Allosteric-Activation Mechanism of Bovine Chymosin Revealed by Bias-Exchange Metadynamics and Molecular Dynamics Simulations

Abstract

The aspartic protease, bovine chymosin, catalyzes the proteolysis of κ-casein proteins in milk. The bovine chymosin-κ-casein complex is of industrial interest as the enzyme is used extensively 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.

On the effect of mutations in bovine or camel chymosin on the thermodynamics of binding κ‐caseins

“…The α‐helical region containing Asp112Glu has been implicated in allosteric activation of bovine chymosin by the P8‐P4 residues of bovine κ‐casein . In this mechanism, the His‐Pro cluster (HPHPH in P8‐P4 of bovine κ‐casein) interacts with the α‐helix, which both allows the β‐hairpin flap in residues 72–84 of chymosin to twist and causes the side‐chain of Phe114 to vacate a pocket that is occupied by Tyr77 in the open conformation.…”

“…The results suggest that the Gln residue observed in wildtype bovine enzyme is more favored for binding than the Arg residue from its camel counterpart. The role of the residues in the S9 pocket has not previously been defined, but it may be to help orientate the neighboring P8‐P4 residues of κ‐casein, which have been implicated by both experimental and modeling studies in the allosteric activation of chymosin …”

“…The side chains of the catalytic aspartic acid residues extend toward each other in a planar geometry, which is stabilized by a network of hydrogen bonds with two threonine residues, referred to as “ the fireman's grip ” . Similar features are found in other homologous aspartic proteases …”

Allosteric Regulation at the Crossroads of New Technologies: Multiscale Modeling, Networks, and Machine Learning

“…These NMR-guided simulation techniques have enabled adequate sampling of the conformational space and robust structure reconstruction using experimental constraints (Robustelli et al, 2010 ; Cavalli et al, 2011 ; Granata et al, 2013 ). NMR chemical shift observables can be also used in combination with other collective variables in meta-dynamics simulations to guide the efficient exploration of allosteric states and functional transitions (Kimanius et al, 2015 ; Ansari et al, 2016 ).…”

Exploring high‐dimensional free energy landscapes of chemical reactions

“…A number of studies used BEMetaD to study protein folding . Literature of BEMetaD also includes conformational sampling in α ‐helical glycoproteins, Bovine Chymosin, peptides, conformational sampling of intrinsically disordered as well as other proteins, conduction through ion channels, and protein aggregation . The method has also helped to resolve the structure of large and complex systems such as protein–RNA complex, and membrane inserted influenza fusion peptide .…”