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F429 Regulation of Tunnels in Cytochrome P450 2B4: A Top Down Study of Multiple Molecular Dynamics Simulations.

Mancini G, Zazza C - PLoS ONE (2015)

Bottom Line: Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties.The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels.Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization.

View Article: PubMed Central - PubMed

Affiliation: Scuola Normale Superiore di Pisa, Piazza dei Cavalieri 7, 56126, Pisa, Italy, and Istituto Nazionale di Fisica Nucleare (INFN) sezione di Pisa, Largo Bruno Pontecorvo 3, 56127, Pisa, Italy.

ABSTRACT
The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization.

No MeSH data available.


a,b) Cartoon representation of the CYP-2B4 crystal structure (PDB ID 1SUO) with specific domains coloured and labeled according to Scott et al.;[12] the heme group, the peroxide anion and Cys426 are shown as ball and stick. The enzyme is shown looking from the distal (a) or proximal (b) side of the heme group. To be coherent with the literature, the same color map reported in the original experimental work has been used. (c,d) Detail of the active site.The protein backbone is shown as a transparent cyan ribbon, while Cpd0 is shown in and stick style, together with the various rotamers of residue 429. Cys 436, residue 429 and the A and D propionate groups are labeled.
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pone.0137075.g002: a,b) Cartoon representation of the CYP-2B4 crystal structure (PDB ID 1SUO) with specific domains coloured and labeled according to Scott et al.;[12] the heme group, the peroxide anion and Cys426 are shown as ball and stick. The enzyme is shown looking from the distal (a) or proximal (b) side of the heme group. To be coherent with the literature, the same color map reported in the original experimental work has been used. (c,d) Detail of the active site.The protein backbone is shown as a transparent cyan ribbon, while Cpd0 is shown in and stick style, together with the various rotamers of residue 429. Cys 436, residue 429 and the A and D propionate groups are labeled.

Mentions: Initial coordinates were obtained by the 1SUO[9] crystal structure (at a resolutions of 1.9 Å, see Fig 2A and 2B). At first, the enzyme was modified in order to form the Cpd 0 species, replacing the inhibitor present in the crystal ctructure with a O-OH covalent moiety.[43] The 4 mutations (Ala, Glu, His and Leu) were generated using the Dunbrack rotamer library[45]. In Fig 2 (panels a, b), we report a pictorial view of the WT form using the original crystal (1SUO.pdb) structure, with various domain shown in different colours; Fig 2C shows the active site with the with the mutated residue 429,which is located at the proximal side of the CYP 2B4 active site, and in particular the region adjacent to the cysteine residue as the fifth ligand to heme iron atom. Missing hydrogen atoms were added by MolProbity[46] and protonation states counter-checked with H++ code[47]. Simulations were carried out with GROMACS, version 4.6.6.[48] Afterward, the entire enzyme was placed at the center of a square box of size 10.76 nm, subsequently filled with SPC[49] water molecules at a density of 1000 kg/m3; Cl- anions were added to ensure the overall electrical neutrality of each system. The GROMOS 54A7 force field[50] was adopted for modeling the enzymes. Electrostatic interactions were accounted for by means of the Particle Mesh Ewald method[51] using a cutoff of 1.5 nm for the real space and Van der Waals interactions. The LINCS algorithm[52] was used to constrain all bond lengths and angles. Relaxation of solvent molecules and Cl- anions was initially performed keeping solute atoms restrained to their initial positions with a force constant of 1000 kJ/(mol • nm2), for 3.0 ns in a NPT ensemble at 1 bar using the Parrinello-Rahman barostat [53] and a coupling constant of 1.0 ps; temperature was increased in 50 K steps from 0 to 298.15 K using the velocity rescale method [54] and a coupling constant of 0.1 ps; the integration time step was increased progressively from 0.1 to 1.0 fs. Each system was carried back to 0 K and then heated again to 298.15 K in a NVT ensemble using the same stepwise fashion (for a total of 3.0 ns of further thermalization) without restrains on protein atoms. The five systems were then simulated for 150 ns each in a NVT ensemble with a time step of 2.0 fs (updating the neighbor list every 10 steps). Additional short (10 ns) simulation were also carried out for each system generating new random velocities. In total, 2 short simulations were carried out for each system. Thus, 3 simulations were carried out for each system or 18 simulations in total. The starting coordinates for these trajectories were selected observing the Root Mean Square Deviation graph (see Section 3.1 and S1 Fig).


