Dynamics of CYP51: implications for function and inhibitor design

Sterol 14α‐demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane‐bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane‐bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen‐bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes. Copyright © 2015 John Wiley & Sons, Ltd.

[1]  M. Waterman,et al.  Structural Insights into Inhibition of Sterol 14α-Demethylase in the Human Pathogen Trypanosoma cruzi* , 2010, The Journal of Biological Chemistry.

[2]  S. Kawato,et al.  Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study. , 1992, Biochemistry.

[3]  D. Tieleman,et al.  The MARTINI force field: coarse grained model for biomolecular simulations. , 2007, The journal of physical chemistry. B.

[4]  S. Sligar,et al.  Structural differences between soluble and membrane bound cytochrome P450s. , 2012, Journal of inorganic biochemistry.

[5]  M. Waterman,et al.  Crystal Structures of Trypanosoma brucei Sterol 14α-Demethylase and Implications for Selective Treatment of Human Infections*♦ , 2009, The Journal of Biological Chemistry.

[6]  Jeremy C. Smith,et al.  Coupled flexibility change in cytochrome P450cam substrate binding determined by neutron scattering, NMR, and molecular dynamics simulation. , 2012, Biophysical journal.

[7]  Berk Hess,et al.  GROMACS 3.0: a package for molecular simulation and trajectory analysis , 2001 .

[8]  R. Larson,et al.  The MARTINI Coarse-Grained Force Field: Extension to Proteins. , 2008, Journal of chemical theory and computation.

[9]  Karel Berka,et al.  Dynamics and hydration of the active sites of mammalian cytochromes P450 probed by molecular dynamics simulations. , 2012, Current drug metabolism.

[10]  Y. Yoshida.  Cytochrome P450 of fungi: primary target for azole antifungal agents. , 1988, Current topics in medical mycology.

[11]  William L. Jorgensen,et al.  Quantum and statistical mechanical studies of liquids. 25. Solvation and conformation of methanol in water , 1983 .

[12]  K. Berka,et al.  Behavior of human cytochromes P450 on lipid membranes. , 2013, The journal of physical chemistry. B.

[13]  A. Mark,et al.  Coarse grained model for semiquantitative lipid simulations , 2004 .

[14]  M. Waterman,et al.  Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol[S] , 2012, Journal of Lipid Research.

[15]  Victor Guallar,et al.  Peripheral heme substituents control the hydrogen-atom abstraction chemistry in cytochromes P450 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[16]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[17]  J Deisenhofer,et al.  Structure and function of cytochromes P450: a comparative analysis of three crystal structures. , 1995, Structure.

[18]  D. Nelson,et al.  Cytochrome P450 and the individuality of species. , 1999, Archives of biochemistry and biophysics.

[19]  Gerrit Groenhof,et al.  GROMACS: Fast, flexible, and free , 2005, J. Comput. Chem..

[20]  Piotr Cieplak,et al.  The R.E.D. tools: advances in RESP and ESP charge derivation and force field library building. , 2010, Physical chemistry chemical physics : PCCP.

[21]  Emad Tajkhorshid,et al.  Characterizing the Membrane-Bound State of Cytochrome P450 3A4: Structure, Depth of Insertion, and Orientation , 2013, Journal of the American Chemical Society.

[22]  I. Pikuleva.  Putative F–G loop is involved in association with the membrane in P450scc (P450 11A1) , 2004, Molecular and Cellular Endocrinology.

[23]  O. Gotoh,et al.  Sterol 14-demethylase P450 (CYP51) provides a breakthrough for the discussion on the evolution of cytochrome P450 gene superfamily. , 2000, Biochemical and biophysical research communications.

[24]  V. Hornak,et al.  Comparison of multiple Amber force fields and development of improved protein backbone parameters , 2006, Proteins.

[25]  S D Black,et al.  Membrane topology of liver microsomal cytochrome P450 2B4 determined via monoclonal antibodies directed to the halt-transfer signal. , 1994, Biochemistry.

[26]  Rebecca C Wade,et al.  The ins and outs of cytochrome P450s. , 2007, Biochimica et biophysica acta.

[27]  Andrei L. Lomize,et al.  OPM: Orientations of Proteins in Membranes database , 2006, Bioinform..

[28]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[29]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[30]  P. Kollman,et al.  A well-behaved electrostatic potential-based method using charge restraints for deriving atomic char , 1993 .

[31]  Carsten Kutzner,et al.  GROMACS 4:  Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. , 2008, Journal of chemical theory and computation.

[32]  Alessandra Magistrato,et al.  Influence of the Membrane Lipophilic Environment on the Structure and on the Substrate Access/Egress Routes of the Human Aromatase Enzyme. A Computational Study , 2012, J. Chem. Inf. Model..

