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Lauber, Thomas ; Tidten, Naomi ; Matecko, Irena ; Zeeb, Markus ; Rösch, Paul ; Marx, Ute C.:
Design and Characterization of a Soluble Fragment of the Extracellular Ligand Binding Domain of the Peptide Hormone Receptor Guanylyl Cyclase-C.
In: Protein Engineering, Design & Selection.
Bd. 22
(2009)
Heft 1
.
- S. 1-7.
ISSN 1741-0134
DOI: https://doi.org/10.1093/protein/gzn062
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.