F429 Regulation of Tunnels in Cytochrome P450 2B4: A Top Down Study of Multiple Molecular Dynamics Simulations.

Mancini G, Zazza C - PLoS ONE (2015)

a,b) Cartoon representation of the CYP-2B4 crystal structure (PDB ID 1SUO) with specific domains coloured and labeled according to Scott et al.;[12] the heme group, the peroxide anion and Cys426 are shown as ball and stick. The enzyme is shown looking from the distal (a) or proximal (b) side of the heme group. To be coherent with the literature, the same color map reported in the original experimental work has been used. (c,d) Detail of the active site.The protein backbone is shown as a transparent cyan ribbon, while Cpd0 is shown in and stick style, together with the various rotamers of residue 429. Cys 436, residue 429 and the A and D propionate groups are labeled.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4587367&req=5

pone.0137075.g002: a,b) Cartoon representation of the CYP-2B4 crystal structure (PDB ID 1SUO) with specific domains coloured and labeled according to Scott et al.;[12] the heme group, the peroxide anion and Cys426 are shown as ball and stick. The enzyme is shown looking from the distal (a) or proximal (b) side of the heme group. To be coherent with the literature, the same color map reported in the original experimental work has been used. (c,d) Detail of the active site.The protein backbone is shown as a transparent cyan ribbon, while Cpd0 is shown in and stick style, together with the various rotamers of residue 429. Cys 436, residue 429 and the A and D propionate groups are labeled.
Mentions: Initial coordinates were obtained by the 1SUO[9] crystal structure (at a resolutions of 1.9 Å, see Fig 2A and 2B). At first, the enzyme was modified in order to form the Cpd 0 species, replacing the inhibitor present in the crystal ctructure with a O-OH covalent moiety.[43] The 4 mutations (Ala, Glu, His and Leu) were generated using the Dunbrack rotamer library[45]. In Fig 2 (panels a, b), we report a pictorial view of the WT form using the original crystal (1SUO.pdb) structure, with various domain shown in different colours; Fig 2C shows the active site with the with the mutated residue 429,which is located at the proximal side of the CYP 2B4 active site, and in particular the region adjacent to the cysteine residue as the fifth ligand to heme iron atom. Missing hydrogen atoms were added by MolProbity[46] and protonation states counter-checked with H++ code[47]. Simulations were carried out with GROMACS, version 4.6.6.[48] Afterward, the entire enzyme was placed at the center of a square box of size 10.76 nm, subsequently filled with SPC[49] water molecules at a density of 1000 kg/m3; Cl- anions were added to ensure the overall electrical neutrality of each system. The GROMOS 54A7 force field[50] was adopted for modeling the enzymes. Electrostatic interactions were accounted for by means of the Particle Mesh Ewald method[51] using a cutoff of 1.5 nm for the real space and Van der Waals interactions. The LINCS algorithm[52] was used to constrain all bond lengths and angles. Relaxation of solvent molecules and Cl- anions was initially performed keeping solute atoms restrained to their initial positions with a force constant of 1000 kJ/(mol • nm2), for 3.0 ns in a NPT ensemble at 1 bar using the Parrinello-Rahman barostat [53] and a coupling constant of 1.0 ps; temperature was increased in 50 K steps from 0 to 298.15 K using the velocity rescale method [54] and a coupling constant of 0.1 ps; the integration time step was increased progressively from 0.1 to 1.0 fs. Each system was carried back to 0 K and then heated again to 298.15 K in a NVT ensemble using the same stepwise fashion (for a total of 3.0 ns of further thermalization) without restrains on protein atoms. The five systems were then simulated for 150 ns each in a NVT ensemble with a time step of 2.0 fs (updating the neighbor list every 10 steps). Additional short (10 ns) simulation were also carried out for each system generating new random velocities. In total, 2 short simulations were carried out for each system. Thus, 3 simulations were carried out for each system or 18 simulations in total. The starting coordinates for these trajectories were selected observing the Root Mean Square Deviation graph (see Section 3.1 and S1 Fig).

Bottom Line: Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties.The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels.Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization.

View Article: PubMed Central - PubMed

Affiliation: Scuola Normale Superiore di Pisa, Piazza dei Cavalieri 7, 56126, Pisa, Italy, and Istituto Nazionale di Fisica Nucleare (INFN) sezione di Pisa, Largo Bruno Pontecorvo 3, 56127, Pisa, Italy.

ABSTRACT
The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization.

No MeSH data available.