RAF, and ZIK). These subfamilies were further confirmed by conserved domain/motif analysis and gene structure analysis. Based on the expression profiles of all identified EuMAPK cascades in various organs at different developmental stages, three genes (EuRAF22-2, EuRAF34-1, and EuRAF33-2) with stable expression patterns at all stages of fruit or leaf development, three genes (EuRAF2-3, EuMPK11, and EuMEKK21) with differential expression patterns, and two highly expressed genes (EuZIK1 and EuMKK2) were screened and validated by qRT-PCR. Overall, our results could be used for further research on the precise role of MAPK cascades during organ development in E. ulmoides.

PLCD3, a flotillin2-interacting protein, is involved in proliferation, migration and invasion of nasopharyngeal carcinoma cells.

Phospholipase C (PLC) is a pivotal enzyme in the phosphoinositide pathway that promotes the second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), to participate in eukaryotic signal transduction. Several PLC isozymes are associated with cancer, such as PLC-β1, PLC-δ1, PLC-ε and PLC-γ1. However, the role of PLC-δ3 (PLCD3) in nasopharyngeal carcinoma (NPC) has not been investigated to date. In our previous study, we demonstrated that flotillin2 (Flot2) plays a pro-neoplastic role in NPC and is involved in tumour progression and metastasis. In the present study, we screened the interacting proteins of Flot2 using the yeast two-hybrid (Y2H) method and verified the interaction between PLCD3 and Flot2 by co-immunoprecipitation. We also investigated the biological functions of PLCD3 in NPC. Inhibition of PLCD3 expression impaired the malignant potential of 5–8F, a highly metastatic NPC cell line, by restraining its growth, proliferation, mobility and migration. The present study demonstrated that PLCD3 may be an oncogenic protein in NPC and that it plays an important role in the progression of NPC partially by interacting with Flot2.

Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide Variety of Non-canonical Specificities

SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways. The majority of SH3 domains studied thus far act by binding to proline-rich sequences in partner proteins, but a growing number of studies have revealed alternative recognition mechanisms. We have comprehensively surveyed the specificity landscape of human SH3 domains in an unbiased manner using peptide-phage display and deep sequencing. Based on ∼70,000 unique binding peptides, we obtained 154 specificity profiles for 115 SH3 domains, which reveal that roughly half of the SH3 domains exhibit non-canonical specificities and collectively recognize a wide variety of peptide motifs, most of which were previously unknown. Crystal structures of SH3 domains with two distinct non-canonical specificities revealed novel peptide-binding modes through an extended surface outside of the canonical proline-binding site. Our results constitute a significant contribution toward a complete understanding of the mechanisms underlying SH3-mediated cellular responses.

The Arabidopsis Mitogen-Activated Protein Kinase Kinase Kinase 20 (MKKK20) Acts Upstream of MKK3 and MPK18 in Two Separate Signaling Pathways Involved in Root Microtubule Functions

Mitogen-activated protein kinase (MAPK) signaling networks represent important means of signal transduction in plants and other eukaryotes, controlling intracellular signaling by linking perception of environmental or developmental cues to downstream targets. In the Arabidopsis MEKK subfamily, the MKKK19, 20, and 21 form a highly supported clade with the Solanaceous Fertilization-Related Kinases. In Arabidopsis, little is known about this group, except for MKKK20, which is involved in osmotic stress. Using a directed MKKK-MKK yeast two-hybrid (Y2H) screen, MKKK20 was found to interact only with MKK3, while a MKKK20 large-scale Y2H screen retrieved MPK18 as a direct interactant. In vitro phosphorylation assays showed that MKKK20 phosphorylates both MKK3 and MPK18. However, when all three kinases are combined, no synergistic effect is observed on MPK18 phosphorylation, suggesting a direct access to MPK18, consistent with the absence of interaction between MKK3 and MPK18 in protein–protein interaction assays. Since mpk18 mutant plants were previously shown to be defective in microtubule-related functions, phenotypes of mkkk20 single and mkkk20/mpk18 double mutants were investigated to determine if MKKK20 acts upstream of MPK18. This was the case, as mkkk20 root length was shorter than WT in media containing microtubule-disrupting drugs as previously observed for mpk18 plants. Surprisingly, mkk3 plants were also similarly affected, suggesting the presence of two non-complementary pathways involved in Arabidopsis cortical microtubule function, the first including MKKK20, MKK3 and an unknown MPK; the second, a non-canonical MAPK cascade made of MKKK20 and MPK18 that bypasses the need for an MKK intermediate.

