Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics.

Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gßγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.

ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering.

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Light-induced Trpin/Metout Switching During BLUF Domain Activation in ATP-bound Photoactivatable Adenylate Cyclase OaPAC.

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed.

Engineering Bacteriophytochrome-coupled Photoactivated Adenylyl Cyclases for Enhanced Optogenetic cAMP Modulation.

Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors.

Unraveling the inhibitory mechanism of adenylyl cyclase 8E: New insights into regulatory pathways of cAMP signal integration.

Adenylyl Cyclase 8E (AC8E), which lacks part of M1 transmembrane domain, has been previously shown to dimerize with AC3 and down-regulate its activity, but the molecular mechanism of this inhibitory effect has remained elusive. Here, we first show that AC8E also inhibits AC2 and AC6, highlighting the functional importance of this novel regulatory mechanism in the cAMP signaling pathway across AC families. We then completed the partial structure of Bos taurus AC9 using combinations of comparative modeling and fold recognition methods, and used this as a template to build the first full 3D-models of AC8 and AC8E. These models evidenced that the lack of M1 transmembrane domain of AC8E shifts the N-terminal domain, which impacts the orientation of the helical domains, thus affecting the catalytic site. This was confirmed in living cells with cAMP imaging, where we showed that the N-terminal domain is required for reducing cAMP production. Our data also show that AC8E prevents the translocation of other ACs towards the plasma membrane, further reducing the cAMP responsiveness to extracellular signals. This newly discovered dual inhibitory mechanism provides an additional level of regulation of cAMP-dependent signals integration.

Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State.

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent 'on' state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.

Structural insights into membrane adenylyl cyclases, initiators of cAMP signaling.

Membrane adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. As effector proteins of G protein-coupled receptors and other signaling pathways, ACs receive and amplify signals from the cell surface, translating them into biochemical reactions in the intracellular space and integrating different signaling pathways. Despite their importance in signal transduction and physiology, our knowledge about the structure, function, regulation, and molecular interactions of ACs remains relatively scarce. In this review, we summarize recent advances in our understanding of these membrane enzymes.

A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.

Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.

Soluble adenylyl cyclase coordinates intracellular pH homeostasis and biomineralization in calcifying cells of a marine animal.

Biomineralizing cells concentrate dissolved inorganic carbon (DIC) and remove protons from the site of mineral precipitation. However, the molecular regulatory mechanisms that orchestrate pH homeostasis and biomineralization of calcifying cells are poorly understood. Here, we report that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) coordinates intracellular pH (pHi) regulation in the calcifying primary mesenchyme cells (PMCs) of sea urchin larvae. Single-cell transcriptomics, in situ hybridization, and immunocytochemistry elucidated the spatiotemporal expression of sAC during skeletogenesis. Live pHi imaging of PMCs revealed that the downregulation of sAC activity with two structurally unrelated small molecules inhibited pHi regulation of PMCs, an effect that was rescued by the addition of cell-permeable cAMP. Pharmacological sAC inhibition also significantly reduced normal spicule growth and spicule regeneration, establishing a link between PMC pHi regulation and biomineralization. Finally, increased expression of sAC mRNA was detected during skeleton remineralization and exposure to CO2-induced acidification. These findings suggest that transcriptional regulation of sAC is required to promote remineralization and to compensate for acidic stress. This work highlights the central role of sAC in coordinating acid-base regulation and biomineralization in calcifying cells of a marine animal.

Twin cyclic mononucleotide cyclase and phosphodiesterase domain architecture as a common feature in complex plant proteins.

