title: Role of Enzyme Flexibility in Ligand Access and Egress to Active Site: Bias-Exchange Metadynamics Study of 1,3,7-Trimethyluric Acid in Cytochrome P450 3A4.
abstract: Although the majority of enzymes have buried active sites, very little is known about the energetics and mechanisms associated with substrate and product channeling in and out. Gaining direct information about these processes is a challenging task both for experimental and theoretical techniques. Here, we present a methodology that enables following of a ligand during its passage to the active site of cytochrome P450 (CYP) 3A4 and mapping of the free energy associated with this process. The technique is based on a combination of a bioinformatics tool for identifying access channels and bias-exchange metadynamics and provides converged free energies in good agreement with experimental data. In addition, it identifies the energetically preferred escape routes, limiting steps, and amino acids residues lining the channel. The approach was applied to mapping of a complex channel network in a complex environment, i.e., CYP3A4 attached to a lipid bilayer mimicking an endoplasmic reticulum membrane. The results provided direct information about the energetics and conformational changes associated with the ligand channeling. The methodology can easily be adapted to study channeling through other flexible biomacromolecular channels.
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc , tř. 17 Listopadu 12, 771 46 Olomouc, Czech Republic.
authors.forename: Markéta
authors.initials: M
authors.lastname: Paloncýová
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc , tř. 17 Listopadu 12, 771 46 Olomouc, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Navrátilová
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc , tř. 17 Listopadu 12, 771 46 Olomouc, Czech Republic.
authors.forename: Karel
authors.initials: K
authors.lastname: Berka
authors.affiliation: SISSA - Scuola Internazionale Superiore di Studi Avanzati, via Bonomea 265, 34136 Trieste, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Laio
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc , tř. 17 Listopadu 12, 771 46 Olomouc, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Otyepka
mesh_terms: D020134:Catalytic Domain; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D013816:Thermodynamics; D014527:Uric Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D014527:Uric Acid; C039222:1,3,7-trimethyluric acid; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human