Metal coordination in kinases and pseudokinases.

Protein phosphorylation, mediated by protein kinases, is a key event in the regulation of eukaryotic signal transduction. The majority of eukaryotic protein kinases perform phosphoryl transfer, assisted by two divalent metal ions. About 10% of all human protein kinases are, however, thought to be catalytically inactive. These kinases lack conserved residues of the kinase core and are classified as pseudokinases. Yet, it has been demonstrated that pseudokinases are critically involved in biological functions. Here, we show how pseudokinases have developed strategies by modifying amino acid residues in order to achieve stable, active-like conformations. This includes binding of the co-substrate ATP in a two metal-, one metal- or even no metal-binding mode. Examples of the respective pseudokinases are provided on a structural basis and compared with a canonical protein kinase, Protein Kinase A. Moreover, the functional roles of both independent metal-binding sites, Me1 and Me2, are discussed. Lack of phosphotransferase activity does not implicate a loss of function and can easily point to alternative roles of pseudokinases, i.e. acting as switches or scaffolds, and having evolved as components crucial for cellular cross-talk and signaling. Interestingly, pseudokinases are present in all kingdoms of life and their specific roles remain enigmatic. More studies are needed to unravel the crucial functions of those interesting proteins.

Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A.

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.

Structural and functional annotation of the MADS-box transcription factor family in grapevine.

BackgroundMADS-box genes encode transcription factors that are involved in developmental control and signal transduction in eukaryotes. In plants, they are associated to numerous development processes most notably those related to reproductive development: flowering induction, specification of inflorescence and flower meristems, establishment of flower organ identity, as well as regulation of fruit, seed and embryo development. Genomic analyses of MADS-box genes in different plant species are providing new relevant information on the function and evolution of this transcriptional factor family. We have performed a true genome-wide analysis of the complete set of MADS-box genes in grapevine (Vitis vinifera), analyzed their expression pattern and establish their phylogenetic relationships (including MIKC* and type I MADS-box) with genes from 16 other plant species. This study was integrated to previous works on the family in grapevine.ResultsA total of 90 MADS-box genes were detected in the grapevine reference genome by completing current gene annotations with a genome-wide analysis based on sequence similarity. We performed a thorough in-depth curation of all gene models and combined the results with gene expression information including RNAseq data to clarifying the expression of newly identified genes and improve their functional characterization. Curated data were uploaded to the ORCAE database for grapevine in the frame of the grapevine genome curation effort. This approach resulted in the identification of 30 additional MADS box genes. Among them, ten new MIKCC genes were identified, including a potential new group of short proteins similar to the SVP protein subfamily. The MIKC* subgroup contains six genes in grapevine that can be grouped in the S (4 genes) and P (2 genes) clades, showing less redundancy than that observed in Arabidopsis thaliana. Expression pattern of these genes in grapevine is compatible with a role in male gametophyte development. Most of the identified new genes belong to the type I MADS-box genes and were classified as members of the Mα and Mγ subclasses. Ours analyses indicate that only few members of type I genes in grapevine have homology in other species and that species-specific clades appeared both in the Mα and Mγ subclasses. On the other hand, as deduced from the phylogenetic analysis with other plant species, genes that can be crucial for development of central cell, endosperm and embryos seems to be conserved in plants.ConclusionsThe genome analysis of MADS-box genes in grapevine, the characterization of their pattern of expression and the phylogenetic analysis with other plant species allowed the identification of new MADS-box genes not yet described in other plant species as well as basic characterization of their possible role, particularly in the case of type I and MIKC* genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2398-7) contains supplementary material, which is available to authorized users.

The untapped potential of tyrosine-based G protein signaling

Tyrosine-based and trimeric G protein-based signaling are the two most widely studied and distinct mechanisms for signal transduction in eukaryotes. How each of them relay signals across the plasma membrane independently of each other has been extensively characterized; however, an understanding of how they work together remained obscure. Recently, a rapidly emerging paradigm has revealed that tyrosine based signals are relayed via G proteins, and that the cross-talk between the two hubs are more robustly and sophisticatedly integrated than was previously imagined. More importantly, by straddling the two signaling hubs that are most frequently targeted for their therapeutic significance, the tyrosine-based G-protein signaling pathway has its own growing list of pathophysiologic importance, both as therapeutic target in a variety of disease states, and by paving the way for personalized medicine. The fundamental principles of this emerging paradigm and its pharmacologic potential are discussed.

Genome-Wide Identification of Calcium Dependent Protein Kinase Gene Family in Plant Lineage Shows Presence of Novel D-x-D and D-E-L Motifs in EF-Hand Domain.

Calcium ions are considered ubiquitous second messengers in eukaryotic signal transduction pathways. Intracellular Ca2+ concentration are modulated by various signals such as hormones and biotic and abiotic stresses. Modulation of Ca2+ ion leads to stimulation of calcium dependent protein kinase genes (CPKs), which results in regulation of gene expression and therefore mediates plant growth and development as well as biotic and abiotic stresses. Here, we reported the CPK gene family of 40 different plant species (950 CPK genes) and provided a unified nomenclature system for all of them. In addition, we analyzed their genomic, biochemical and structural conserved features. Multiple sequence alignment revealed that the kinase domain, auto-inhibitory domain and EF-hands regions of regulatory domains are highly conserved in nature. Additionally, the EF-hand domains of higher plants were found to contain four D-x-D and two D-E-L motifs, while lower eukaryotic plants had two D-x-D and one D-x-E motifs in their EF-hands. Phylogenetic analysis showed that CPK genes are clustered into four different groups. By studying the CPK gene family across the plant lineage, we provide the first evidence of the presence of D-x-D motif in the calcium binding EF-hand domain of CPK proteins.

Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction.

Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process.

Parasite Calcineurin Regulates Host Cell Recognition and Attachment by Apicomplexans.

Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse-genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition, and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans.

Putative Role of Glutathione S‐transferases as Mediators of Cell Growth, Development, and Differentiation in the Eukaryotic Model Organism, Dictyostelium discoideum

In addition to their role in detoxification, Glutathione S‐transferase (GST) enzymes influence signal transduction, development, and differentiation in many eukaryotic cells. Transcriptome analysis of the cellular slime mold, Dictyostelium discoideum (strain AX4) expresses five GST (α1‐α5 class) transcripts whose function(s) remain unknown. This study characterized the activity and expression of GST isozymes in D. discoideum at various stages of growth and development. A GSH‐agarose pull down assay of the lysates from vegetative amoebae identified two protein species of ~24kDa; mass spectrophotometric analysis identified GSTα3> GSTα1>GSTα2 as predominant species. Measurements of GST activity in amoeba using the substrate 1‐chloro‐2‐4‐dinitrobenzene (CDNB) revealed functional enzyme activity, which was significantly reduced by as much as 50% in the presence of 500 nM ethacrynic acid (EA, a GSTs inhibitor). Ethacrynic acid also delayed the formation of aggregates and multicellular development. Levels of GSTs activity differed significantly between vegetative amoebae and multicellular stages of development (aggregate, slug, culmination, fruiting body), indicating the importance of GSTs to influence cell signaling and gene expression during morphogenesis and differentiation. Current studies aim to determine: a) the levels of each isozyme and their substrate specificities; b) the impact of GST null mutants on growth and development of the organism. These studies will contribute to elucidating the expanding the role and function of GST enzymes in eukaryotic signal transduction that regulates cell growth, chemotaxis, and differentiation.

