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

Abstract

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, salt-bridge 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.

Fig. 1.

Structural determinants of CYP21A2 mutations causing salt-wasting CAH. (A) Residues that are within 3.5 Å of heme- or substrate-binding site, when mutated result in SW CAH. R91, R426, and H365 form hydrogen bonds with the propionate chain of heme and, thus, prevent misalignment in an enclosed environment. (B) Mutations of residues that result in disruption of the tertiary structure results in SW CAH. (Ba) V139E mutation is not tolerated in a small hydrophobic pocket. (Bb) Disruption of the π-π stacking interactions destabilizes the structural elements. (Bc) A bulkier leucine side chain is not accepted for P386. (Bd) Q481P results in the extensive loss of hydrogen-bonding networks. (C) Mutation of residues forming salt-bridge interactions and hydrogen-bonding networks that enable maintenance of tertiary structural elements when mutated result in SW CAH. Salt-bridge interactions between R316-D407 (Ca), R366-D111 (Cb), R444-E140 (Cc), and hydrogen bonding between the highly conserved Glu-Xaa-Xaa-Arg (EXXR) motif formed by E351-R354-R408 (Cd) are shown.

Fig. 2.

Structural determinants of CYP21A2 mutations causing SV and NC CAH. (A) I77 and I317, which are surrounded by hydrophobic residues. Mutations of I77 or I317 (cyan sticks) result in disruption of hydrophobicity. Whereas mutation I77T results in SV CAH, L317M/V causes NC CAH (because valine and methionine are both hydrophobic residues that can be tolerated at this position). (B) Positively charged resides that have been implicated in the binding of oxidoreductase. Mutation of these residues causes NC CAH. All of these residues are present on the same face of CYP21A2. (C) Disruption of salt-bridge and hydrogen bonding interactions that result in localized destabilization of structure causes NC CAH. Salt-bridge interactions between R149-E162 (Ca), R479-E163 (Cb), R136-D407 (Cc), R435-E431 (Cd), R483-D322 (Ce), and hydrogen-bonding network between G35-H62 and G67 (Cf).

Fig. 3.

Previously unreported CYP21A2 mutations. Previously unreported missense mutations, namely S113Y/F, V305D, F306V, L307V, R316L, L321P, E351K, R366H, G381S, P386L, F404L, R408H, R426P, L433P, and R444P are shown. The location of the mutated residue and the impact each mutation has on protein structure and the predicted clinical outcomes are shown.