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Protein Science (2004), 13:626-632. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Directed discovery of bivalent peptide ligands to an SH3 domain

Monique R. Ferguson1,3,4, Xiuzhen Fan1,3, Munia Mukherjee1,3, Jinquan Luo1,5, Raza Khan1,6, Josephine C. Ferreon1, Vincent J. Hilser1, Robert E. Shope2 and Robert O. Fox1

1 Department of Human Biological Chemistry and Genetics, and Sealy Center for Structural Biology, and
2 Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, and Sealy Center for Vaccine Development, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555, USA

(RECEIVED October 2, 2003; FINAL REVISION December 5, 2003; ACCEPTED December 5, 2003)



Abstract

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G4-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the {beta}-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67phox SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G4-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.

Keywords: SH3 domain; signal transduction; phage display; combinatorial library; peptide ligand; bivalent ligand; NMR spectroscopy; non-PXXP binding site

Reprint requests to: Robert O. Fox, Department of Human Biological Chemistry and Genetics, 301 University Blvd., Mail Route 0647, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-0647, USA; e-mail: fox{at}bloch.utmb.edu; fax: (409) 747-4745.

3 These authors contributed equally to this work.

4 Present addresses: The Department of Internal Medicine, The Division of Infectious Diseases, The University of Texas Medical Branch, Galveston, TX 77555-0435, USA;

5 Centocor, Inc., Johnson and Johnson, 200 Great Valley Parkway, M/S R-3-1, Malvern, PA 19355, USA;

6 Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03470504.


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