A cyclic peptidic serine protease inhibitor: increasing affinity by increasing Peptide flexibility

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Standard

A cyclic peptidic serine protease inhibitor : increasing affinity by increasing Peptide flexibility. / Zhao, Baoyu; Xu, Peng; Jiang, Longguang; Paaske, Berit; Kromann-Hansen, Tobias; Jensen, Jan K; Sørensen, Hans Peter; Liu, Zhuo; Nielsen, Jakob Toudahl; Christensen, Anni; Hosseini, Masood; Sørensen, Kasper K; Nielsen, Niels Christian; Jensen, Knud J.; Huang, Mingdong; Andreasen, Peter A.

I: PLOS ONE, Bind 9, Nr. 12, e115872, 2014.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Harvard

Zhao, B, Xu, P, Jiang, L, Paaske, B, Kromann-Hansen, T, Jensen, JK, Sørensen, HP, Liu, Z, Nielsen, JT, Christensen, A, Hosseini, M, Sørensen, KK, Nielsen, NC, Jensen, KJ, Huang, M & Andreasen, PA 2014, 'A cyclic peptidic serine protease inhibitor: increasing affinity by increasing Peptide flexibility', PLOS ONE, bind 9, nr. 12, e115872. https://doi.org/10.1371/journal.pone.0115872

APA

Zhao, B., Xu, P., Jiang, L., Paaske, B., Kromann-Hansen, T., Jensen, J. K., ... Andreasen, P. A. (2014). A cyclic peptidic serine protease inhibitor: increasing affinity by increasing Peptide flexibility. PLOS ONE, 9(12), [e115872]. https://doi.org/10.1371/journal.pone.0115872

CBE

Zhao B, Xu P, Jiang L, Paaske B, Kromann-Hansen T, Jensen JK, Sørensen HP, Liu Z, Nielsen JT, Christensen A, Hosseini M, Sørensen KK, Nielsen NC, Jensen KJ, Huang M, Andreasen PA. 2014. A cyclic peptidic serine protease inhibitor: increasing affinity by increasing Peptide flexibility. PLOS ONE. 9(12). https://doi.org/10.1371/journal.pone.0115872

MLA

Vancouver

Author

Zhao, Baoyu ; Xu, Peng ; Jiang, Longguang ; Paaske, Berit ; Kromann-Hansen, Tobias ; Jensen, Jan K ; Sørensen, Hans Peter ; Liu, Zhuo ; Nielsen, Jakob Toudahl ; Christensen, Anni ; Hosseini, Masood ; Sørensen, Kasper K ; Nielsen, Niels Christian ; Jensen, Knud J. ; Huang, Mingdong ; Andreasen, Peter A. / A cyclic peptidic serine protease inhibitor : increasing affinity by increasing Peptide flexibility. I: PLOS ONE. 2014 ; Bind 9, Nr. 12.

Bibtex

@article{e074fe196c6b4085a42cdec1e6558b95,
title = "A cyclic peptidic serine protease inhibitor: increasing affinity by increasing Peptide flexibility",
abstract = "Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.",
author = "Baoyu Zhao and Peng Xu and Longguang Jiang and Berit Paaske and Tobias Kromann-Hansen and Jensen, {Jan K} and S{\o}rensen, {Hans Peter} and Zhuo Liu and Nielsen, {Jakob Toudahl} and Anni Christensen and Masood Hosseini and S{\o}rensen, {Kasper K} and Nielsen, {Niels Christian} and Jensen, {Knud J.} and Mingdong Huang and Andreasen, {Peter A}",
year = "2014",
doi = "10.1371/journal.pone.0115872",
language = "English",
volume = "9",
journal = "P L o S One",
issn = "1932-6203",
publisher = "public library of science",
number = "12",

}

RIS

TY - JOUR

T1 - A cyclic peptidic serine protease inhibitor

T2 - increasing affinity by increasing Peptide flexibility

AU - Zhao, Baoyu

AU - Xu, Peng

AU - Jiang, Longguang

AU - Paaske, Berit

AU - Kromann-Hansen, Tobias

AU - Jensen, Jan K

AU - Sørensen, Hans Peter

AU - Liu, Zhuo

AU - Nielsen, Jakob Toudahl

AU - Christensen, Anni

AU - Hosseini, Masood

AU - Sørensen, Kasper K

AU - Nielsen, Niels Christian

AU - Jensen, Knud J.

AU - Huang, Mingdong

AU - Andreasen, Peter A

PY - 2014

Y1 - 2014

N2 - Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

AB - Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

U2 - 10.1371/journal.pone.0115872

DO - 10.1371/journal.pone.0115872

M3 - Journal article

C2 - 25545505

VL - 9

JO - P L o S One

JF - P L o S One

SN - 1932-6203

IS - 12

M1 - e115872

ER -