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The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid

The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid
The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid
The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.
0022-2836
1282-1295
Findeisen, Felix
c6f157a7-30ed-4400-bf15-c6603024aaed
Linder, Jürgen U
3baa084f-7034-4d06-990f-1b2c5cb77612
Schultz, Anita
88dff698-b107-4aa0-9476-cb88d6f26015
Schultz, Joachim E
7e091719-97bf-4742-a0c2-4b8213642b05
Brügger, Britta
7d92eca4-9a68-41a5-b69c-99de253914be
Wieland, Felix
fb91d08d-6ea7-469b-a705-2610fb1d5795
Sinning, Irmgard
fbc3f199-8a3b-47a6-9ee7-00bfc472e079
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Findeisen, Felix
c6f157a7-30ed-4400-bf15-c6603024aaed
Linder, Jürgen U
3baa084f-7034-4d06-990f-1b2c5cb77612
Schultz, Anita
88dff698-b107-4aa0-9476-cb88d6f26015
Schultz, Joachim E
7e091719-97bf-4742-a0c2-4b8213642b05
Brügger, Britta
7d92eca4-9a68-41a5-b69c-99de253914be
Wieland, Felix
fb91d08d-6ea7-469b-a705-2610fb1d5795
Sinning, Irmgard
fbc3f199-8a3b-47a6-9ee7-00bfc472e079
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd

Findeisen, Felix, Linder, Jürgen U, Schultz, Anita, Schultz, Joachim E, Brügger, Britta, Wieland, Felix, Sinning, Irmgard and Tews, Ivo (2007) The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid. Journal of Molecular Biology, 369 (5), 1282-1295. (doi:10.1016/j.jmb.2007.04.013). (PMID:17482646)

Record type: Article

Abstract

The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.

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Published date: 22 June 2007
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 200595
URI: http://eprints.soton.ac.uk/id/eprint/200595
ISSN: 0022-2836
PURE UUID: c449c69a-3045-4431-b8e8-093d1df7cdc7
ORCID for Ivo Tews: ORCID iD orcid.org/0000-0002-4704-1139

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Date deposited: 01 Nov 2011 14:04
Last modified: 17 Dec 2019 01:41

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