Abstract
The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.
Highlights
Infections with enterotoxigenic strains of Escherichia coli are the cause of traveler’s diarrhea, but are a major cause of infant mortality in many developing countries (Levine et al, 1977; Field and Semrad, 1993; Farthing, 1994; Orndorff et al, 1996; WHO Fact Sheets, 1996; Qadri et al, 2005)
Activation of guanylyl cyclase-C (GC-C) in the intestinal mucosa stimulates transepithelial Cl2 and HCO32 secretion via cGMP as a second messenger through the opening of apical CFTR Cl2 channels, and causes inhibition of Na+ absorption (Gianella, 1995; Vaandrager, 2002). These effects are accompanied by an enhanced secretion of fluid into the intestinal lumen, providing an explanation for the severe diarrhea caused by bacterial stable enterotoxins (STa), which stimulate GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin (Currie et al, 1992; Carpick and Gariepy, 1993; Klodt et al, 1997; Forte, 1999; Qadri et al, 2005)
The structural characterization of the guanylin(uroguanylin/ STa)/GC-C system is of high pharmacological interest
Summary
Infections with enterotoxigenic strains of Escherichia coli are the cause of traveler’s diarrhea, but are a major cause of infant mortality in many developing countries (Levine et al, 1977; Field and Semrad, 1993; Farthing, 1994; Orndorff et al, 1996; WHO Fact Sheets, 1996; Qadri et al, 2005). Based on our model structure of the GC-CECD, we have designed, constructed, overexpressed and characterized a soluble and properly folded receptor fragment (miniGC-C, 197 amino acid residues) that is able to bind the ligand STa-(5-17) with nanomolar affinity. These results provide the basis for the investigation of a peptide hormone/receptor system under membrane-free conditions
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