title: Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a beta-strand/loop/beta-strand structure secured by hydrophobic interactions and predictive of activity.
abstract: A novel fragment of chromogranin A, known as 'catestatin' (bovine chromogranin A344-364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide's structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin's primary structure bore significant (29-35.5% identity, general alignment score 44-57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, IPKM, and 2IG2. Each of these sequences exists in nature as a beta-strand/loop/beta-strand structure, stabilized by hydrophobic interactions between the beta-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic beta-strand. Circular dichroism in the far ultraviolet showed substantial (63%) beta-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg351, Arg353, and Arg358), though not for two arginine residues in the strand region toward the amino-terminus. [125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was 'docked' with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the beta and delta subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist.
authors.affiliation: Department of Medicine, Center for Molecular Genetics, University of California, San Diego, USA.
authors.forename: I
authors.initials: I
authors.lastname: Tsigelny
authors.forename: S K
authors.initials: SK
authors.lastname: Mahata
authors.forename: L
authors.initials: L
authors.lastname: Taupenot
authors.forename: N E
authors.initials: NE
authors.lastname: Preece
authors.forename: M
authors.initials: M
authors.lastname: Mahata
authors.forename: I
authors.initials: I
authors.lastname: Khan
authors.forename: R J
authors.initials: RJ
authors.lastname: Parmer
authors.forename: D T
authors.initials: DT
authors.lastname: O'Connor
mesh_terms: D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism*; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry* / Q000494:pharmacology; D002942:Circular Dichroism; D008958:Models, Molecular; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D055672:Static Electricity
chemical_list: D002395:Catecholamines; D053379:Chromogranin A; D002864:Chromogranins; D010446:Peptide Fragments; C109084:chromogranin A (344-364); D002217:Carbachol