Explorations of Lipid Effects in Cyclic Nucleotide-Gated Ion Channels using a Nanodisc Platform

The lipid membranes of cells are complex and dynamic mosaic environments that can alter the activity of membrane proteins including cyclic nucleotide-gated (CNG) ion channels. CNG channels are cation channels modulated by cytoplasmic cyclic nucleotides, which play important roles in sensory signal transduction in eukaryotes. Cell-based assays have accumulated evidence for lipid-modulation of CNG ion channels. To isolate and systematically study the effect of lipids on CNG channels, we reconstituted a model CNG channel into lipid bilayer nanodiscs composed of phosphocholine, phosphoglycerol and phosphoethanolamine lipids. We characterized these nanodisc preparations using size exclusion chromatography, electron microscopy and dynamic light scattering to ensure the formation of nanosized discoidal protein/lipid complexes. The reconstitution of CNG channels into nanodiscs permits the direct thermodynamic measurement of cAMP binding to the channel using isothermal titration calorimetry (ITC). This platform allowed us to probe the binding of cyclic nucleotides to CNG channels in the presence of different lipids, an important step in understanding the molecular mechanism of lipid modulation of CNG channel gating.

The modulation of ABC transporter-mediated multidrug resistance in cancer: a review of the past decade.

ATP-binding cassette (ABC) transporters represent one of the largest and oldest families of membrane proteins in all extant phyla from prokaryotes to humans, which couple the energy derived from ATP hydrolysis essentially to translocate, among various substrates, toxic compounds across the membrane. The fundamental functions of these multiple transporter proteins include: (1) conserved mechanisms related to nutrition and pathogenesis in bacteria, (2) spore formation in fungi, and (3) signal transduction, protein secretion and antigen presentation in eukaryotes. Moreover, one of the major causes of multidrug resistance (MDR) and chemotherapeutic failure in cancer therapy is believed to be the ABC transporter-mediated active efflux of a multitude of structurally and mechanistically distinct cytotoxic compounds across membranes. It has been postulated that ABC transporter inhibitors known as chemosensitizers may be used in combination with standard chemotherapeutic agents to enhance their therapeutic efficacy. The current paper reviews the advance in the past decade in this important domain of cancer chemoresistance and summarizes the development of new compounds and the re-evaluation of compounds originally designed for other targets as transport inhibitors of ATP-dependent drug efflux pumps.

The GPCR repertoire in the demosponge Amphimedon queenslandica: insights into the GPCR system at the early divergence of animals.

BackgroundG protein-coupled receptors (GPCRs) play a central role in eukaryotic signal transduction. However, the GPCR component of this signalling system, at the early origins of metazoans is not fully understood. Here we aim to identify and classify GPCRs in Amphimedon queenslandica (sponge), a member of an earliest diverging metazoan lineage (Porifera). Furthermore, phylogenetic comparisons of sponge GPCRs with eumetazoan and bilaterian GPCRs will be essential to our understanding of the GPCR system at the roots of metazoan evolution.ResultsWe present a curated list of 220 GPCRs in the sponge genome after excluding incomplete sequences and false positives from our initial dataset of 282 predicted GPCR sequences obtained using Pfam search. Phylogenetic analysis reveals that the sponge genome contains members belonging to four of the five major GRAFS families including Glutamate (33), Rhodopsin (126), Adhesion (40) and Frizzled (3). Interestingly, the sponge Rhodopsin family sequences lack orthologous relationships with those found in eumetazoan and bilaterian lineages, since they clustered separately to form sponge specific groups in the phylogenetic analysis. This suggests that sponge Rhodopsins diverged considerably from that found in other basal metazoans. A few sponge Adhesions clustered basal to Adhesion subfamilies commonly found in most vertebrates, suggesting some Adhesion subfamilies may have diverged prior to the emergence of Bilateria. Furthermore, at least eight of the sponge Adhesion members have a hormone binding motif (HRM domain) in their N-termini, although hormones have yet to be identified in sponges. We also phylogenetically clarified that sponge has homologs of metabotropic glutamate (mGluRs) and GABA receptors.ConclusionOur phylogenetic comparisons of sponge GPCRs with other metazoan genomes suggest that sponge contains a significantly diversified set of GPCRs. This is evident at the family/subfamily level comparisons for most GPCR families, in particular for the Rhodopsin family of GPCRs. In summary, this study provides a framework to perform future experimental and comparative studies to further verify and understand the roles of GPCRs that predates the divergence of bilaterian and eumetazoan lineages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-014-0270-4) contains supplementary material, which is available to authorized users.

