Dataset Information

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment.

ABSTRACT: In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3',5'-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.

SUBMITTER: Steegborn C

PROVIDER: S-EPMC3644947 | biostudies-literature | 2005 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment.

Steegborn Clemens C Litvin Tatiana N TN Levin Lonny R LR Buck Jochen J Wu Hao H

Nature structural & molecular biology 20041226 1

In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3',5'-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotid ...[more]

PMID: 15619637

Similar Datasets

Project description:The diterpene forskolin (FS) binds to, and activates, mammalian membranous adenylyl cyclase (AC) isoforms I-VIII. Diterpenes without C(1)-OH group do not activate ACs. The C(1)-OH group forms a hydrogen bond with the backbone oxygen of Val506 of the C1 catalytic subunit of AC (isoform V numbering). To better understand the mechanism of AC activation we examined the interactions of FS and eight FS analogs with purified catalytic AC subunits C1 (AC V) and C2 (AC II) by fluorescence spectroscopy, using 2',3'-O-(N-methylanthraniloyl)-guanosine 5'-triphosphate (MANT-GTP) as fluorescent reporter probe, and by enzymatic activity. FS analogs induced C1/C2 assembly as assessed by fluorescence resonance energy transfer from Trp1020 of C2 to MANT-GTP and by increased direct MANT-GTP fluorescence in the order of efficacy FS approximately 7-deacetyl-FS approximately 6-acetyl-7-deacetyl-FS approximately 9-deoxy-FS>7-deacetyl-7-(N-methylpiperazino-gamma-butyryloxy)-FS>1-deoxy-FS approximately 1,9-dideoxy-FS approximately 7-deacetyl-1-deoxy-FS approximately 7-deacetyl-1,9-dideoxy-FS. In contrast, FS analogs activated catalysis in the order of efficacy FS>7-deacety-FS approximately 6-acetyl-7-deacetyl-FS approximately 9-deoxy-FS>7-deacetyl-7-(N-methylpiperazino-gamma-butyryloxy)-FS>>1-deoxy-FS, 1,9-dideoxy-FS, 7-deacetyl-1-deoxy-FS and 7-deacetyl-1,9-dideoxy-FS (all ineffective). 1-Deoxy-FS analogs inhibited FS-stimulated catalysis by an apparently non-competitive mechanism. Our data suggest a two-step mechanism of AC activation by diterpenes. In the first step, diterpenes, regardless of their substitution pattern, promote C1/C2 assembly. In the second and yet poorly understood step, diterpenes that form a hydrogen bond between C(1)-OH and Val506 promote a conformational switch that results in activation of catalysis. The apparent non-competitive interaction of FS with 1-deoxy-FS analogs is explained by impaired ligand exchange due to strong hydrophobic interactions with C1/C2.

Project description:Membrane guanylate cyclase (MGC) is a ubiquitous multi-switching cyclic GMP generating signaling machine linked with countless physiological processes. In mammals it is encoded by seven distinct homologous genes. It is a single transmembrane spanning multi-modular protein; composed of integrated blocks and existing in homo-dimeric form. Its core catalytic domain (CCD) module is a common transduction center where all incoming signals are translated into the production of cyclic GMP, a cellular signal second messenger. Crystal structure of the MGC's CCD does not exist and its precise identity is ill-defined. Here, we define it at a sub-molecular level for the phototransduction-linked MGC, the rod outer segment guanylate cyclase type 1, ROS-GC1. (1) The CCD is a conserved 145-residue structural unit, represented by the segment V820-P964. (2) It exists as a homo-dimer and contains seven conserved catalytic elements (CEs) wedged into seven conserved motifs. (3) It also contains a conserved 21-residue neurocalcin ?-modulated structural domain, V836-L857. (4) Site-directed mutagenesis documents that each of the seven CEs governs the cyclase's catalytic activity. (5) In contrast to the soluble and the bacterium MGC which use Mn2+-GTP substrate for catalysis, MGC CCD uses the natural Mg2+-GTP substrate. (6) Strikingly, the MGC CCD requires anchoring by the Transmembrane Domain (TMD) to exhibit its major (?92%) catalytic activity; in isolated form the activity is only marginal. This feature is not linked with any unique sequence of the TMD; there is minimal conservation in TMD. Finally, (7) the seven CEs control each of four phototransduction pathways- -two Ca2+-sensor GCAPs-, one Ca2+-sensor, S100B-, and one bicarbonate-modulated. The findings disclose that the CCD of ROS-GC1 has built-in regulatory elements that control its signal translational activity. Due to conservation of these regulatory elements, it is proposed that these elements also control the physiological activity of other members of MGC family.

Dataset Information

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment.

Publications

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets