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

doi:10.1016/j.jmb.2011.07.028

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

16 Citations (Scopus)

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.

Original language	English
Journal	Journal of Molecular Biology
Volume	412
Issue number	2
Pages (from-to)	235-250
Number of pages	16
ISSN	0022-2836
DOIs	https://doi.org/10.1016/j.jmb.2011.07.028
Publication status	Published - 16 Sept 2011

Keywords

Amino Acid Sequence
Humans
Molecular Sequence Data
Peptides, Cyclic
Protein Binding
Serine Proteinase Inhibitors
Surface Plasmon Resonance

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10.1016/j.jmb.2011.07.028

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Jiang, L., Svane, A. S. P., Sørensen, H. P., Jensen, J. K., Hosseini, M., Chen, Z., Weydert, C., Nielsen, J. T., Christensen, A., Yuan, C., Jensen, K. J., Nielsen, N. C., Malmendal, A., Huang, M., & 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

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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.",

keywords = "Amino Acid Sequence, Humans, Molecular Sequence Data, Peptides, Cyclic, Protein Binding, Serine Proteinase Inhibitors, Surface Plasmon Resonance",

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",

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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

PY - 2011/9/16

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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.

KW - Amino Acid Sequence

KW - Humans

KW - Molecular Sequence Data

KW - Peptides, Cyclic

KW - Protein Binding

KW - Serine Proteinase Inhibitors

KW - Surface Plasmon Resonance

U2 - 10.1016/j.jmb.2011.07.028

DO - 10.1016/j.jmb.2011.07.028

M3 - Journal article

C2 - 21802428

SN - 0022-2836

VL - 412

SP - 235

EP - 250

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 2

ER -

The binding mechanism of a peptidic cyclic serine protease inhibitor

Abstract

Keywords

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