The binding mechanism of a peptidic cyclic serine protease inhibitor

Longguang Jiang, Anna Sigrid P. Svane, Hans Peter Sørensen, Jan K Jensen, Masood Hosseini, Zhuo Chen, Caroline Weydert, Jakob Toudahl Nielsen, Anni Christensen, Cai Yuan, Knud Jørgen Jensen, Niels Chr Nielsen, Anders Malmendal, Mingdong Huang, Peter Andreasen

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Abstract

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1 are strongly influenced by the addition of three to four amino acids long N-terminal and C-terminal extensions to the core, disulfide-bond-constrained sequence: The C-terminal extension stabilises the solution structure compared to the core peptide alone, and the protease-bound structure of the peptide is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high specificity and new inhibitory mechanisms.
OriginalsprogEngelsk
TidsskriftJournal of Molecular Biology
Vol/bind412
Udgave nummer2
Sider (fra-til)235-250
Antal sider16
ISSN0022-2836
DOI
StatusUdgivet - 2011
Udgivet eksterntJa

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