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Structural basis of activation and inhibition of the Ca2+/calmodulin-sensitive adenylyl cyclase 8 (LiP-MS)

ABSTRACT: Membrane adenylyl cyclases (ACs) catalyze the conversion of ATP to cAMP, a second messenger involved in different signaling pathways. AC8 is one of the 9 membrane-bound isoforms, present in the brain and implicated in cognitive functions. AC8 is regulated by Ca2+/CaM and different G proteins but structural evidence on its regulation is scarce. We solved the structure of full-length AC8 in complex with Gas and CaM. ACs contain large stretches of disordered/highly flexible domains that cannot be resolved with cryo-EM. To overcome this limitation, we have studied AC8's interaction with Gas and Gbg using limited proteolysis-coupled mass spectrometry (LiP-MS). A previously published data set on the interaction of AC8 and CaM was included in the analysis (PXD039520).

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Dina Schuster

LAB HEAD: Volodymyr M. Korkhov

PROVIDER: PXD040303 | Pride | 2024-02-14

REPOSITORIES: Pride

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			Action	DRS
	201202_WLABFU_dschuster_P04_416.raw	Raw
	201202_WLABFU_dschuster_P04_417.raw	Raw
	201202_WLABFU_dschuster_P04_418.raw	Raw
	201202_WLABFU_dschuster_P04_419.raw	Raw
	201202_WLABFU_dschuster_P04_420.raw	Raw

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Structural basis of activation and inhibition of the Ca2+/calmodulin-sensitive adenylyl cyclase 8 (XL-MS)

Project description:Membrane adenylyl cyclases (ACs) catalyze the conversion of ATP to cAMP, a second messenger involved in different signaling pathways. AC8 is one of the 9 membrane-bound isoforms, present in the brain and implicated in cognitive functions. AC8 is regulated by Ca2+/CaM and different G proteins but structural evidence on its regulation is scarce. We solved the structure of full-length AC8 in complex with Gas and CaM. ACs contain large stretches of disordered/highly flexible domains that cannot be resolved with cryo-EM. To overcome this limitation, we have studied AC8's interaction with CaM, Gas and Gbg using crosslinking mass spectrometry (XL-MS).

2024-02-14 | PXD040374 | Pride

Adenylyl cyclase 8, calmodulin - absolute quantification

Project description:The aim of this experiment was to absolutely quantify adenylyl cyclase 8 (AC8) and the co-purified interactor calmodulin (CaM) to assess their stoichiometry.

2024-02-14 | PXD044766 | Pride

Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: comparison with membranous adenylyl cyclase I.

Project description:The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2'-(N-methylanthraniloyl)-3'-deoxy-adenosine 5'-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.

| S-EPMC3915356 | biostudies-literature

Temporal and regional regulation of gene expression by calcium-stimulated adenylyl cyclase activity during fear memory

Project description:Mice with the two calcium-stmulated adenylyl cyclase isoforms (AC1 and AC8; DKO mice) knocked-out show conditioned fear memory deficits. We assessed gene expression changes at baseline and several time points after conditioned fear learning to assess transcriptional changes at different stages of learning. Transcriptional changes were assessed in the amydgdala and hippocampus of DKO and wild-type mice.

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Structural impact of TM domain mutations on mycobacterial adenylyl cyclase Rv1625c (LiP-MS)

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2022-08-19 | PXD033826 | Pride

Transcriptional changes associated with chemical induction of adenylyl cyclase activity in Mtb

Project description:Goal: Assess transcriptional changes in Mtb associated with activation of adenylyl cyclase activity in the bacterium, by treatment with the Rv1625c agonist V-59 or activation of the TetOn-cAMP construct. Specifically, address changes in transcription of cholestrrol utilization genes, during growth of Mtb in cholesterol media. Method: WT, Rv1625c knockout, and Rv1625c Complement strains of Mtb were grown in cholesterol-based media and treated with V-59 or vehicle control (DMSO). V-59 is known to increase cAMP synthesis in WT, but not in Rv1625c knockout Mtb. V-59 increases cAMP synthesis above that observed in WT in the Complement strain, due to Rv1625c overexpression in this strain. Also, utilized TetOn-cAMP Mtb strain, to induce cAMP synthesis independent of V-59 and Rv1625c. Treatment with Atc induces expression of catalytic domain of Rv1264 in this strain. We grew the TetOn-cAMP strain in cholesterol-based media, treated with Atc or EtOH (vehicle control). Conclusions: Transcriptional changes to cholesterol utilization genes associated with V-59 treatment in WT Mtb were similar to those associated with TetOn-cAMP induction. The transcriptional changes associated with blockade of cholesterol degradation following V-59 treatment in WT Mtb were not observed in the Rv1625c knockout strain. The Rv1625c knockout strain had intrinsic defects in induction of cholesterol utilization genes. The Complement strain showed enhanced transcriptional changes in response to V-59 treatment.

2021-12-16 | GSE190875 | GEO

Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins.

Project description:Edema factor (EF) and CyaA are calmodulin (CaM)-activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance spectroscopy analyses, we show that low Ca(2+) concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca(2+) concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca(2+) concentrations. Despite the similarity in Ca(2+) sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100-fold higher affinity for CaM than EF. CaM has N- and C-terminal globular domains, each binding two Ca(2+) ions. CyaA can be fully activated by CaM mutants with one defective C-terminal Ca(2+)-binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca(2+)-CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.

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Transcriptional changes associated with chemical induction of adenylyl cyclase activity in Mtb

Project description:Transcriptional changes associated with chemical induction of adenylyl cyclase activity in Mtb

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Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin.

Project description:CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.

| S-EPMC1224690 | biostudies-literature

Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

Project description:Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

| S-EPMC4110316 | biostudies-literature

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