Molecular Dynamics Simulations to Investigate the Influences of Amino Acid Mutations on Protein Three-Dimensional Structures of Cytochrome P450 2D6.1, 2, 10, 14A, 51, and 62

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl’s side chain tightens and relaxes the opening to the channel in conjunction with the arginyl’s, though the latter’s side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.

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Deciphering Structural Alterations Associated with Activity Reductions of Genetic Polymorphisms in Cytochrome P450 2A6 Using Molecular Dynamics Simulations

International Journal of Molecular Sciences ◽

10.3390/ijms221810119 ◽

2021 ◽

Vol 22 (18) ◽

pp. 10119

Author(s):

Koichi Kato ◽

Tomoki Nakayoshi ◽

Rika Nokura ◽

Hiroki Hosono ◽

Masahiro Hiratsuka ◽

...

Keyword(s):

Molecular Dynamics ◽

Cytochrome P450 ◽

Molecular Dynamics Simulations ◽

Genetic Polymorphisms ◽

Structural Changes ◽

Three Dimensional ◽

Smoking Behavior ◽

Enzymatic Activities ◽

Access Channel ◽

Dynamics Simulations

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities of the allelic variants of CYP2A6 were analyzed in previous studies. However, the three-dimensional structures of the mutants were not investigated, and the mechanisms underlying activity reduction remain unknown. In this study, to investigate the structural changes involved in the reduction in enzymatic activities, we performed molecular dynamics simulations for ten allelic mutants of CYP2A6. For the calculated wild type structure, no significant structural changes were observed in comparison with the experimental structure. On the other hand, the mutations affected the interaction with heme, substrates, and the redox partner. In CYP2A6.44, a structural change in the substrate access channel was also observed. Those structural effects could explain the alteration of enzymatic activity caused by the mutations. The results of simulations provide useful information regarding the relationship between genotype and phenotype.

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Prediction of three-dimensional structures and structural flexibilities of wild-type and mutant cytochrome P450 1A2 using molecular dynamics simulations

Journal of Molecular Graphics and Modelling ◽

10.1016/j.jmgm.2016.06.006 ◽

2016 ◽

Vol 68 ◽

pp. 48-56 ◽

Cited By ~ 7

Author(s):

Yurie Watanabe ◽

Shuichi Fukuyoshi ◽

Masahiro Hiratsuka ◽

Noriyuki Yamaotsu ◽

Shuichi Hirono ◽

...

Keyword(s):

Molecular Dynamics ◽

Cytochrome P450 ◽

Molecular Dynamics Simulations ◽

Three Dimensional ◽

Wild Type ◽

Cytochrome P450 1A2 ◽

Dynamics Simulations

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Cytochrome P450 3A4 Inhibition by Ketoconazole: Tackling the Problem of Ligand Cooperativity Using Molecular Dynamics Simulations and Free-Energy Calculations

Journal of Chemical Information and Modeling ◽

10.1021/ci300118x ◽

2012 ◽

Vol 52 (6) ◽

pp. 1573-1582 ◽

Cited By ~ 59

Author(s):

Urban Bren ◽

Chris Oostenbrink

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Cytochrome P450 ◽

Molecular Dynamics Simulations ◽

Free Energy Calculations ◽

Cytochrome P450 3A4 ◽

Energy Calculations ◽

Dynamics Simulations

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New CHARMM force field parameters for dehydrated amino acid residues, the key to lantibiotic molecular dynamics simulations

RSC Advances ◽

10.1039/c4ra09897h ◽

2014 ◽

Vol 4 (89) ◽

pp. 48621-48631 ◽

Cited By ~ 9

Author(s):

Eleanor R. Turpin ◽

Sam Mulholland ◽

Andrew M. Teale ◽

Boyan B. Bonev ◽

Jonathan D. Hirst

Keyword(s):

Molecular Dynamics ◽

Amino Acid ◽

Force Field ◽

Molecular Dynamics Simulations ◽

Amino Acid Residues ◽

Charmm Force Field ◽

Force Field Parameters ◽

Dynamics Simulations

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Rheology of Two- and Three-dimensional Granular Mixtures Under Uniform Shear Flow: Enskog Kinetic Theory Versus Molecular Dynamics Simulations

