Predicted effects of missense mutations on native-state stability account for phenotypic outcome in phenylketonuria, a paradigm of misfolding diseases

Abstract

Phenylketonuria (PKU) is a genetic disease caused by mutations in human phenylalanine hydroxylase (PAH). Most missense mutations result in misfolding of PAH, increased protein turnover, and a loss of enzymatic function. We studied the prediction of the energetic impact on PAH native-state stability of 318 PKU-associated missense mutations, using the protein-design algorithm FoldX. For the 80 mutations for which expression analyses have been performed in eukaryote systems, in most cases we found substantial overall correlations between the mutational energetic impact and both in vitro residual activities and patient metabolic phenotype. This finding confirmed that the decrease in protein stability is the main molecular pathogenic mechanism in PKU and the determinant for phenotypic outcome. Metabolic phenotypes have been shown to be better predicted than in vitro residual activities, probably because of greater stringency in the phenotyping process. Finally, all the remaining 238 PKU missense mutations compiled at the PAH locus knowledgebase (PAHdb) were analyzed, and their phenotypic outcomes were predicted on the basis of the energetic impact provided by FoldX. Residues in exons 7-9 and in interdomain regions within the subunit appear to play an important structural role and constitute hotspots for destabilization. FoldX analysis will be useful for predicting the phenotype associated with rare or new mutations detected in patients with PKU. However, additional factors must be considered that may contribute to the patient phenotype, such as possible effects on catalysis and interindividual differences in physiological and metabolic processes.

Figure 1.

Mutation-dependent destabilization and in vitro residual activity. A, Plot of calculated ΔΔG (in kcal/mol dimer, at a penalty of 5 kcal/mol) versus in vitro residual activity calculated for the structure of the dephosphorylated dimeric form for 79 individual mutations (all compiled in table 1 except A447P, which is not included in this structure). Seventy-four of these mutations (unblackened triangles), grouped in types I, II, and III according to the residual activities (table 1), were included in the calculation of the mean±SD ΔΔG values (blackened circles) for types I, II, and III (4.3±2.8 kcal/mol, 5.7±4.8 kcal/mol, and 14.2±11.7 kcal/mol, respectively). Five mutations were defined as outliers (blackened triangles; R68G is also indicated by an arrow; also see main text for details) and were not included in the calculation of the mean ΔΔG values. The line is only to guide the eye and has no formal significance. B and C, Means±SDs of m and y₀ values for the different in vitro activity groups calculated using individual fits for each mutation. P values are obtained from one-way ANOVA; P<.05 is considered statistically significant.

Figure 2.

ΔΔG values (in kcal/mol dimer) predicted by FoldX with the use of different structural models. Data were obtained from table 5 for the unphosphorylated dimer (based on PDB 2PHM; 74 mutations) (blackened circles), phosphorylated dimer (based on PDB 1PHZ; 74 mutations) (unblackened circles), and the N-terminal truncated tetramer (2PAH; 55 mutations) (blackened triangles). A, Plot of ΔΔG versus in vitro residual activity (types I, II, and II as defined in table 1) (with penalty of 5 kcal/mol). B, Plot of ΔΔG (with penalty of 5 kcal/mol) for PKU mutations classified by phenotypic association found in patients with PKU (as defined in table 2). C and D, Dependence of ΔΔG on different energetic penalties for PKU mutations classified by in vitro activity (C) or association with in vivo phenotypes (D).

Figure 3.

Effect of the energetic penalizations applied (5–20 kcal/mol) on the ΔΔG values calculated for the unphosphorylated dimeric model (based on PDB 2PHM). A, Seventy-four PKU mutations classified into three mutant types according to their in vitro residual activity (as defined in table 1). Lines are linear fits as follows: type I (blackened triangles), y₀=3.9±0.1 kcal/mol, m=0.07±0.01; type II (unblackened circles), y₀=3.6±0.1 kcal/mol, m=0.44±0.01; type III (blackened circles), y₀=8.4±0.4 kcal/mol, m=1.21±0.03. B, Forty-one PKU mutations classified into three phenotypic groups according to their associated patient phenotypes (as defined in table 2). Lines are linear fits as follows: severe PKU (blackened triangles), y₀=7.2±0.5 kcal/mol, m=1.19±0.04; mild PKU (unblackened circles), y₀=4.8±0.1 kcal/mol, m=0.19±0.01; and MHP (blackened circles), y₀=2.6±0.1 kcal/mol, m=0.00±0.01.

Figure 4.

Mutation-dependent destabilization and in vivo patient phenotype. A, Calculated effect on ΔΔG (in kcal/mol dimer) for 46 PKU mutants classified by phenotypic groups at a 5-kcal/mol penalty. The mean±SD ΔΔG values (blackened circles) for the three phenotypic groups, calculated using 41 mutations (unblackened triangles), were 2.8±2.2 kcal/mol, 5.7±2.7 kcal/mol, and 13.0±9.5 kcal/mol for MHP (group 1), mild (group 2), and severe (group 3) phenotypes, respectively. Five outliers (blackened triangles) have been removed (see text for details) for the calculation of the mean values. B and C, Means±SDs of m and y₀ values for the different phenotypic groups, calculated using individual fits for each mutation. P values are obtained from one-way ANOVA; P<.05 is considered statistically significant.

Figure 5.

Mutation-dependent destabilization represented on the dimeric structure (PDB 2PHM). All mutations summarized in table 7 have been represented according to the following criteria. Mutated residues are colored according to (A) the predicted phenotype: MHP (blue), mild (yellow), and severe (red); (B) the y₀ parameter: y₀⩽3 kcal/mol (blue), 3<y₀⩽7 kcal/mol (yellow), and y₀>7 kcal/mol (red); and (C) the m parameter: m⩽0.1 (blue), 0.1<m⩽0.3 (yellow), and m>0.3 (red). For the cases with several mutations in the same residue, that position has been colored according to the mutation with the most severe effect.

Figure 6.

Hotspots for destabilization. The modeled composite subunit structure of human PAH is shown in ribbon representation, with regions 235–281 corresponding to exon 7 (green) and regions 282–330 including exons 8 and 9 (orange). Some residues important for interdomain interactions in the monomer (Ser70–Arg71, P122, Leu308–L311, and Arg408) are shown in dark red. The nonheme iron at the active site is shown as a yellow sphere, and the coordinating residues His285, His290, and Glu330 are shown as orange sticks. See the main text for details.