5VC0

Crystal structure of human CYP3A4 bound to ritonavir

Summary for 5VC0

• Entry DOI: 10.2210/pdb5vc0/pdb
• Related: 5VCC 5VCD 5VCE 5VCG
• Related PRD ID: PRD_001001
• Descriptor: Cytochrome P450 3A4, PROTOPORPHYRIN IX CONTAINING FE, RITONAVIR (3 entities in total)
• Functional Keywords: cytochrome p450, cyp3a4, monooxygenase, ritonavir, inhibitor, oxidoreductase-inhibitor complex, oxidoreductase/inhibitor
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 1
• Total formula weight: 57095.24

Authors

Sevrioukova, I. (deposition date: 2017-03-30, release date: 2017-05-31, Last modification date: 2023-10-04)

Primary citation

Sevrioukova, I.F.
High-Level Production and Properties of the Cysteine-Depleted Cytochrome P450 3A4.
Biochemistry, 56:3058-3067, 2017

PubMed Abstract: Human drug-metabolizing cytochrome P450 3A4 (CYP3A4) is a dynamic enzyme with a large and highly malleable active site that can fit structurally diverse compounds. Despite extensive investigations, structure-function relationships and conformational dynamics in CYP3A4 are not fully understood. This study was undertaken to engineer a well-expressed and functionally active cysteine-depleted CYP3A4 that can be used in biochemical and biophysical studies. cDNA codon optimization and screening mutagenesis were utilized to boost the level of bacterial expression of CYP3A4 and identify the least harmful substitutions for all six non-heme-ligating cysteines. The C58A/C64M/C98A/C239T/C377A/C468S (Cys-less) mutant was found to be expressed as highly as the optimized wild-type (opt-WT) CYP3A4. The high-resolution X-ray structures of opt-WT and Cys-less CYP3A4 revealed that gene optimization leads to a different folding in the Phe108 and Phe189 regions and promotes binding of the active site glycerol that interlocks Ser119 and Arg212, critical for ligand association, and the hydrophobic cluster adjacent to Phe108. Crowding and decreased flexibility of the active site, as well as structural alterations observed at the C64M, C239T, and C468S mutational sites, might be responsible for the distinct ligand binding behavior of opt-WT and Cys-less CYP3A4. Nonetheless, the Cys-less mutant could be used for structure-function investigations because it orients bromoergocryptine and ritonavir (a high-affinity substrate and a high-potency inhibitor, respectively) like the WT and has a higher activity toward 7-benzyloxy(4-trifluoromethyl)coumarin.

PubMed: 28590129
DOI: 10.1021/acs.biochem.7b00334

Experimental method

X-RAY DIFFRACTION (2.7 Å)