The SEP domain of p47 acts as a reversible competitive inhibitor of cathepsin L

Edited by Amy McGough

Received 20 July 2004; received in revised form 13 September 2004; accepted 13 September 2004. published online 27 September 2004.

Abstract

The solution structure of the human p47 SEP domain in a construct comprising residues G1-S2-p47(171–270) was determined by NMR spectroscopy. A structure-derived hypothesis about the domains' function was formulated and pursued in binding experiments with cysteine proteases. The SEP domain was found to be a reversible competitive inhibitor of cathepsin L with a Ki of 1.5 μM. The binding of G1-S2-p47(171–270) to cathepsin L was mapped by biochemical assays and the binding interface was investigated by NMR chemical shift perturbation experiments.

Abbreviations: NaPi, sodium phosphate buffer, SEP, Saccharomyces cerevisiae SHP1, Drosophila melanogaster eyes closed gene, vertebrate p47, NOE, nuclear Overhauser effect, CSP, chemical shift perturbations

Keywords: SEP, p97, p47, Cathepsin L, NMR, Protein structure

a Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle Str. 10, D-13125 Berlin, Germany

b Proteinstrukturfabrik, Berlin, Germany

c Max Planck Institut für Molekulare Genetik, Berlin, Germany

d Freie Universitaet Berlin, Berlin, Germany

e Department of Clinical Chemistry and Clinical Biochemistry, LMU Munich, Germany

Corresponding author. Fax: +493094793169

1 Present address: Structural Genomics Consortium, University of Oxford, Botnar Research Centre, OX37LD, UK.

PII: S0014-5793(04)01153-6

doi:10.1016/j.febslet.2004.09.037

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