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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.


Related in: MedlinePlus

Root Mean Square Fluctuations in clusters and specific systems.a) Cα atom RMSF in the 3 clusters obtained by clustering. Cyan dashed line: cluster #1 (i.e. frames from the F429A, F429E and F429H simulations); full black line: cluster #2 (WT); magenta dot-dashed line: cluster #3 (F429L). b) difference of RMSF calculated from the trajectories of 4 mutants with respect to the WT trajectory. Full blue line: F429A; magenta dashed line: F429E; orange dot-dashed line: F429H; green dotted line: F429L.
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pone.0137075.g005: Root Mean Square Fluctuations in clusters and specific systems.a) Cα atom RMSF in the 3 clusters obtained by clustering. Cyan dashed line: cluster #1 (i.e. frames from the F429A, F429E and F429H simulations); full black line: cluster #2 (WT); magenta dot-dashed line: cluster #3 (F429L). b) difference of RMSF calculated from the trajectories of 4 mutants with respect to the WT trajectory. Full blue line: F429A; magenta dashed line: F429E; orange dot-dashed line: F429H; green dotted line: F429L.

Mentions: Following the investigation of global structural changes induced by the 4 point mutations with respect to the active site we proceeded with investigating the possible role of single residues in generating these differences, by comparing the Root Mean Square Fluctuation (RMSF) Cα atoms. We have compared in Fig 5 the RMSF calculated obtained from the frames of clusters #1, #2 and #3 (13940 frames in total) trajectories (panel a) with the RMSF difference obtained by directly subtracting the RMSF yielded by the wild-type enzyme to those obtained from the 4 mutants trajectories (panel b) (using configurations from frame set #2). Three regions, centered around M137, K225 and G418 showed conserved high fluctuations in all clusters as well as in the original simulations. A number of relevant differences in the fluctuation patterns could be observed; for instance, the first cluster showed larger fluctuations around residues A92, S277, R343 and K433. In general, the region 430–435 near the C436 and the hem distal side features high fluctuations in the Ala/Glu/His mutants as compared to the WT enzyme or F429L. Inspection of panel b shows that in general F429A and F429E showed the greatest variation as compared to WT while F429L produced the most similar fluctuations. It can also be seen that the fluctuation in A92 is mostly due to configurations from the F429E and F429H trajectories while that of S277 and R343 to F429A and F429E; peaks associated to E424 were observable in the WT and F429L trajectories (and thus in clusters #2 and #3).


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

Mancini G, Zazza C - PLoS ONE (2015)

Root Mean Square Fluctuations in clusters and specific systems.a) Cα atom RMSF in the 3 clusters obtained by clustering. Cyan dashed line: cluster #1 (i.e. frames from the F429A, F429E and F429H simulations); full black line: cluster #2 (WT); magenta dot-dashed line: cluster #3 (F429L). b) difference of RMSF calculated from the trajectories of 4 mutants with respect to the WT trajectory. Full blue line: F429A; magenta dashed line: F429E; orange dot-dashed line: F429H; green dotted line: F429L.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0137075.g005: Root Mean Square Fluctuations in clusters and specific systems.a) Cα atom RMSF in the 3 clusters obtained by clustering. Cyan dashed line: cluster #1 (i.e. frames from the F429A, F429E and F429H simulations); full black line: cluster #2 (WT); magenta dot-dashed line: cluster #3 (F429L). b) difference of RMSF calculated from the trajectories of 4 mutants with respect to the WT trajectory. Full blue line: F429A; magenta dashed line: F429E; orange dot-dashed line: F429H; green dotted line: F429L.
Mentions: Following the investigation of global structural changes induced by the 4 point mutations with respect to the active site we proceeded with investigating the possible role of single residues in generating these differences, by comparing the Root Mean Square Fluctuation (RMSF) Cα atoms. We have compared in Fig 5 the RMSF calculated obtained from the frames of clusters #1, #2 and #3 (13940 frames in total) trajectories (panel a) with the RMSF difference obtained by directly subtracting the RMSF yielded by the wild-type enzyme to those obtained from the 4 mutants trajectories (panel b) (using configurations from frame set #2). Three regions, centered around M137, K225 and G418 showed conserved high fluctuations in all clusters as well as in the original simulations. A number of relevant differences in the fluctuation patterns could be observed; for instance, the first cluster showed larger fluctuations around residues A92, S277, R343 and K433. In general, the region 430–435 near the C436 and the hem distal side features high fluctuations in the Ala/Glu/His mutants as compared to the WT enzyme or F429L. Inspection of panel b shows that in general F429A and F429E showed the greatest variation as compared to WT while F429L produced the most similar fluctuations. It can also be seen that the fluctuation in A92 is mostly due to configurations from the F429E and F429H trajectories while that of S277 and R343 to F429A and F429E; peaks associated to E424 were observable in the WT and F429L trajectories (and thus in clusters #2 and #3).

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.


Related in: MedlinePlus