Allosteric activation mechanism of bovine chymosin revealed by bias-exchange metadynamics and molecular dynamics simulations

Samiul M. Ansari, Andrea Coletta, Katrine Kirkeby Skeby, Jesper Sørensen, Birgit Schiøtt, David S. Palmer

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

The aspartic protease, bovine chymosin, catalyses the proteolysis of κ-casein proteins in milk. The bovine chymosin–κ-casein complex is of industrial interest as the enzyme is widely employed in the manufacturing of processed dairy products. The apo form of the enzyme adopts a self-inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis and crystallographic data, it has been widely reported that a HPHPH sequence in the P8-P4 residues of the natural substrate κ-casein acts as the allosteric activator, but the mechanism by which this occurs has not previously been elucidated due to the challenges associated with studying this process by experimental methods. Here we have employed two computational techniques, molecular dynamics and bias exchange metadynamics simulations, to study the mechanism of allosteric activation and to compute the free energy surface for the process. The simulations reveal that allosteric activation is initiated by interactions between the HPHPH sequence of κ-casein and a small α-helical region of chymosin (residues 112-116). A small conformational change in the α-helix causes the side chain of Phe114 to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open transition is significantly altered by the presence of the HPHPH sequence of κ-casein.
LanguageEnglish
Pages10453-10462
Number of pages10
JournalJournal of Physical Chemistry B
Volume120
Issue number40
Early online date14 Sep 2016
DOIs
Publication statusE-pub ahead of print - 14 Sep 2016

Fingerprint

Chymosin
chymosin
Casein
kappa-casein
molecular dynamics
Molecular Dynamics Simulation
Caseins
Molecular dynamics
Ion exchange
Chemical activation
activation
enzymes
cattle
Computer simulation
free energy
mutagenesis
protease
milk
simulation
Free energy

Keywords

  • chymosin
  • molecular dynamics
  • bias-exchange metadynamics
  • cheese production
  • computational chemistry
  • aspartic protease
  • enzyme
  • proteolysis
  • k-casein
  • milk
  • casein micelles

Cite this

Ansari, Samiul M. ; Coletta, Andrea ; Kirkeby Skeby, Katrine ; Sørensen, Jesper ; Schiøtt, Birgit ; Palmer, David S. / Allosteric activation mechanism of bovine chymosin revealed by bias-exchange metadynamics and molecular dynamics simulations. In: Journal of Physical Chemistry B. 2016 ; Vol. 120, No. 40. pp. 10453-10462.
@article{a5466205950148fc969f599b166f28cd,
title = "Allosteric activation mechanism of bovine chymosin revealed by bias-exchange metadynamics and molecular dynamics simulations",
abstract = "The aspartic protease, bovine chymosin, catalyses the proteolysis of κ-casein proteins in milk. The bovine chymosin–κ-casein complex is of industrial interest as the enzyme is widely employed in the manufacturing of processed dairy products. The apo form of the enzyme adopts a self-inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis and crystallographic data, it has been widely reported that a HPHPH sequence in the P8-P4 residues of the natural substrate κ-casein acts as the allosteric activator, but the mechanism by which this occurs has not previously been elucidated due to the challenges associated with studying this process by experimental methods. Here we have employed two computational techniques, molecular dynamics and bias exchange metadynamics simulations, to study the mechanism of allosteric activation and to compute the free energy surface for the process. The simulations reveal that allosteric activation is initiated by interactions between the HPHPH sequence of κ-casein and a small α-helical region of chymosin (residues 112-116). A small conformational change in the α-helix causes the side chain of Phe114 to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open transition is significantly altered by the presence of the HPHPH sequence of κ-casein.",
keywords = "chymosin, molecular dynamics, bias-exchange metadynamics, cheese production, computational chemistry, aspartic protease, enzyme, proteolysis, k-casein, milk, casein micelles",
author = "Ansari, {Samiul M.} and Andrea Coletta and {Kirkeby Skeby}, Katrine and Jesper S{\o}rensen and Birgit Schi{\o}tt and Palmer, {David S.}",
note = "This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Physical Chemistry B, copyright {\circledC} American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acs.jpcb.6b07491",
year = "2016",
month = "9",
day = "14",
doi = "10.1021/acs.jpcb.6b07491",
language = "English",
volume = "120",
pages = "10453--10462",
journal = "Journal of Physical Chemistry B",
issn = "1520-6106",
publisher = "American Chemical Society",
number = "40",

