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
1PHZ 1998-11-11T00:00:00+0000 2.2 STRUCTURE OF PHOSPHORYLATED PHENYLALANINE HYDROXYLASE
5DEN 2015-08-25T00:00:00+0000 2.9 The First Structure of a Full-Length Mammalian Phenylalanine Hydroxylase Reveals the Architecture of an Auto-inhibited Tetramer
2PHM 1998-11-11T00:00:00+0000 2.6 STRUCTURE OF PHENYLALANINE HYDROXYLASE DEPHOSPHORYLATED
5FGJ 2015-12-20T00:00:00+0000 3.6 Structure of tetrameric rat phenylalanine hydroxylase, residues 1-453
5EGQ 2015-10-27T00:00:00+0000 2.5 Structure of tetrameric rat phenylalanine hydroxylase mutant R270K, residues 25-453
2PAH 1998-05-26T00:00:00+0000 3.1 TETRAMERIC HUMAN PHENYLALANINE HYDROXYLASE
1DMW 1999-12-15T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF DOUBLE TRUNCATED HUMAN PHENYLALANINE HYDROXYLASE WITH BOUND 7,8-DIHYDRO-L-BIOPTERIN
6N1K 2018-11-08T00:00:00+0000 3.06 Full-length human phenylalanine hydroxylase (PAH) in the resting state
6HPO 2018-09-21T00:00:00+0000 1.67 Crystallographic structure of the catalytic domain of Human Phenylalanine Hydroxylase (hPAH CD) in complex with iron at 1.6 Angstrom
4PAH 1998-08-20T00:00:00+0000 2.0 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND NOR-ADRENALINE INHIBITOR

Relevant UniProtKB Entries

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