Three-dimensional Structure of Steroid 21-Hydroxylase (Cytochrome P450 21A2) with Two Substrates Reveals Locations of Disease-associated Variants

Zhao, Bin; Lei, Li; Kagawa, Norio; Sundaramoorthy, Munirathinam; Banerjee, Surajit; Nagy, Leslie D.; Guengerich, F. Peter; Waterman, Michael R.

doi:10.1074/jbc.m111.323501

Cited by 74 publications

(97 citation statements)

References 40 publications

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“…Bovine and the human sequences share 79% sequence identity. The human model of CYP21A2 was constructed by aligning bovine and human sequences and extracting structural features from the bovine template (17). An alignment of six cytochrome P450 proteins involved in steroid biosynthesis was also carried out to identify conserved structural elements (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Both charged and hydrophobic residues on the proximal surface of cytochrome P450 are involved in interaction with POR (24). The basic residues on the surface of CYP21A2, namely K121, R132, R341, R339, and R369, are known to interact with the acidic residues on POR (8,17). All of these positively charged residues are also aligned to one face of the protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…P08686). The sequence from the bovine CYP21A2 crystal structure [Protein Data Bank ID code 3QZ1 (17)] was extracted and aligned with the human sequence using ClustalX (33) (overall sequence similarity, 79%). The sequence conservation in the heme-binding cavity, between human and bovine, is 95%.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, the bovine CYP21A2 crystal structure has become available (17). We have, thus, constructed a humanized model of CYP21A2 and mapped disease-causing mutations to investigate the structural role of all known mutations in CYP21A2.…”

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confidence: 99%

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Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Haider

Islam

D’Atri

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

130

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Mutations in the cytochrome p450 (CYP)21A2 gene, which encodes the enzyme steroid 21-hydroxylase, cause the majority of cases in congenital adrenal hyperplasia, an autosomal recessive disorder. To date, more than 100 CYP21A2 mutations have been reported. These mutations can be associated either with severe salt-wasting or simple virilizing phenotypes or with milder nonclassical phenotypes. Not all CYP21A2 mutations have, however, been characterized biochemically, and the clinical consequences of these mutations remain unknown. Using the crystal structure of its bovine homolog as a template, we have constructed a humanized model of CYP21A2 to provide comprehensive structural explanations for the clinical manifestations caused by each of the known disease-causing missense mutations in CYP21A2. Mutations that affect membrane anchoring, disrupt heme and/or substrate binding, or impair stability of CYP21A2 cause complete loss of function and salt-wasting disease. In contrast, mutations altering the transmembrane region or conserved hydrophobic patches cause up to a 98% reduction in enzyme activity and simple virilizing disease. Mild nonclassical disease can result from interference in oxidoreductase interactions, saltbridge and hydrogen-bonding networks, and nonconserved hydrophobic clusters. A simple in silico evaluation of previously uncharacterized gene mutations could, thus, potentially help predict the often diverse phenotypes of a monogenic disorder.structure-phenotype correlation | molecular modeling | CYP21A2 structural analysis

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Haider

Islam

D’Atri

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

130

View full text Add to dashboard Cite

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“…For x-ray crystallographic experiments, zebrafish P450 17A1 and 17A2 were expressed and purified as described previously for E. coli recombinant bovine P450 21A2 (36), including Ni 2ϩ -nitrilotriacetate, DEAE, and SP-Sepharose chromatography steps. The proteins were each eluted from an SP-Sepharose Fast Flow FPLC column (GE Healthcare) with 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol (v/v), 0.1 mM dithiothreitol, 0.1 mM EDTA, 0.004% (w/v) 3,6,9,12,15,18,21,24,27-nonaoxanonatriacontan-1-ol (C12E9) detergent (Anatrace, Maumee, OH), and either 500 mM NaCl (for P450 17A1) or 250 mM NaCl (for P450 17A2).…”

Section: Methodsmentioning

confidence: 99%

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

Pallan

Nagy

Lei

et al. 2015

Journal of Biological Chemistry

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Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase reactions. A second fish P450, 17A2 (51% identical), catalyzes only 17␣-hydroxylation. Results: Crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1 are very similar. Conclusion: In kinetic analysis, the two-step oxidation of progesterone is more distributive than for pregnenolone. Significance: Small structural differences are associated with activities of the two fish P450s.

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In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish

Lei

Egli

2016

CP Protein Science

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Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins impossible or challenging. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins.

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Three-dimensional Structure of Steroid 21-Hydroxylase (Cytochrome P450 21A2) with Two Substrates Reveals Locations of Disease-associated Variants

Cited by 74 publications

References 40 publications

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish

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