}

Allosteric activation mechanism of bovine chymosin revealed by bias-exchange metadynamics and molecular dynamics simulations. / Ansari, Samiul M.; Coletta, Andrea; Kirkeby Skeby, Katrine; Sørensen, Jesper; Schiøtt, Birgit; Palmer, David S.

In: Journal of Physical Chemistry B, Vol. 120, No. 40, 14.09.2016, p. 10453-10462.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Allosteric activation mechanism of bovine chymosin revealed by bias-exchange metadynamics and molecular dynamics simulations

AU - Ansari, Samiul M.

AU - Coletta, Andrea

AU - Kirkeby Skeby, Katrine

AU - Sørensen, Jesper

AU - Schiøtt, Birgit

AU - Palmer, David S.

N1 - This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Physical Chemistry B, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acs.jpcb.6b07491

PY - 2016/9/14

Y1 - 2016/9/14

N2 - The aspartic protease, bovine chymosin, catalyses the proteolysis of κ-casein proteins in milk. The bovine chymosin–κ-casein complex is of industrial interest as the enzyme is widely employed in the manufacturing of processed dairy products. The apo form of the enzyme adopts a self-inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis and crystallographic data, it has been widely reported that a HPHPH sequence in the P8-P4 residues of the natural substrate κ-casein acts as the allosteric activator, but the mechanism by which this occurs has not previously been elucidated due to the challenges associated with studying this process by experimental methods. Here we have employed two computational techniques, molecular dynamics and bias exchange metadynamics simulations, to study the mechanism of allosteric activation and to compute the free energy surface for the process. The simulations reveal that allosteric activation is initiated by interactions between the HPHPH sequence of κ-casein and a small α-helical region of chymosin (residues 112-116). A small conformational change in the α-helix causes the side chain of Phe114 to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open transition is significantly altered by the presence of the HPHPH sequence of κ-casein.

AB - The aspartic protease, bovine chymosin, catalyses the proteolysis of κ-casein proteins in milk. The bovine chymosin–κ-casein complex is of industrial interest as the enzyme is widely employed in the manufacturing of processed dairy products. The apo form of the enzyme adopts a self-inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis and crystallographic data, it has been widely reported that a HPHPH sequence in the P8-P4 residues of the natural substrate κ-casein acts as the allosteric activator, but the mechanism by which this occurs has not previously been elucidated due to the challenges associated with studying this process by experimental methods. Here we have employed two computational techniques, molecular dynamics and bias exchange metadynamics simulations, to study the mechanism of allosteric activation and to compute the free energy surface for the process. The simulations reveal that allosteric activation is initiated by interactions between the HPHPH sequence of κ-casein and a small α-helical region of chymosin (residues 112-116). A small conformational change in the α-helix causes the side chain of Phe114 to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open transition is significantly altered by the presence of the HPHPH sequence of κ-casein.

KW - chymosin

KW - molecular dynamics

KW - bias-exchange metadynamics

KW - cheese production

KW - computational chemistry

KW - aspartic protease

KW - enzyme

KW - proteolysis

KW - k-casein

KW - milk

KW - casein micelles

UR - http://pubs.acs.org/journal/jpcbfk

U2 - 10.1021/acs.jpcb.6b07491

DO - 10.1021/acs.jpcb.6b07491

M3 - Article

VL - 120

SP - 10453

EP - 10462

JO - Journal of Physical Chemistry B

T2 - Journal of Physical Chemistry B

JF - Journal of Physical Chemistry B

SN - 1520-6106

IS - 40

ER -