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

Resumé

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

Citer dette

Jiang, L., Svane, A. S. P., Sørensen, H. P., Jensen, J. K., Hosseini, M., Chen, Z., ... Andreasen, P. (2011). The binding mechanism of a peptidic cyclic serine protease inhibitor. Journal of Molecular Biology, 412(2), 235-250. https://doi.org/10.1016/j.jmb.2011.07.028
Jiang, Longguang ; Svane, Anna Sigrid P. ; Sørensen, Hans Peter ; Jensen, Jan K ; Hosseini, Masood ; Chen, Zhuo ; Weydert, Caroline ; Nielsen, Jakob Toudahl ; Christensen, Anni ; Yuan, Cai ; Jensen, Knud Jørgen ; Nielsen, Niels Chr ; Malmendal, Anders ; Huang, Mingdong ; Andreasen, Peter. / The binding mechanism of a peptidic cyclic serine protease inhibitor. I: Journal of Molecular Biology. 2011 ; Bind 412, Nr. 2. s. 235-250.
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title = "The binding mechanism of a peptidic cyclic serine protease inhibitor",
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.",
author = "Longguang Jiang and Svane, {Anna Sigrid P.} and S{\o}rensen, {Hans Peter} and Jensen, {Jan K} and Masood Hosseini and Zhuo Chen and Caroline Weydert and Nielsen, {Jakob Toudahl} and Anni Christensen and Cai Yuan and Jensen, {Knud J{\o}rgen} and Nielsen, {Niels Chr} and Anders Malmendal and Mingdong Huang and Peter Andreasen",
note = "Copyright {\circledC} 2011 Elsevier Ltd. All rights reserved.",
year = "2011",
doi = "10.1016/j.jmb.2011.07.028",
language = "English",
volume = "412",
pages = "235--250",
journal = "Journal of Molecular Biology",
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Jiang, L, Svane, ASP, Sørensen, HP, Jensen, JK, Hosseini, M, Chen, Z, Weydert, C, Nielsen, JT, Christensen, A, Yuan, C, Jensen, KJ, Nielsen, NC, Malmendal, A, Huang, M & Andreasen, P 2011, 'The binding mechanism of a peptidic cyclic serine protease inhibitor', Journal of Molecular Biology, bind 412, nr. 2, s. 235-250. https://doi.org/10.1016/j.jmb.2011.07.028

The binding mechanism of a peptidic cyclic serine protease inhibitor. / Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter; Jensen, Jan K; Hosseini, Masood; Chen, Zhuo; Weydert, Caroline; Nielsen, Jakob Toudahl; Christensen, Anni; Yuan, Cai; Jensen, Knud Jørgen; Nielsen, Niels Chr; Malmendal, Anders; Huang, Mingdong; Andreasen, Peter.

I: Journal of Molecular Biology, Bind 412, Nr. 2, 2011, s. 235-250.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - The binding mechanism of a peptidic cyclic serine protease inhibitor

AU - Jiang, Longguang

AU - Svane, Anna Sigrid P.

AU - Sørensen, Hans Peter

AU - Jensen, Jan K

AU - Hosseini, Masood

AU - Chen, Zhuo

AU - Weydert, Caroline

AU - Nielsen, Jakob Toudahl

AU - Christensen, Anni

AU - Yuan, Cai

AU - Jensen, Knud Jørgen

AU - Nielsen, Niels Chr

AU - Malmendal, Anders

AU - Huang, Mingdong

AU - Andreasen, Peter

N1 - Copyright © 2011 Elsevier Ltd. All rights reserved.

PY - 2011

Y1 - 2011

N2 - 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.

AB - 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.

U2 - 10.1016/j.jmb.2011.07.028

DO - 10.1016/j.jmb.2011.07.028

M3 - Journal article

VL - 412

SP - 235

EP - 250

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -

Jiang L, Svane ASP, Sørensen HP, Jensen JK, Hosseini M, Chen Z et al. The binding mechanism of a peptidic cyclic serine protease inhibitor. Journal of Molecular Biology. 2011;412(2):235-250. https://doi.org/10.1016/j.jmb.2011.07.028