KLK4 Inhibition by Cyclic and Acyclic Peptides

Structural and Dynamical Insights into Standard-Mechanism Protease Inhibitors

Blake T. Riley, Olga Ilyichova, Simon J. De Veer, Joakim E. Swedberg, Emily Wilson, David E. Hoke, Jonathan M. Harris, Ashley M. Buckle

Research output: Contribution to journalArticleResearchpeer-review

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Abstract

Sunflower trypsin inhibitor (SFTI-1) is a 14 amino acid serine protease inhibitor. The dual antiparallel β-sheet arrangement of SFTI-1 is stabilized by an N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favor initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between the cleaved and uncleaved inhibitor in the presence of a protease. We have determined, at 1.15 Å resolution, the X-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14) and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of the altered contact sequence, intramolecular hydrogen bonding network, and backbone cyclization in altering the state of SFTI's scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition and suggest that modifications on the non-contact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.

Original languageEnglish
Pages (from-to)2524-2533
Number of pages10
JournalBiochemistry
Volume58
Issue number21
DOIs
Publication statusPublished - 28 May 2019

Cite this

Riley, Blake T. ; Ilyichova, Olga ; De Veer, Simon J. ; Swedberg, Joakim E. ; Wilson, Emily ; Hoke, David E. ; Harris, Jonathan M. ; Buckle, Ashley M. / KLK4 Inhibition by Cyclic and Acyclic Peptides : Structural and Dynamical Insights into Standard-Mechanism Protease Inhibitors. In: Biochemistry. 2019 ; Vol. 58, No. 21. pp. 2524-2533.
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title = "KLK4 Inhibition by Cyclic and Acyclic Peptides: Structural and Dynamical Insights into Standard-Mechanism Protease Inhibitors",
abstract = "Sunflower trypsin inhibitor (SFTI-1) is a 14 amino acid serine protease inhibitor. The dual antiparallel β-sheet arrangement of SFTI-1 is stabilized by an N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favor initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between the cleaved and uncleaved inhibitor in the presence of a protease. We have determined, at 1.15 {\AA} resolution, the X-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14) and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of the altered contact sequence, intramolecular hydrogen bonding network, and backbone cyclization in altering the state of SFTI's scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition and suggest that modifications on the non-contact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.",
author = "Riley, {Blake T.} and Olga Ilyichova and {De Veer}, {Simon J.} and Swedberg, {Joakim E.} and Emily Wilson and Hoke, {David E.} and Harris, {Jonathan M.} and Buckle, {Ashley M.}",
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KLK4 Inhibition by Cyclic and Acyclic Peptides : Structural and Dynamical Insights into Standard-Mechanism Protease Inhibitors. / Riley, Blake T.; Ilyichova, Olga; De Veer, Simon J.; Swedberg, Joakim E.; Wilson, Emily; Hoke, David E.; Harris, Jonathan M.; Buckle, Ashley M.

In: Biochemistry, Vol. 58, No. 21, 28.05.2019, p. 2524-2533.

Research output: Contribution to journalArticleResearchpeer-review

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T2 - Structural and Dynamical Insights into Standard-Mechanism Protease Inhibitors

AU - Riley, Blake T.

AU - Ilyichova, Olga

AU - De Veer, Simon J.

AU - Swedberg, Joakim E.

AU - Wilson, Emily

AU - Hoke, David E.

AU - Harris, Jonathan M.

AU - Buckle, Ashley M.

PY - 2019/5/28

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N2 - Sunflower trypsin inhibitor (SFTI-1) is a 14 amino acid serine protease inhibitor. The dual antiparallel β-sheet arrangement of SFTI-1 is stabilized by an N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favor initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between the cleaved and uncleaved inhibitor in the presence of a protease. We have determined, at 1.15 Å resolution, the X-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14) and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of the altered contact sequence, intramolecular hydrogen bonding network, and backbone cyclization in altering the state of SFTI's scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition and suggest that modifications on the non-contact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.

AB - Sunflower trypsin inhibitor (SFTI-1) is a 14 amino acid serine protease inhibitor. The dual antiparallel β-sheet arrangement of SFTI-1 is stabilized by an N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favor initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between the cleaved and uncleaved inhibitor in the presence of a protease. We have determined, at 1.15 Å resolution, the X-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14) and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of the altered contact sequence, intramolecular hydrogen bonding network, and backbone cyclization in altering the state of SFTI's scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition and suggest that modifications on the non-contact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.

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U2 - 10.1021/acs.biochem.9b00191

DO - 10.1021/acs.biochem.9b00191

M3 - Article

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EP - 2533

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JF - Biochemistry

SN - 0006-2960

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