DNAJC12 stabilizes activated phenylalanine hydroxylase and reduces the concentration of L-Phe needed for activation

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH4)-dependent enzyme that converts L-phenylalanine (L-Phe) to L-tyrosine. PAH dysfunction leads to the accumulation of L-Phe in the blood (hyperphenylalaninemia; HPA), which may reach neurotoxic levels, resulting in phenylketonuria (PKU). PKU is associated with pathogenic variants in PAH, most causing misfolding and instability, leading to decreased levels of PAH protein and activity. Recently, variants in the class C J-domain protein DNAJC12 have also been associated with HPA in patients, demonstrating the importance of protein homeostasis regulation for proper PAH function. DNAJC12 and PAH have previously been reported to interact, but the molecular and structural mechanisms behind complex formation have remained unclear. In this work, we show that DNAJC12 binds to PAH, but presents higher affinity for its L-Phe activated form, which resembles the conformation of unliganded tyrosine hydroxylase, a structurally and functionally-related enzyme that also binds to DNAJC12. At saturation, four monomers of DNAJC12 bind and stabilize the PAH tetramer, protecting it from aggregation and lowering the L-Phe concentration necessary for substrate-induced activation, without affecting the interaction of the enzyme with its cofactor BH4. Importantly, DNAJC12 also stabilizes and delays the aggregation of the PKU-associated variant PAH-p.R261Q. Furthermore, L-Phe activated wild-type or variant PAH is required to stimulate Hsc70 ATPase activity. SIGNIFICANCE STATEMENTDeficiencies in the cochaperone DNAJC12 have recently been linked to hyperphenylalaninemia, dystonia and intellectual disabilities as DNAJC12 regulates the proteostasis of the aromatic amino acid hydroxylases, including phenylalanine hydroxylase (PAH). This study explores the mechanisms of the PAH:DNAJC12 interaction and examines the functional effects of their complex formation on PAH activity and stability. These findings enhance our understanding on the pathogenic mechanisms behind DNAJC12 variants and provide insights that could guide the development of drugs targeting this protein-protein interaction.