Reactive centre loop dynamics and serpin specificity

Emilia M. Marijanovic, James Fodor, Blake T. Riley, Benjamin T. Porebski, Mauricio G.S. Costa, Itamar Kass, David E. Hoke, Sheena McGowan, Ashley M. Buckle

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α 1 -antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

Original languageEnglish
Article number3870
Number of pages15
JournalScientific Reports
Volume9
Issue number1
DOIs
Publication statusPublished - 7 Mar 2019

Keywords

  • blood proteins
  • computational biophysics
  • molecular conformation
  • protein design

Cite this

Marijanovic, Emilia M. ; Fodor, James ; Riley, Blake T. ; Porebski, Benjamin T. ; Costa, Mauricio G.S. ; Kass, Itamar ; Hoke, David E. ; McGowan, Sheena ; Buckle, Ashley M. / Reactive centre loop dynamics and serpin specificity. In: Scientific Reports. 2019 ; Vol. 9, No. 1.
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Reactive centre loop dynamics and serpin specificity. / Marijanovic, Emilia M.; Fodor, James; Riley, Blake T.; Porebski, Benjamin T.; Costa, Mauricio G.S.; Kass, Itamar; Hoke, David E.; McGowan, Sheena; Buckle, Ashley M.

In: Scientific Reports, Vol. 9, No. 1, 3870, 07.03.2019.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Reactive centre loop dynamics and serpin specificity

AU - Marijanovic, Emilia M.

AU - Fodor, James

AU - Riley, Blake T.

AU - Porebski, Benjamin T.

AU - Costa, Mauricio G.S.

AU - Kass, Itamar

AU - Hoke, David E.

AU - McGowan, Sheena

AU - Buckle, Ashley M.

PY - 2019/3/7

Y1 - 2019/3/7

N2 - Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α 1 -antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

AB - Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α 1 -antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

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M3 - Article

VL - 9

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

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Marijanovic EM, Fodor J, Riley BT, Porebski BT, Costa MGS, Kass I et al. Reactive centre loop dynamics and serpin specificity. Scientific Reports. 2019 Mar 7;9(1). 3870. https://doi.org/10.1038/s41598-019-40432-w