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

Autor:	Khanppnavar B; Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland.; Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland., Schuster D; Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland.; Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland.; Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland., Lavriha P; Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland., Uliana F; Department of Biology, Institute of Biological Chemistry, ETH Zurich, Zurich, Switzerland., Özel M; Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland., Mehta V; Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland., Leitner A; Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland., Picotti P; Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland., Korkhov VM; Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland. volodymyr.korkhov@psi.ch.; Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland. volodymyr.korkhov@psi.ch.
Jazyk:	angličtina
Zdroj:	EMBO reports [EMBO Rep] 2024 Mar; Vol. 25 (3), pp. 1513-1540. Date of Electronic Publication: 2024 Feb 13.
DOI:	10.1038/s44319-024-00076-y
Abstrakt:	Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca 2+ 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 Ca 2+ /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 Gβγ, 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. (© 2024. The Author(s).)
Databáze:	MEDLINE
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