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PEDS Advance Access originally published online on September 9, 2004
Protein Engineering Design and Selection 2004 17(8):647-657; doi:10.1093/protein/gzh073
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Protein Engineering, Design & Selection vol. 17 no. 8 © Oxford University Press 2004; all rights reserved

Rational design and selection of bivalent peptide ligands of thrombin incorporating P4–P1 tetrapeptide sequences: from good substrates to potent inhibitors

Zhengding Su, Anna Vinogradova, Anatol Koutychenko, Dmitri Tolkatchev and Feng Ni1

Biomolecular NMR and Protein Research Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2

1 To whom correspondence should be addressed. E-mail: feng.ni{at}bri.nrc.ca

The tetrapeptide Phe-Asn-Pro-Arg is a structurally optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or some variants to a C-terminal fragment of hirudin, we were able to generate a series of new bivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P3 and P'3 sites of the active-site directed sequence, Phe(P4)-Xaa(P3)-Pro(P2)-Arg(P1)-Pro(P'1)-Gln(P'2)-Yaa(P'3). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P'3 sites for bivalent and optimized two-site binding. Very significantly, panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-clotting activities in the low nanomolar range and yet interfering only partially the catalytic active site of thrombin. Modes of action of the newly discovered bivalent inhibitors are rationalized in light of the allosteric properties of thrombin, especially the interplay between the proteolytic action and regulatory binding occurring at thrombin surfaces remote from the catalytic active site.

Received August 30, 2004; accepted September 6, 2004.

Edited by Andre Menez


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