Role of Disulfide Bonds for the Structure and Folding of Proguanylin

Abstract

The intestinal peptide hormone guanylin circulates mainly as its corresponding prohormone of 94 amino acids and is the first identified endogenous ligand of intestinal guanylyl cyclase C. While the prohormone is biologically inactive, it is processed to the fully functional form with 15 amino acid residues corresponding to the COOH terminus of the precursor protein. In addition to this inactivation of the hormone region, the prosequence makes an essential contribution to the disulfide-coupled folding of the hormone. On the basis of the recently determined solution structure of proguanylin, explanations for these functions of the prosequence were found, indicating that interstrand contacts between the NH 2 -terminal -hairpin of the prosequence and the COOH-terminal hormone region are crucial for formation of the correct disulfide bonds of guanylin. To further investigate the role of individual disulfide bonds upon stabilization of the overall three-dimensional structure of proguanylin and to test the assumption of a direct effect of the prosequence on the structure of the hormone region, we studied the cysteine double mutant proteins proguanylin-C48S/C61S and proguanylin-C86S/C94S. Disulfide determination as well as CD and NMR spectroscopy of proguanylin-C48S/C61S reveals an essential function of the Cys48-Cys61 disulfide bond for the stability of the hydrophobic core and thereby for the stability of the overall three-dimensional structure of proguanylin. Furthermore, sequence specific resonance assignment of the second disulfide deletion mutant, proguanylin-C86S/C94S, and comparison of the NMR spectra of this protein with those of the wild-type protein demonstrate that the rigid helical core structure of proguanylin is not affected by the mutation. Additionally, analysis of the interstrand contacts between the termini reveals a direct effect of the prosequence on the conformation of the hormone region. On the basis of these results, we propose a cooperative mechanism that leads to formation of the correct disulfide pattern of guanylin.The peptide hormone guanylin is the first identified endogenous ligand of intestinal guanylyl cyclase C (GC-C) (1) and is part of a newly discovered entero-endocrine axis connecting intestine and kidney by regulating electrolyte and fluid secretion in the intestine (2) as well as kaliuresis and diuresis in the kidney (3-6). In the intestine, binding of guanylin to GC-C leads to an increased level of intracellular cGMP that subsequently activates the cystic fibrosis transmembrane conductance regulator chloride channel, resulting in an increased level of secretion of Cl -, HCO 3 -, and water into the intestinal lumen, and simultaneously inhibits sodium absorption (7,8). For GC-C binding and activation, formation of the correct 1-3/2-4 disulfide bond pattern of guanylin is required (1, 9), allowing formation of two interconvertable topological stereoisomers (10), with only one of them (Aisomer) showing biological activity (11).The main circulating form of guanylin is the cor...

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