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

ABSTRACT: LiP-MS was used to assess the structural impact of transmembrane domain mutations on the overall conformation of the mycobacterial cycase Rv1625c.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli Mycobacterium Tuberculosis Sumu001

TISSUE(S): Cell Culture

SUBMITTER: Dina Schuster

LAB HEAD: Volodymyr Korkhov

PROVIDER: PXD033826 | Pride | 2022-08-19

REPOSITORIES: Pride

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			Action	DRS
	20210614_104457_dschuster_TC.sne	Other
	20210614_104551_dschuster_Report.setup_TC.txt	Txt
	20210614_104551_dschuster_Report_TC.csv	Csv
	20210614_104551_dschuster_Report_TC.log.txt	Txt
	20210614_104551_dschuster_Report_TC.params	Other

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Publications

Structure of <i>Mycobacterium tuberculosis</i> Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases.

Mehta Ved V Khanppnavar Basavraj B Schuster Dina D Kantarci Ilayda I Vercellino Irene I Kosturanova Angela A Iype Tarun T Stefanic Sasa S Picotti Paola P Korkhov Volodymyr M VM

eLife 20220818

<i>Mycobacterium tuberculosis</i> adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilita ...[more]

PMID: 35980026

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Adenylyl cyclase Rv1625c of Mycobacterium tuberculosis: a progenitor of mammalian adenylyl cyclases.

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Structural basis of adenylyl cyclase 9 activation.

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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.

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