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Identification of a haem domain in human soluble adenylate cyclase.

Middelhaufe S, Leipelt M, Levin LR, Buck J, Steegborn C - Biosci. Rep. (2012)

Bottom Line: The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer.Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide).Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, Bochum, Germany.

ABSTRACT
The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

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Testing of potential axial haem ligands(A) Spectra of sAC-HD at pH 7.8 (black line), pH 10.0 (dotted line) and pH 5.5 (grey line). Acidic pH causes a shift of the Soret peak from 418 (pH 7.8) to 414 nm (pH 5.5) indicating histidine or lysine as probably axial haem ligands. (B) Sequence alignment of sAC-HD and homologous fragments from other sACs reveals six histidine residues (●) with different levels of conservation as putative axial haem ligands. Further, a putative binding motive for covalently bound haem is indicated (▲). The residue numbers and predicted secondary structure elements refer to human sAC-HD. Blue-coloured residues indicate high conservation of physicochemical properties and red colour reflects medium conservation. The secondary structure prediction was generated with Jpred [48].
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Figure 4: Testing of potential axial haem ligands(A) Spectra of sAC-HD at pH 7.8 (black line), pH 10.0 (dotted line) and pH 5.5 (grey line). Acidic pH causes a shift of the Soret peak from 418 (pH 7.8) to 414 nm (pH 5.5) indicating histidine or lysine as probably axial haem ligands. (B) Sequence alignment of sAC-HD and homologous fragments from other sACs reveals six histidine residues (●) with different levels of conservation as putative axial haem ligands. Further, a putative binding motive for covalently bound haem is indicated (▲). The residue numbers and predicted secondary structure elements refer to human sAC-HD. Blue-coloured residues indicate high conservation of physicochemical properties and red colour reflects medium conservation. The secondary structure prediction was generated with Jpred [48].

Mentions: Typical transporters for diatomic gas ligands belong to the globin family, which features an all-α-fold. Secondary structure prediction for sAC-HD suggests a mixed α–β-fold, and UV–CD spectroscopy indeed indicates approx. 34% α-helix and 30% β-sheet content (Figure 3A). While this distribution suggests no similarity to globins, it instead indicates a potential structural resemblance to the mixed-α–β HDs of the H-NOX (haem-NO/oxygen) family, which comprises the sGC HD and the gas sensing PAS (Per-Ant-Sim) domains [30–32]. Both families are highly degenerate on sequence level, but manual alignment of predicted sAC-HD secondary structure elements with two structurally characterized haem-containing PAS domains [33] uncovered weak similarity (Figure 3B). The alignment indicated His966 of sAC-HD as a potential axial haem ligand, i.e. one of the possibly two haem iron ligands not provided by the porphyrin system. Positions of soret and α-peak for sAC-HD indeed indicate a six-fold co-ordination [21], which implies the presence of two such axial amino acid ligands. However, when we mutated this histidine residue to alanine, spectra of the sAC-HD-H966A variant showed negligible deviations from wild-type (Table 1). The lack of an additional absorption band above 600 nm also excluded methionine as axial ligand [28], and a Soret shift at acidic pH indicated histidine or lysine and ruled out tyrosine (Figure 4A). Mutating six histidine residues conserved in sAC from different species (Figure 4B) also did not result in significant spectral deviation from wild-type (Table 1). Thus determining the fold and elucidating haem interaction details of sAC-HD will require further structural studies.


Identification of a haem domain in human soluble adenylate cyclase.

Middelhaufe S, Leipelt M, Levin LR, Buck J, Steegborn C - Biosci. Rep. (2012)

Testing of potential axial haem ligands(A) Spectra of sAC-HD at pH 7.8 (black line), pH 10.0 (dotted line) and pH 5.5 (grey line). Acidic pH causes a shift of the Soret peak from 418 (pH 7.8) to 414 nm (pH 5.5) indicating histidine or lysine as probably axial haem ligands. (B) Sequence alignment of sAC-HD and homologous fragments from other sACs reveals six histidine residues (●) with different levels of conservation as putative axial haem ligands. Further, a putative binding motive for covalently bound haem is indicated (▲). The residue numbers and predicted secondary structure elements refer to human sAC-HD. Blue-coloured residues indicate high conservation of physicochemical properties and red colour reflects medium conservation. The secondary structure prediction was generated with Jpred [48].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475452&req=5

Figure 4: Testing of potential axial haem ligands(A) Spectra of sAC-HD at pH 7.8 (black line), pH 10.0 (dotted line) and pH 5.5 (grey line). Acidic pH causes a shift of the Soret peak from 418 (pH 7.8) to 414 nm (pH 5.5) indicating histidine or lysine as probably axial haem ligands. (B) Sequence alignment of sAC-HD and homologous fragments from other sACs reveals six histidine residues (●) with different levels of conservation as putative axial haem ligands. Further, a putative binding motive for covalently bound haem is indicated (▲). The residue numbers and predicted secondary structure elements refer to human sAC-HD. Blue-coloured residues indicate high conservation of physicochemical properties and red colour reflects medium conservation. The secondary structure prediction was generated with Jpred [48].
Mentions: Typical transporters for diatomic gas ligands belong to the globin family, which features an all-α-fold. Secondary structure prediction for sAC-HD suggests a mixed α–β-fold, and UV–CD spectroscopy indeed indicates approx. 34% α-helix and 30% β-sheet content (Figure 3A). While this distribution suggests no similarity to globins, it instead indicates a potential structural resemblance to the mixed-α–β HDs of the H-NOX (haem-NO/oxygen) family, which comprises the sGC HD and the gas sensing PAS (Per-Ant-Sim) domains [30–32]. Both families are highly degenerate on sequence level, but manual alignment of predicted sAC-HD secondary structure elements with two structurally characterized haem-containing PAS domains [33] uncovered weak similarity (Figure 3B). The alignment indicated His966 of sAC-HD as a potential axial haem ligand, i.e. one of the possibly two haem iron ligands not provided by the porphyrin system. Positions of soret and α-peak for sAC-HD indeed indicate a six-fold co-ordination [21], which implies the presence of two such axial amino acid ligands. However, when we mutated this histidine residue to alanine, spectra of the sAC-HD-H966A variant showed negligible deviations from wild-type (Table 1). The lack of an additional absorption band above 600 nm also excluded methionine as axial ligand [28], and a Soret shift at acidic pH indicated histidine or lysine and ruled out tyrosine (Figure 4A). Mutating six histidine residues conserved in sAC from different species (Figure 4B) also did not result in significant spectral deviation from wild-type (Table 1). Thus determining the fold and elucidating haem interaction details of sAC-HD will require further structural studies.

Bottom Line: The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer.Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide).Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, Bochum, Germany.

ABSTRACT
The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

Show MeSH
Related in: MedlinePlus