The majority of proteins in both prokaryote and eukaryote proteomes consist of two or more functional centers, which allows for intramolecular tuning of protein functions. Such architecture, as opposed to animal orthologs, applies to the plant cyclases (CNC) and phosphodiesterases (PDEs), the vast majority of which are part of larger multifunctional proteins. In plants, until recently, only two cases of combinations of CNC-PDE in one protein were reported. Here we propose that in plants, multifunctional proteins in which the PDE motif has been identified, the presence of the additional CNC center is common. Searching the Arabidopsis thaliana proteome with a combined PDE-CNC motif allowed the creation of a database of proteins with both activities. One such example is methylenetetrahydrofolate dehydrogenase, in which we determined the activities of adenylate cyclase (AC) and PDE. Based on biochemical and mutagenesis analyses we assessed the impact of the AC and PDE catalytic centers on the dehydrogenase activity. This allowed us to propose additional regulatory mechanism that govern folate metabolism by cAMP. It is therefore conceivable that the combined CNC-PDE architecture is a common regulatory configuration, where control of the level of cyclic nucleotides (cNMP) influences other catalytic activities of the protein.

Molecular modelling of novel ADCY3 variant predicts a molecular target for tackling obesity.

Severe early­onset obesity is mainly attributed to single gene variations of the hypothalamic leptin­melanocortin system, which is critical for controlling the balance between appetite and energy expenditure. Adenylate cyclase 3 (ADCY3), a transmembrane enzyme localized in primary neuronal cilia, is a key genetic candidate, which appears to have an essential role in regulating body weight. The present study aimed to identify ADCY3 genetic variants in severely obese young patients of Greek­Cypriot origin by genomic sequencing. Apart from previously reported variants, the novel and probably pathogenic variant c.349T>A, causing a p.Leu117Met substitution within one of the two pseudo­symmetric halves of the transmembrane part of the protein, was reported. Molecular modelling analysis used to delineate bonding interactions within the mutated protein structure strongly suggested a change in interactive forces and energy levels affecting the pseudo­twofold symmetry of the transmembrane domain of the protein and probably its catalytic function. These results support the involvement of ADCY3 in the pathology of the disease and point towards the requirement of defining protein function and evaluating the clinical significance of the detected variants.

Direct Observation of Ultrafast Proton Rocking in the BLUF Domain.

We present direct observation of ultrafast proton rocking in the central motif of a BLUF domain protein scaffold. The mutant design has taken consideration of modulating the proton-coupled electron transfer (PCET) driving forces by replacing Tyr in the original motif with Trp, in order to remove the interference of a competing electron transfer pathway. Using femtosecond pump-probe spectroscopy and detailed kinetics analysis, we resolved an electron-transfer-coupled Grotthuss-type forward and reverse proton rocking along the FMN-Gln-Trp proton relay chain. The rates of forward and reverse proton transfer are determined to be very close, namely 51 ps vs. 52 ps. The kinetic isotope effect (KIE) constants associated with the forward and reverse proton transfer are 3.9 and 5.3, respectively. The observation of ultrafast proton rocking is not only a crucial step towards revealing the nature of proton relay in the BLUF domain, but also provides a new paradigm of proton transfer in proteins for theoretical investigations.

Identification of the Catalytic Residues in the Cyclase Domain of the Class IV Lanthipeptide Synthetase SgbL.

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are subdivided into different classes based on their processing enzymes. The three-domain class IV lanthipeptide synthetases (LanL enzymes) consist of N-terminal lyase, central kinase, and C-terminal cyclase domains. While the catalytic residues of the kinase domains (mediating ATP-dependent Ser/Thr phosphorylations) and the lyase domains (carrying out subsequent phosphoserine/phosphothreonine (pSer/pThr) eliminations to yield dehydroalanine/dehydrobutyrine (Dha/Dhb) residues) have been characterized previously, such studies are missing for LanL cyclase domains. To close this gap of knowledge, this study reports on the identification and validation of the catalytic residues in the cyclase domain of the class IV lanthipeptide synthetase SgbL, which facilitate the nucleophilic attacks by Cys thiols on Dha/Dhb residues for the formation of ß-thioether crosslinks.

Photoreaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes.

The photochemical reaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes PCC 7420 (mPAC), which consists of a Per-Arnt-Sim (PAS), a light­oxygene-voltage (LOV), and an adenylate cyclase (AC) domain, was investigated mainly using the time-resolved transient grating method. An absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 0.66 µs. After this reaction, a significant diffusion coefficient (D)-change was observed with a time constant of 38 ms. The determined D-value was concentration-dependent indicating a rapid equilibrium between the dimer and tetramer. Combining the results of size exclusion chromatography and CD spectroscopy, we concluded that the photoinduced D-change was mainly attributed to the equilibrium shift from the dimer rich to the tetramer rich states upon light exposure. Since the reaction rate does not depend on concentration, the rate determining step of the tetramer formation is not the collision of proteins by diffusion, but a conformation change. The roles of the PAS and AC domains as well as the N- and C-terminal flanking helices of the LOV domain (A'α- and Jα-helices) were investigated using various truncated mutants. The PAS domain was found to be a strong dimerization site and is related to efficient signal transduction. It was found that simultaneous existence of the A'α- and Jα-helices in mPAC is important for the light-induced conformation change to lead the conformation change which induces the tetramer formation. The results suggest that the angle changes of the coiled-coil structures in the A'α and Jα-helices are essential for this conformation change. The reaction scheme of mPAC is proposed.

The lipolytic activity of LipJ, a stress-induced enzyme, is regulated by its C-terminal adenylate cyclase domain.

Aim: The confirmation of lipolytic activity and role of Rv1900c in the Mycobacterium physiology Methods:rv1900c/N-terminus domain (rv1900NT) were cloned in pET28a/Escherichia coli, purified by affinity chromatography and characterized. Results: A zone of clearance on tributyrin-agar and activity with pNP-decanoate confirmed the lipolytic activity of Rv1900c. The Rv1900NT demonstrated higher enzyme specific activity, Vmax and kcat, but Rv1900c was more thermostable. The lipolytic activity of Rv1900c decreased in presence of ATP. Mycobacterium smegmatis expressed rv1900c/rv1900NT-altered colony morphology, growth, cell surface properties and survival under stress conditions. The effect was more prominent with Rv1900NT as compared with Rv1900c. Conclusion: The study confirmed the lipolytic activity of Rv1900c and suggested its regulation by the adenylate cyclase domain and role in the intracellular survival of bacteria.

Lay abstract Tuberculosis (TB) remains the top contagious/infectious killer in the world. It is caused by the bacteria Mycobacterium tuberculosis. The bacteria resides/replicates in the immune cell that normally has to eradicate infectious microorganisms. Though the treatment of TB is available, the emergence of drug-resistant bacteria is of major concern. The treatment of drug-resistant TB has been reported to be more difficult due to lengthy and complex treatment regimens. Therefore, there is an urgent need for new and better drugs to treat TB/drug-resistant TB. For this purpose understanding the role of each protein in the physiology of mycobacteria is required. Lipids play a critical role in the intracellular survival of this pathogen in the host. Our study demonstrated that LipJ supported the intracellular survival of bacteria. Therefore, it could be a potential drug target.

A Potent N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide Inhibitor of Adenylyl Cyclase of G. lamblia: Biological Evaluation and Molecular Modelling Studies.

In this work, we report a derivative of N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide as a new inhibitor for adenylyl cyclase of Giardia lamblia which was obtained from a study using structural data of the nucleotidyl cyclase 1 (gNC1) of this parasite. For such a study, we developed a model for this specific enzyme by using homology techniques, which is the first model reported for gNC1 of G. lamblia. Our studies show that the new inhibitor has a competitive mechanism of action against this enzyme. 2-Hydroxyestradiol was used as the reference compound for comparative studies. Results in this work are important from two points of view. on the one hand, an experimentally corroborated model for gNC1 of G. lamblia obtained by molecular modelling is presented; on the other hand, the new inhibitor obtained is an undoubtedly excellent starting structure for the development of new metabolic inhibitors for G. lamblia.

Gαi1 inhibition mechanism of ATP-bound adenylyl cyclase type 5.

