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PEDS Advance Access originally published online on November 5, 2008
Protein Engineering Design and Selection 2009 22(1):1-7; doi:10.1093/protein/gzn062
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C

T. Lauber1,5, N. Tidten1,5, I. Matecko1, M. Zeeb2,3, P. Rösch1,6 and U.C. Marx1,4,6

1Lehrstuhl für Biopolymere and Forschungszentrum für Bio-Makromoleküle 2Lehrstuhl für Biochemie, Universität Bayreuth 95440, Bayreuth

6 To whom correspondence should be addressed. E-mail: u.marx{at}imb.uq.edu.au (U.C.M.)/paul.roesch{at}uni-bayreuth.de (P.R.)

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.

Keywords: guanylate cyclase-C/heat-stable enterotoxin STa/membrane protein/peptide hormone receptor/uroguanylin

Received February 19, 2008; revised October 7, 2008; accepted October 8, 2008.

3 Present address: Department of Lead Discovery and Structural Research, Boehringer Ingelheim Pharma GmbH & Co. KG, D-88397 Biberach an der Riss, Germany

4 Present address: Institute for Molecular Bioscience, SRC for Functional and Applied Genomics, The University of Queensland, Brisbane, QLD 4072, Australia

5 Both authors contributed equally to this work.


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