Research Resource: Correlating Human Cytochrome P450 21A2 Crystal Structure and Phenotypes of Mutations in Congenital Adrenal Hyperplasia

Abstract

Cytochrome P450 21A2 is a key player in steroid 21-hydroxylation and converts progesterone to 11-deoxycorticosterone and 17α-hydroxy progesterone to 11-deoxycortisol. More than 100 mutations in P450 21A2 have been established in patients thus far; these account for the vast majority of occurrences of congenital adrenal hyperplasia (CAH), which is among the most common heritable metabolic diseases in humans. CAH phenotypes range from the most severe, salt-wasting (SW), to the simple virilizing (SV), and nonclassical (NC) CAH forms. We recently determined the crystal structure of human P450 21A2 in complex with progesterone. To gain more insight into the structural and stability changes underlying the phenotypes of individual mutations, we analyzed 24 SW, SV, and NC mutants in the context of the crystal structure of the human enzyme. Our analysis reveals clear differences in the localization of SW, SV, and NC mutations, with many of the first type mapping to the active site and near the heme and/or substrate and mostly resulting in complete loss of enzyme activity. Conversely, NC mutations are often found near the periphery and close to the surface of the protein, and mutant enzymes retain partial activity. The main conclusion from the mutation-structure-activity study is that the severity of the CAH clinical manifestations can be directly correlated with the degree of mutation-induced damage in terms of protein fold stability and active site changes in the structural model. Thus, the NC phenotype is typically associated with mutations that have a compensatory effect, ie, H-bonding replacing hydrophobic interactions and vice versa.

Figure 1.

Crystal structure of human P450 21A2 in complex with progesterone (PDB ID code 4Y8W) (17). The protein main chain is shown in ribbon mode and rainbow coloring, from N terminus (blue) to C terminus (red). Carbon, nitrogen, and oxygen atoms of the heme moiety are colored in black, blue, and red, respectively, and Fe³⁺ is shown as an orange sphere. Carbon and oxygen atoms of progesterone are colored in magenta and red, respectively. Secondary structure elements are labeled and the residue ranges for α-helices A to M and β-strands 1 to 9 are as follows (every 10th residue in the chain is labeled): β1 32–36, A 45–56, β2 59–64, β3 67–72, B 75–84, B́ 86–89, C 115–131, D 136–153, E 161–178, F 188–202, F́ 205–212, G 224–246, H 257–264, I 279–310, J 312–322, K 344–356, β4 364–368, β5 372–374, β6 377–379, β7 384–387, L 389–394, M 432–447, β8 450–454, and β9 479–483.

Figure 2.

Amino acids whose mutation gives rise to the (A) SW, (B) SV, and (C) NC CAH phenotypes mapped in the crystal structure of P450 21A2. Carbon atoms of wt residues are highlighted in blue (SW), green (SV), and purple (NC). The orientation of P450 in the top panels corresponds approximately to that in Figure 1.

Figure 3.

Activities of hP450 21A2 mutant enzymes giving rise to the (A) SW, (B) SV, and (C) NC phenotypes. See also Supplemental Tables 1–3.

Figure 4.

Comparisons between amino acid side chain interactions in the crystal structure of the human P450 21A2 progesterone complex (17) and the humanized model of the enzyme based on the crystal structure of bovine 21A2 (insets) (7). Notice the difference of 1 between residue numbers in the calculated and experimental models. Predicted and actual configurations of salt bridges involving (A) D408, (B) D112, (C) E141, and (D) E352. E, The V305D mutant was assumed to result in salt bridge formation with K473 and increased rigidity (7). However, in the crystal structure of hP450 21A2, K473 and V305 are spaced far apart with M474 inserted between them. Selected contacts in the experimental model are indicated with thin solid lines and distances in Å.

Figure 5.

Selected mutations causing the SW phenotype. (A) P31Q, (B) G65E, (C) L108R, and (D) V140E. Side chain carbon atoms of mutated amino acids are turquois and selected residues are labeled.

Figure 6.

Selected mutations causing the SV phenotype. (A) I78T, (B) C148R, (C) C170R, and (D) V282G.

Figure 7.

Selected mutations causing the NC phenotype. (A) H120R, (B) K121Q, (C) I172N, and (D) I195N.