Conversion of adenosine triphosphate (ATP) to the second messenger cyclic adenosine monophosphate (cAMP) is an essential reaction mechanism that takes place in eukaryotes, triggering a variety of signal transduction pathways. ATP conversion is catalyzed by the enzyme adenylyl cyclase (AC), which can be regulated by binding inhibitory, Gαi, and stimulatory, Gαs subunits. In the past twenty years, several crystal structures of AC in isolated form and complexed to Gαs subunits have been resolved. Nevertheless, the molecular basis of the inhibition mechanism of AC, induced by Gαi, is still far from being fully understood. Here, classical molecular dynamics simulations of the isolated holo AC protein type 5 and the holo binary complex AC5:Gαi have been analyzed to investigate the conformational impact of Gαi association on ATP-bound AC5. The results show that Gαi appears to inhibit the activity of AC5 by preventing the formation of a reactive ATP conformation.

Crystal structure and enzymatic characterization of the putative adenylyl cyclase HpAC1 from Hippeastrum reveal dominant triphosphatase activity.

HpAC1, a protein from Hippeastrum hybrid cultivars, was previously suggested to be a plant adenylyl cyclase. We describe a structural and enzymatic characterization of HpAC1. A crystal structure of HpAC1 in complex with a non-hydrolyzable GTP analog confirms a generic CYTH architecture, comprising a ß-barrel with an internal substrate site. The structure reveals significant active site differences to AC proteins with CYTH fold, however, and we find that HpAC1 lacks measurable AC activity. Instead, HpAC1 has substantial triphosphatase activity, indicating this protective activity or a related activity as the protein's physiological function.

Allosteric Inhibition of Adenylyl Cyclase Type 5 by G-Protein: A Molecular Dynamics Study.

Adenylyl cyclases (ACs) have a crucial role in many signal transduction pathways, in particular in the intricate control of cyclic AMP (cAMP) generation from adenosine triphosphate (ATP). Using homology models developed from existing structural data and docking experiments, we have carried out all-atom, microsecond-scale molecular dynamics simulations on the AC5 isoform of adenylyl cyclase bound to the inhibitory G-protein subunit Gαi in the presence and in the absence of ATP. The results show that Gαi has significant effects on the structure and flexibility of adenylyl cyclase, as observed earlier for the binding of ATP and Gsα. New data on Gαi bound to the C1 domain of AC5 help explain how Gαi inhibits enzyme activity and obtain insight on its regulation. Simulations also suggest a crucial role of ATP in the regulation of the stimulation and inhibition of AC5.

AC6 regulates the microtubule-depolymerizing kinesin KIF19A to control ciliary length in mammals.

Motile cilia are hairlike structures that line the respiratory and reproductive tracts and the middle ear and generate fluid flow in these organs via synchronized beating. Cilium growth is a highly regulated process that is assumed to be important for flow generation. Recently, Kif19a, a kinesin residing at the cilia tip, was identified to be essential for ciliary length control through its microtubule depolymerization function. However, there is a lack of information on the nature of proteins and the integrated signaling mechanism regulating growth of motile cilia. Here, we report that adenylate cyclase 6 (AC6), a highly abundant AC isoform in airway epithelial cells, inhibits degradation of Kif19a by inhibiting autophagy, a cellular recycling mechanism for damaged proteins and organelles. Using epithelium-specific knockout mice of AC6, we demonstrated that AC6 knockout airway epithelial cells have longer cilia compared with the WT cells because of decreased Kif19a protein levels in the cilia. We demonstrated in vitro that AC6 inhibits AMP-activated kinase (AMPK), an important modulator of cellular energy-conserving mechanisms, and uncouples its binding with ciliary kinesin Kif19a. In the absence of AC6, activation of AMPK mobilizes Kif19a into autophagosomes for degradation in airway epithelial cells. Lower Kif19a levels upon pharmacological activation of AMPK in airway epithelial cells correlated with elongated cilia and vice versa. In all, the AC6-AMPK pathway, which is tunable to cellular cues, could potentially serve as one of the crucial ciliary growth checkpoints and could be channeled to develop therapeutic interventions for cilia-associated disorders.