Protein-tyrosine phosphorylation interaction network in Bacillus subtilis reveals new substrates, kinase activators and kinase cross-talk.

Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis.

Effects of deletion of different PP2C protein phosphatase genes on stress responses in Saccharomyces cerevisiae.

A key mechanism of signal transduction in eukaryotes is reversible protein phosphorylation, mediated through protein kinases and protein phosphatases (PPases). Modulation of signal transduction by this means regulates many biological processes. Saccharomyces cerevisiae has 40 PPases, including seven protein phosphatase 2C (PP2C PPase) genes (PTC1-PTC7). However, their precise functions remain poorly understood. To elucidate their cellular functions and to identify those that are redundant, we constructed 127 strains with deletions of all possible combinations of the seven PP2C PPase genes. All 127 disruptants were viable under nutrient-rich conditions, demonstrating that none of the combinations induced synthetic lethality under these conditions. However, several combinations exhibited novel phenotypes, e.g. the Δptc5Δptc7 double disruptant and the Δptc2Δptc3Δptc5Δptc7 quadruple disruptant exhibited low (13°C) and high (37°C) temperature-sensitive growth, respectively. Interestingly, the septuple disruptant Δptc1Δptc2Δptc3Δptc4Δptc5Δptc6Δptc7 showed an essentially normal growth phenotype at 37°C. The Δptc2Δptc3Δptc5Δptc7 quadruple disruptant was sensitive to LiCl (0.4 m). Two double disruptants, Δptc1Δptc2 and Δptc1Δptc4, displayed slow growth and Δptc1Δptc2Δptc4 could not grow on medium containing 1.5 m NaCl. The Δptc1Δptc6 double disruptant showed increased sensitivity to caffeine, congo red and calcofluor white compared to each single deletion. Our observations indicate that S. cerevisiae PP2C PPases have a shared and important role in responses to environmental stresses. These disruptants also provide a means for exploring the molecular mechanisms of redundant PTC gene functions under defined conditions.

Differential regulation of staphylococcal virulence by the sensor kinase SaeS in response to neutrophil-derived stimuli.

Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the histidine kinase SaeS recognizes specific host stimuli is unknown. After mutagenizing the predicted extracellular loop of SaeS, we discovered one methionine residue (M31) was essential for the ability of S. aureus to transcribe sae target genes, including hla, lukAB/lukGH, and hlgA. This single M31A mutation also significantly reduced cytotoxicity in human neutrophils to levels observed in cells following interaction with ΔsaeS. Another important discovery was that mutation of two aromatic anchor residues (W32A and F33A) disrupted the normal basal signaling of SaeS in the absence of inducing signals, yet both mutant kinases had appropriate activation of effector genes following exposure to neutrophils. Although the transcriptional profile of aromatic mutation W32A was consistent with that of WT in response to human α-defensin 1, mutant kinase F33A did not properly transcribe the γ-toxin genes in response to this stimulus. Taken together, our results provide molecular evidence for how SaeS recognizes host signals and triggers activation of select virulence factors to facilitate evasion of innate immunity. These findings have important implications for signal transduction in prokaryotes and eukaryotes due to conservation of aromatic anchor residues across both of these domains and the important role they play in sensor protein structure and function.

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera's transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.

Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1

Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly because of a lack of direct structural information. Here we report ligand-induced conformational changes in full-length MloK1, a cyclic nucleotide-modulated potassium channel from the bacterium Mesorhizobium loti, analysed by electron crystallography and atomic force microscopy. Upon cAMP binding, the cyclic nucleotide-binding domains move vertically towards the membrane, and directly contact the S1–S4 voltage sensor domains. This is accompanied by a significant shift and tilt of the voltage sensor domain helices. In both states, the inner pore-lining helices are in an ‘open’ conformation. We propose a mechanism in which ligand binding can favour pore opening via a direct interaction between the cyclic nucleotide-binding domains and voltage sensors. This offers a simple mechanistic hypothesis for the coupling between ligand gating and voltage sensing in eukaryotic HCN channels.