The N-Terminal Sequence of Tyrosine Hydroxylase Is a Conformationally Versatile Motif That Binds 14-3-3 Proteins and Membranes

Abstract

Tyrosine hydroxylase (TH) catalyzes the rate limiting step in the synthesis of catecholamine neutrotransmitters, and a reduction of TH activity is associated with several neurological diseases. Human TH is regulated, among other mechanisms, by Ser19-phosphorylation-dependent interaction with 14-3-3 proteins. The N-terminal sequence (residues 1-43), which corresponds to an extension to the TH regulatory domain, also interacts with negatively charged membranes. By using X-ray crystallography together with molecular dynamics simulations and structural bioinformatics analysis, we have probed the conformations of the Ser19-phosphorylated N-terminal peptide (THp-(1-43)), bound to 14-3-3γ, free in solution and bound to a phospholipid bilayer, and of the unphosphorylated peptide TH-(1-43) both free and bilayer-bound. As seen in the crystal structure of THp-(1-43) complexed with 14-3-3γ, the region surrounding pSer19 adopts an extended conformation in the bound state, whereas THp-(1-43) adopts a bent conformation when free in solution, with higher content of secondary structure and higher number of internal hydrogen bonds. TH-(1-43) in solution presents the highest mobility and least defined structure of all forms studied, and it shows an energetically more favorable interaction with membranes relative to THp-(1-43). Cationic residues, notably Arg15 and Arg16, which are the recognition sites of the kinases phosphorylating at Ser19, are also contributing to the interaction with the membrane. Our results reveal the structural flexibility of this region of TH, in accordance with the functional versatility and conformational adaptation to different partners. Furthermore, this structural information has potential relevance for the development of therapeutics for neurodegenerative disorders, through modulation of TH-partner interactions.

Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity

“…21 and this work). Experimental20 and computational28 studies on TH-(1-43) peptide revealed an increased α-helical content in this stretch when bound to negatively charged membranes, a change that may be less easy to observe in the full-length enzyme, a tetramer of 497 amino acids per subunit. Many other protein-membrane interactions appear to involve amphipathic helices3, and altogether, our results support a model in which hTH1 associates to the membrane via the α-helical N-terminus and aggregates through inter-tetramer cross-β interactions involving the catalytic domains (Fig.…”

“…Previous studies have determined the interaction of TH and its N-terminal sequence with negatively charged membranes202128, however the effect of this interaction on membrane integrity has not been investigated. We assessed the effect of TH on the integrity of negatively charged membranes, using purified recombinant hTH1, which is the most abundant splicing isoform of TH in brain and adrenal medulla17, and measuring the fluorescence of released 8-aminonaphtalene-1,3,6-trisulphonic acid, disodium salt (ANTS) from LUVs, which will also be referred here to as liposomes.…”

De Novo Mutations in YWHAG Cause Early-Onset Epilepsy

“…57,60 An individual (ND27637) with Lennox-Gastaut syndrome and a de novo p.Asp129Glu variant is present in the Epi4K-EPGP dataset. 4 The Asp129 residue is also located within the 14-3-3g binding groove ( Figure 1) and plays an important role in determining the orientation of the phosphorylated peptides.…”

Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins

“…Lines indicate protein-glycopeptide hydrogen bonds (Left), and blue mesh indicates the initial Fo-Fc map, calculated before glycopeptide addition, contoured at 2.9 σ (14-3-3γ) or 2.5 σ (14-3-3β/α) (Right). (F) Overlay of 14-3-3γ-glycopeptide (from D) and 14-3-3γphosphopeptide from tyrosine hydroxylase (magenta protein, red phosphopeptide; PDB 4J6S)(75). O-GlcNAc and O-phosphate moieties are highlighted by transparent surfaces.…”