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.

Fig. 1

See lengthwise print. Alignment of the catestatin region of chromogranin A with and homologous regions of proteins with partial amino acid sequence identity and known three-dimensional structure, as determined by xray crystallography: 8FAB (a human myeloma immunogobulin), 1PKM (cat muscle pyruvate kinase), and 2IG2 (a monoclonal human immunogobulin). Sequence homologies were detected by the algorithms BLAST and FASTA, while three-dimensional structures were found in the Protein Data Bank (PDB). Homology modeling was performed using the program HOMOLOGY (Molecular Simulations, Inc., San Diego, CA). Columns in bold contain residues found not only in the majority of catestatin regions but also in at least one of the homologous proteins, in the FASTA alignment, the gap penalty was -12/-2. Human chromogranin A sequence (1): Konecki, D.S., U.M. Benedum, H.H. Gerdes, and W.B. Huttner. 1987. The primary structure of human chromogranin A and pancreastatin. J. Biol. Chem. 262:17026-17030. Human chromogranin A sequence (2): Helman, L.J., T.G. Ahn, M.A. Levine, A. Allison, P.S. Cohen, M.J. Cooper, D.V. Cohn, M.A. Israel. 1988. Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA. J. Biol. Chem. 263:11559-11563. For other chromogranin A sequences in the catestatin region, see Mahata et al, 1997.

Fig. 2

Three-dimensional (stereo) view of the tertiary structures of the homologous regions of proteins 8FAB, 1PKM and 2IG2, in the region of partial sequence identity to catestatin (bovine chromogranin A_344–364). N, amino-terminus; C, carboxy-terminus. Arrows denote the beginning and end of the β-strand/loop/β-strand regions of homology with catestatin.

Fig. 3

Models of catestatin structure, (a) Structure of catestatin: results of homology modeling. The region shown is bovine chromogranin A_342–370, PDRSMRLSFRARGYGFRGPGLQLRRGWRP, thus extending from Pro³⁴² (at its amino-terminus) to Pro³⁷⁰ (at its carboxy-terminus). N, amino-terminus; C, carboxy-terminus. Red, electronegative side chain. Blue, electropositive side chain. Green, hydrophobic side chain. Brown, other hydrophilic side chain. (b) Isopotential surfaces of catestatin (same primary structure, length, and orientation as in panel 3a), as determined by electrostatic energy calculations. Potentials: blue surface, + 2.5 kT/e; red surface, − 2.5 kT/e. Gray spheres: CPK models of atoms. (c) ‘Docking’ of the homology-modeled structure of bovine catestatin (same primary structure and length as in panel 3a) with the extracellular domain of the Torpedo nicotinic cholinergic receptor [34], in the region of the cation pore vestibule. Extracellular portions of the α, β, γ, and δ subunits of the Torpedo nicotinic receptor are shown. Blue CPK atoms in catestatin: basic residues (Arg³⁵³ and Arg³⁵⁸). White stick side chains (basic residues) in nicotinic receptor subunits: Arg⁸⁷ and Lys¹⁰⁴ in α subunit; Arg⁸⁷ in γ subunit. Green stick side chains (hydrophobic residues) in nicotinic receptor subunits: Leu⁸⁷ and Leu¹⁰⁴ in β subunit; Leu¹⁰⁴ in γ subunit; Leu⁸⁹ and Tyr¹⁰⁶ in δ subunit.

Fig. 4

Stability of the catestatin 3-dimensional structure during sustained molecular dynamics simulations in water. Simulations (100 ps, T = 300 K) were conducted for two length versions of the catestatin region of chromogranin A: a longer 29-amino-acid peptide (bovine chromogranin A_342–370; PDRSMRLSFRARGYGFRGPGLQLRRGWRP), and a shorter 21-amino-acid peptide (bovine chromogranin A_344–364; RSMRLSFRARGYGFRGPGLQL). Distances between specified atoms of catestatin are shown as a function of time: (a) Cα (α-carbon) atoms of Arg³⁵⁸ and Arg³⁵³; this distance defines the longest backbone Cα—Cα distance in the tip of the loop, and hence the lower limit of any cleft or cavity which might be accessed by catestatin. (b) Cα (α-carbon) atoms of Arg³⁴⁴ and Pro³⁶⁰; this distance defines the long axis of catestatin. (c) Cζ (terminal carbon in the side chain) atoms of Arg³⁵⁸ and Arg³⁵³; this distance defines the maximum strand-to-strand width of catestatin.

Fig. 5

Hydrophobic moment plot of bovine chromogranin A (431 amino acid mature protein, minus signal peptide), to detect regions of likely amphiphilic β-sheet (alternating polar and apolar residues). The catestatin region is bovine chromogranin A_344–364 (RSMRLSFRARGYGFRGPGLQL). The amphiphilic peak is bounded by residues Ser³⁴⁵ and Gly³⁵⁶, and is maximally amphiphilic at residue Arg³⁵¹. At each residue, results were averaged over an 11-amino-acid window.

Fig. 6

Circular dichroism spectra of catestatin (bovine chromogranin A_334–364; RSMRLSFRARGYGFRGPGLQL; 0.54 mg/ml), as a function of dielectric constant of the solvent, as varied by increasing % composition with an apolar solvent (trifluoroethanol).

Fig. 7

Point mutants of catestatin: functional results of selective alanine substitutions (alanine scan) on nicotinic cholinergic-stimulated catecholamine secretion from PC12 pheochromocytoma cells. IC₅₀, the concentration of peptide required to inhibit 60 μM nicotine-stimulated catecholamine secretion by 50% (derived from dose-response curves determined at 0.1, 1, and 10 μM peptide). Mutant alanine residues are in bold.

Fig. 8

Effect of unlabeled catestatin itself or the nicotinic cholinergic agonist carbachol on equilibrium binding of [¹²⁵I]catestatin to nicotinic receptor-rich Torpedo membranes. Torpedo californica membranes were incubated for 90 min with [¹²⁵I]catestatin in the presence of vehicle (mock), unlabeled catestatin (50 μM) or carbachol (carbamylcholine chloride, 100 μM). Values are given as the means of triplicate determinations±S.E. of one representative experiment.