Granular Matter ◽

10.1007/s10035-006-0001-7 ◽

2006 ◽

Vol 8 (2) ◽

pp. 103-115 ◽

Cited By ~ 22

Author(s):

José María Montanero ◽

Vicente Garzó ◽

Meheboob Alam ◽

Stefan Luding

Keyword(s):

Molecular Dynamics ◽

Shear Flow ◽

Kinetic Theory ◽

Molecular Dynamics Simulations ◽

Three Dimensional ◽

Uniform Shear ◽

Uniform Shear Flow ◽

Granular Mixtures ◽

Theory Versus ◽

Dynamics Simulations

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A Novel Quali-Quantitative Defect of VWF

Blood ◽

10.1182/blood-2021-147899 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1032-1032

Author(s):

Tom Van De Berg ◽

Alice Todaro ◽

Joyce van Beers ◽

Kanin Wichapong ◽

Floor Heubel-Moenen ◽

...

Keyword(s):

Molecular Dynamics ◽

Amino Acid ◽

Genetic Analysis ◽

Molecular Dynamics Simulations ◽

Mrna Level ◽

Bleeding Tendency ◽

Unknown Significance ◽

Dynamics Simulations ◽

Von Willebrand

Abstract Background Von Willebrand Factor (VWF) is a multimeric protein largely involved in both primary and secondary hemostasis. The diagnosis and classification of von Willebrand Disease (VWD) patients can be challenging. In this poster we explore the genetic defects and their structural consequences in a VWD patient with a disproportionately high bleeding phenotype for her VWD severity. A 31-year old female, initially diagnosed with VWD type 1, presented herself with a bleeding tendency (ISTH-BAT 13) disproportionate to the severity of her VWD with antigen levels of 36%. Additional analysis showed decreased FVIII-binding at 28%. The combination of both quantitative and functional defects of VWF was an indication for further genetic analysis in order to better define the subtype of VWD. Aims Genetic and structural analysis of VWF in a patient with a disproportionally high bleeding phenotype with regard to a mild decrease in VWF antigen. Methods Routine laboratory analysis for VWD was performed. Genetic screening was performed by exome sequencing of hemostasis related genes. VWF mRNA analysis was carried out by RT-PCR and Sanger sequencing. The X-ray structure of furin in complex with a peptide-based inhibitor (PDB ID: 6YD7) was used as a template to construct furin-VWF (759HR(R760S)SKRS764) complex. The derived structures (furin in complex with WT/R760S-VWF) were subjected to molecular dynamics (MD) simulations (200ns) and binding free energy (BFE) calculations by using standard parameters and protocols implemented in AMBER20 program. Results Routine analysis showed PFA-ADP and PFA EPI >300 seconds, VWF:ACT of 37% with a VWF:AG of 36%. Collagen binding and FVIII-binding were 46% and 28% respectively. Genetic analysis of the VWF gene disclosed 2 heterozygous variants of unknown significance (VUS): c.2771 G>A (exon 21, p.Arg924Gln) has a 1-2.5% population prevalence and has been previously described in type 1 and 2N VWD. The other VUS (c.2278 C>A; exon 17) is a novel mutation predicting a major amino acid substitution (p.Arg760Ser) in the D2-domain of VWF. Sequencing of exons 17 and 21 in the patient's VWF mRNA revealed homozygosity for the mutated allele at both mutation sites, indicating that the two variants are in cis and that the 'normal' allele is not expressed at mRNA level. Molecular dynamics simulations of the novel c.2278 C>A mutation (Arg760Ser) predicts a markedly decreased binding of furin to its VWF binding site, possibly decreasing or preventing VWF pro-peptide cleavage. This in turn has been shown to lead to reduced FVIII-binding of VWF. Conclusion Genetic analysis shows one polymorphism (c.2771 G>A) and one variation of unknown significance (c.2278 C>A) in the patient's VWF-gene. The polymorphism is known to be of low pathogenicity. The c. 2278 C>A mutation was not known in any of the mutation databases and is a novel VWF mutation. Both mutations were shown to be present on the same allele. As the wild-type allele was not expressed on mRNA level, all of the patient's VWF protein includes both amino acid substitutions. Modeling and molecular dynamics simulations show a markedly decreased affinity of furin to its cleavage site on the VWF protein due to the Arg760Ser substitution, likely resulting in a persistent pro-peptide binding to the mature VWF protein. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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