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/

A novel fragment of chromogranin A, known as 'catestatin' (bovine chro mogranin A(344-364)), inhibits catecholamine release from chromaffin c ells and noradrenergic neurons by acting as a non-competitive nicotini c cholinergic antagonist, and may therefore constitute an endogenous a utocrine feedback regulator of sympathoadrenal activity. To characteri ze how this activity depends on the peptide's structure, we searched f or common 3-dimensional motifs for this primary structure or its homol ogs. Catestatin's primary structure bore significant (29-35.5% identit y, general alignment score 44-57) sequence homology to fragment sequen ces within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, 1PKM, and 2IG2. Each of these sequences e xists in nature as a beta-strand/loop/beta-strand structure, stabilize d by hydrophobic interactions between the beta-strands. The catestatin structure was stable during molecular dynamics simulations. The cates tatin loop contains three Arg residues, whose electropositive side cha ins form the terminus of the structure, and give rise to substantial u ncompensated charge asymmetry in the molecule. A hydrophobic moment pl ot revealed that catestatin is the only segment of chromogranin A pred icted to contain amphiphilic beta-strand. Circular dichroism in the fa r ultraviolet showed substantial (63%) beta-sheet structure, especiall y in a hydrophobic environment. Alanine-substitution mutants of catest atin established a crucial role for the three central arginine residue s in the loop (Arg(351), Arg(353), and Arg(358)), though not for two a rginine residues in the strand region toward the amino-terminus. [I-12 5]Catestatin bound to Torpedo membranes at a site other than the nicot inic agonist binding site. When the catestatin structure was 'docked' with the extracellular domain of the Torpedo nicotinic cholinergic rec eptor, it interacted principally with the beta and delta subunits, in a relatively hydrophobic region of the cation pore extracellular orifi ce, 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 h omology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular s patial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous n icotinic cholinergic antagonist. (C) 1998 Elsevier Science B.V. All ri ghts reserved.