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The N-terminal sequence of tyrosine hydroxylase is a conformationally versatile motif that binds 14-3-3 proteins and membranes - PubMed

  • ️Wed Jan 01 2014

The N-terminal sequence of tyrosine hydroxylase is a conformationally versatile motif that binds 14-3-3 proteins and membranes

Age Aleksander Skjevik et al. J Mol Biol. 2014.

Abstract

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamine neurotransmitters, and a reduction in 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.

Keywords: MD; MM/PBSA; PC; POPS; RU; SPR; TH; X-ray crystallography; free energy of binding; molecular dynamics; molecular dynamics simulations; molecular mechanics Poisson–Boltzmann surface area; palmitoyl-oleoyl phosphatidylserine; phosphatidylcholine; phospholipid bilayers; phosphorylation; response units; surface plasmon resonance; tyrosine hydroxylase.

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Figures

Figure 1
Figure 1. The crystal structure of 14-3-3γ

A) The two 14-3-3γ dimers (chains A–D) in the asymmetric unit (from this work; PDB 4J6S), as ribbons coloured by secondary structure, complexed to the TH peptides, represented by sticks coloured by atom type. B) A dimer of 14-3-3γ (chains A and B in PDB 4J6S), as pale green ribbons, with the TH peptides as bright green sticks, superimposed with the equivalent dimeric structure of 14-3-3γ from PDB 2B05, as pale orange ribbons, with the phosphopeptide (RAIpSLP) represented by red sticks.

Figure 2
Figure 2. Representative structure and interactions from the 14-3-3γ:THp peptide complex simulation

Panel A depicts the 14-3-3γ dimer as a grey surface with THp-(Ala11-Asp22) bound to the left monomer and THp-(Phe14-Ala26) bound to the right monomer. The peptides are coloured as continuous gradients going from blue (N-terminal) to red (C-terminal). In addition, the Ser19 residue in each THp fragment is highlighted in the form of a stick model. Panels B and C (THp-(Ala11-Asp22) and THp-(Phe14-Ala26), respectively) are close-ups revealing the most important hydrogen bond interactions (dashed lines) taking place in the panel A complex structure. 14-3-3γ residues are shown as ball-and-stick representations and THp residues as sticks. Water has been removed for clarity.

Figure 3
Figure 3. MM/PBSA per residue decomposition energies for the crystallographic 14-3-3γ:THp peptide complex after simulation

The calculations were performed for the two TH peptide fragments (Panels A and B) and for the corresponding monomers of 14-3-3γ (Panels C and D), as calculated from 1000 frames representing the last 50 ns of the crystallographic 14-3-3γ:THp peptide complex simulation (System A in Table 1). ΔGdecomp corresponds to the binding enthalpy (ΔG’ in Equation 4 and Table 3) minus the non-decomposable, but most likely negligible, non-polar contribution to the solvation energy (ΔGnon-polar in Equation 4 and Table 3). Panels A and C are sorted by amino acid sequence. Panels B and D are sorted in ascending order based on the decomposition energies for THp-(14-26) and 14-3-3γ monomer 1, respectively. In terms of 14-3-3γ, only the residues making contributions greater than ±0.5 kcal/mol are shown.

Figure 4
Figure 4. B factors for the α-carbons of the N-terminal fragments of TH and representative structure and interactions from the aqueous THp-(1-43) simulation

B factors (Panel A) were calculated from the aqueous simulations for TH-(1-43) (black line) and THp-(1-43) (red line) and from the peptide/membrane simulations for TH-(1-43) (blue line) and THp-(1-43) (green line), and are represented as a function of residue number. Panel B: Representative structure of THp-(1-43) which shows the intrapeptide sidechain-sidechain hydrogen bonds with occupancies >25% (dashed lines) in the aqueous simulation. The peptide is coloured as a continuous gradient going from blue (N-terminal) to red (C-terminal). The last 100 ns of each simulation were used for both analyses.

Figure 5
Figure 5. Binding of TH-(1-43) and THp-(1-43) to POPS liposomes by surface plasmon resonance and circular dichroism

A) Representative sensorgrams for the interaction of TH-(1-43) with POPS liposomes on a L1 sensor chip at increasing concentrations of peptide. B) Peptide concentration dependency of TH-(1-43) (black) and THp-(1-43) (red) when bound to POPS liposomes. RUs were measured as a function of peptide concentration. S0.5 values were extracted from the hyperbolic, single-rectangular, two-parameter curve fitting and resulted in S0.5 of 53.3 ± 9.2 µM and 48.2 ± 7.4 µM for TH-(1-43) and THp-(1-43), respectively. C) Far-UV CD spectra of 40 µM TH-(1-43) (black lines) or THp-(1-43) (red lines) without (solid lines) or with (dotted lines) POPS liposomes (0.6 mM phospholipid). Samples were prepared in 10 mM Na-Hepes, 150 mM NaCl, pH 7.4, and spectra were recorded at 25 °C.

Figure 6
Figure 6. Representative structures and interactions from the peptide/membrane simulations

TH-(1-43)/POPS is presented in panels A and B, and THp-(1-43)/POPS is presented in panels C and D. Panels A and C show the peptide – coloured as a continuous gradient going from blue (N-terminal) to red (C-terminal) – together with most of the peptideinteracting leaflet of the POPS bilayer, the orange spheres representing the head group phosphorus atoms. Panels B and D provide close-ups of the most important hydrogen bond interactions (dashed lines) in each case, where the relevant TH residues are displayed as ball-and-stick models and the POPS lipids as sticks. Water and ions have been removed for clarity.

Figure 7
Figure 7. MM/PBSA per residue decomposition energies for TH-(1-43) and THp-(1-43) from the peptide/membrane simulations

The energies are computed using 1000 frames extracted from the last 100 ns of each peptide/membrane simulation (systems D and E in Table 1). ΔGdecomp = ΔG’ – ΔGnon-polar. The black bars represent the energies derived for each residue in TH-(1-43), and the grey bars are the THp-(1-43) equivalents. A) Sorted by amino acid sequence; B) Sorted in ascending order according to the TH-(1-43) decomposition energies.

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