Toward mechanistic models for genotype-phenotype correlations in phenylketonuria using protein stability calculations.


Abstract

Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild-type PAH of 20 selected PKU-linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild-type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH4 ), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single-site missense variants using the full-length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease-linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.

Submission Details

ID: VTn9KTDA4

Submitter: Shu-Ching Ou

Submission Date: March 22, 2019, 11:40 a.m.

Version: 1

Publication Details
Scheller R;Stein A;Nielsen SV;Marin FI;Gerdes AM;Di Marco M;Papaleo E;Lindorff-Larsen K;Hartmann-Petersen R,Hum Mutat (2019) Toward mechanistic models for genotype-phenotype correlations in phenylketonuria using protein stability calculations. PMID:30648773
Additional Information

Study Summary

Number of data points 8002
Proteins Phenylalanine-4-hydroxylase
Unique complexes 7981
Assays/Quantities/Protocols Computational Protocol: ΔΔG ; Computational Protocol: Enzyme activity (% of WT)
Libraries Variants for PAH

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1J8U 2002-05-22 1.5 Catalytic Domain of Human Phenylalanine Hydroxylase Fe(II) in Complex with Tetrahydrobiopterin
6HPO 2019-06-05 1.67 Crystallographic structure of the catalytic domain of Human Phenylalanine Hydroxylase (hPAH CD) in complex with iron at 1.6 Angstrom
1J8T 2002-05-22 1.7 Catalytic Domain of Human Phenylalanine Hydroxylase Fe(II)
5FII 2016-03-30 1.8 Structure of a human aspartate kinase, chorismate mutase and TyrA domain.
3PAH 1999-04-27 2.0 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND ADRENALINE INHIBITOR
1MMK 2003-09-04 2.0 Crystal structure of ternary complex of the catalytic domain of human phenylalanine hydroxylase ((FeII)) complexed with tetrahydrobiopterin and thienylalanine
4PAH 1999-04-27 2.0 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND NOR-ADRENALINE INHIBITOR
1DMW 2000-03-24 2.0 CRYSTAL STRUCTURE OF DOUBLE TRUNCATED HUMAN PHENYLALANINE HYDROXYLASE WITH BOUND 7,8-DIHYDRO-L-BIOPTERIN
1PAH 1999-01-13 2.0 HUMAN PHENYLALANINE HYDROXYLASE DIMER, RESIDUES 117-424
1MMT 2003-09-04 2.0 Crystal structure of ternary complex of the catalytic domain of human phenylalanine hydroxylase (Fe(II)) complexed with tetrahydrobiopterin and norleucine
5PAH 1999-04-27 2.1 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND DOPAMINE INHIBITOR
1TDW 2004-11-30 2.1 Crystal structure of double truncated human phenylalanine hydroxylase BH4-responsive PKU mutant A313T.
4ANP 2012-04-11 2.11 Crystal structure of human phenylalanine hydroxylase in complex with a pharmacological chaperone
6PAH 1999-04-27 2.15 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND L-DOPA (3,4-DIHYDROXYPHENYLALANINE) INHIBITOR
1TG2 2004-11-30 2.2 Crystal structure of phenylalanine hydroxylase A313T mutant with 7,8-dihydrobiopterin bound
1PHZ 1999-04-30 2.2 STRUCTURE OF PHOSPHORYLATED PHENYLALANINE HYDROXYLASE
1KW0 2003-01-28 2.5 Catalytic Domain of Human Phenylalanine Hydroxylase (Fe(II)) in Complex with Tetrahydrobiopterin and Thienylalanine
5EGQ 2016-05-18 2.5 Structure of tetrameric rat phenylalanine hydroxylase mutant R270K, residues 25-453
2PHM 1999-04-30 2.6 STRUCTURE OF PHENYLALANINE HYDROXYLASE DEPHOSPHORYLATED
5DEN 2016-02-17 2.9 The First Structure of a Full-Length Mammalian Phenylalanine Hydroxylase Reveals the Architecture of an Auto-inhibited Tetramer
6N1K 2019-05-22 3.06 Full-length human phenylalanine hydroxylase (PAH) in the resting state
2PAH 1999-10-06 3.1 TETRAMERIC HUMAN PHENYLALANINE HYDROXYLASE
6HYC 2019-06-05 3.18 The structure of full-length human phenylalanine hydroxylase in complex with the cofactor and negative regulator tetrahydrobiopterin
5FGJ 2016-05-18 3.6 Structure of tetrameric rat phenylalanine hydroxylase, residues 1-453

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
399.2 A,B,C,D Phenylalanine-4-hydroxylase P04176 PH4H_RAT
394.0 A,B,C,D Phenylalanine-4-hydroxylase P16331 PH4H_MOUSE
370.0 A,B,C,D Phenylalanine-4-hydroxylase P00439 PH4H_HUMAN
364.8 A,B,C,D Phenylalanine-4-hydroxylase Q2KIH7 PH4H_BOVIN