[33]  T. Yuen,et al.  Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia , 2013, Proceedings of the National Academy of Sciences.

[34]  L. Waskell,et al.  Theoretical study of the ligand–CYP2B4 complexes: Effect of structure on binding free energies and heme spin state , 2004, Proteins.

[35]  Andrzej J. Rzepiela,et al.  Reconstruction of atomistic details from coarse‐grained structures , 2010, J. Comput. Chem..

[36]  L. Waskell,et al.  Probing the Transmembrane Structure and Topology of Microsomal Cytochrome-P450 by Solid-State NMR on Temperature-Resistant Bicelles , 2013, Scientific Reports.

[37]  P. Kollman,et al.  Automatic atom type and bond type perception in molecular mechanical calculations. , 2006, Journal of molecular graphics & modelling.

[38]  T. Ost,et al.  Phe393 mutants of cytochrome P450 BM3 with modified heme redox potentials have altered heme vinyl and propionate conformations. , 2004, Biochemistry.

[39]  J. Baudry,et al.  Active-site hydration and water diffusion in cytochrome P450cam: a highly dynamic process. , 2011, Biophysical journal.

[40]  Xavier Periole,et al.  Combining an Elastic Network With a Coarse-Grained Molecular Force Field: Structure, Dynamics, and Intermolecular Recognition. , 2009, Journal of chemical theory and computation.

[41]  E. Szczesna-Skorupa,et al.  Identification of membrane-contacting loops of the catalytic domain of cytochrome P450 2C2 by tryptophan fluorescence scanning. , 2006, Biochemistry.

[42]  N. Grishin,et al.  PROMALS3D: a tool for multiple protein sequence and structure alignments , 2008, Nucleic acids research.

[43]  M. Waterman,et al.  CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds. , 2012, International journal for parasitology. Drugs and drug resistance.

[44]  Nathan A. Baker,et al.  PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations , 2004, Nucleic Acids Res..

[45]  S. Sligar,et al.  Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Peter M. Kasson,et al.  GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit , 2013, Bioinform..

[47]  A. Frey,et al.  Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies , 1987, The Journal of cell biology.

[48]  Balázs Jójárt,et al.  Performance of the general amber force field in modeling aqueous POPC membrane bilayers , 2007, J. Comput. Chem..

[49]  William J. Allen,et al.  GridMAT‐MD: A grid‐based membrane analysis tool for use with molecular dynamics , 2009, J. Comput. Chem..

[50]  O. Gotoh,et al.  Sterol 14-demethylase P450 (P45014DM*) is one of the most ancient and conserved P450 species. , 1996, Journal of biochemistry.

[51]  D. van der Spoel,et al.  GROMACS: A message-passing parallel molecular dynamics implementation , 1995 .

[52]  W. David Nes,et al.  CYP51: A Major Drug Target in the Cytochrome P450 Superfamily , 2008, Lipids.

[53]  Junmei Wang,et al.  Development and testing of a general amber force field , 2004, J. Comput. Chem..

[54]  M. Waterman,et al.  VNI cures acute and chronic experimental Chagas disease. , 2013, The Journal of infectious diseases.

[55]  M. Wilce,et al.  The F-G loop region of cytochrome P450scc (CYP11A1) interacts with the phospholipid membrane. , 2003, Biochimica et biophysica acta.

[56]  Mark S. P. Sansom,et al.  Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9 , 2011, PLoS Comput. Biol..

[57]  Piotr Cieplak,et al.  R.E.D. Server: a web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments , 2011, Nucleic Acids Res..

[58]  M. Waterman,et al.  CYP51—the omnipotent P450 , 2004, Molecular and Cellular Endocrinology.

[59]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[60]  R. Stroud,et al.  Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer , 2014, Proceedings of the National Academy of Sciences.

[61]  M. Waterman,et al.  Sterol 14alpha-demethylase (CYP51) as a therapeutic target for human trypanosomiasis and leishmaniasis. , 2011, Current topics in medicinal chemistry.

[62]  M. Waterman,et al.  Substrate Preferences and Catalytic Parameters Determined by Structural Characteristics of Sterol 14α-Demethylase (CYP51) from Leishmania infantum* , 2011, The Journal of Biological Chemistry.

[63]  N. Vermeulen,et al.  Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3 , 2008, Proteins.

[64]  O. Gotoh,et al.  Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. , 1992, The Journal of biological chemistry.

[65]  Karel Berka,et al.  Membrane Position of Ibuprofen Agrees with Suggested Access Path Entrance to Cytochrome P450 2C9 Active Site , 2011, The journal of physical chemistry. A.

[66]  Antonín Pavelka,et al.  CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures , 2012, PLoS Comput. Biol..