Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.
Tyrosine hydroxylase (TH) is the
enzyme catalyzing the rate-limiting
step in the synthesis of dopamine in the brain. Developing enzyme
replacement therapies using TH could therefore be beneficial to patient
groups with dopamine deficiency, and the use of nanocarriers that
cross the blood–brain barrier seems advantageous for this purpose.
Nanocarriers may also help to maintain the structure and function
of TH, which is complex and unstable. Understanding how TH may interact
with a nanocarrier is therefore crucial for the investigation of such
therapeutic applications. This work describes the interaction of TH
with porous silicon nanoparticles (pSiNPs), chosen since they have
been shown to deliver other macromolecular therapeutics successfully
to the brain. Size distributions obtained by dynamic light scattering
show a size increase of pSiNPs upon addition of TH and the changes
observed at the surface of pSiNPs by transmission electron microscopy
also indicated TH binding at pH 7. As pSiNPs are negatively charged,
we also investigated the binding at pH 6, which makes TH less negatively
charged than at pH 7. However, as seen by thioflavin-T fluorescence,
TH aggregated at this more acidic pH. TH activity was unaffected by
the binding to pSiNPs most probably because the active site stays
available for catalysis, in agreement with calculations of the surface
electrostatic potential pointing to the most positively charged regulatory
domains in the tetramer as the interacting regions. These results
reveal pSiNPs as a promising delivery device of enzymatically active
TH to increase local dopamine synthesis.
Tyrosine hydroxylase (TH) is a highly regulated enzyme that catalyses the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines. Mutations and dysfunction in this enzyme lead to DA deficiency and parkinsonisms of different severity. An understanding of TH deficiency at the level of structure and stability has been lacking to date, as only structures of truncated TH forms have been available. Here, we used cryoEM to determine the first high-resolution structure of full-length human tetrameric TH in the absence (3.4 Å) and presence (3.8 Å) of the end-product and feedback inhibitor DA bound to the active site. We show that upon DA binding, an α-helix (residues 39-59) included within the flexible N-terminal tail of the regulatory domain, is internalized in the active site. The observed structural changes reveal the molecular basis of the inhibitory and stabilizing DA effect, reversible by TH S40-phosphorylation, which are crucial regulatory mechanisms for catecholamine and TH homeostasis.
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