Computational protocol: Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

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Protocol publication

[…] SAXS measurements were carried out on the EMBL P12 synchrotron BioSAXS beamline at PETRA III/DESY, Hamburg, Germany on TH1(ZZ) and TH1(MBP) in 20 mM Na-HEPES pH 7.0, 200 mM NaCl (0.5–2.0 mg/mL, 9–36 μM subunit). DA in stoichiometric amounts compared to an enzyme subunit was added, when indicated. Data were collected at 20 °C using an X-ray wavelength of 0.124 nm and an exposure time of 45 ms/frame. 20 consecutive frames were collected for each sample; the sample was flowing through the capillary during the measurement. Frames were controlled for radiation damage and averaged. The data were recorded using a PILATUS 2M detector (Dectris, Baden, Switzerland) at a sample-detector distance of 3.0 m, covering a momentum transfer range, s (4πsinθ/λ) of 0.02–4.8 nm−1, where 2θ is the scattering angle and λ the wavelength and s is in units of nm−1.Data processing and analysis were carried out using the ATSAS package. Solvent scattering was identically measured from the corresponding buffer before and after each sample, and the average background scattering was subtracted with PRIMUS. Rg was determined using Guinier analysis. The maximum particle dimension Dmax and the distance distribution function p(r) were calculated using GNOM. Molecular weights were estimated based on forward scattering I(0) from the sample, compared to standard samples of either glucose isomerase or BSA. [...] To obtain unbiased shape information for full-length TH1, chain-like models were built ab initio using GASBOR. P222 symmetry, as seen in crystal structures of tetrameric species of truncated TH and homologues, was used during modelling.BUNCH was used for SAXS-based rigid body modelling of TH1, using existing crystal and NMR structures as rigid body subunits interconnected by flexible loops. We divided the TH1 subunit chain into 3 rigid bodies interconnected by flexible loops: (1) the tetramerization helix (coordinates from PDB ID 1TOH); (2) catalytic domain (coordinates from PDB ID 1TOH); (3) ACT domain (atomic coordinates obtained from PDB ID 2MDA). To maintain known interfaces (from the crystal and NMR structures) between domains in the simulated annealing protocol we employed distance restraints between (1) two adjacent ACT domains, (2) two adjacent catalytic domains, and (3) the tetramerization bundle. CRYSOL was subsequently used to calculate the final fit to the experimental SAXS data. P222 symmetry and default values for the simulated annealing protocols in BUNCH were used, with 100 temperature steps and a maximum of 24,850 iterations at each temperature.The Ala-rich N-terminal segment Ala45-Ala70 upstream from the ACT domain in TH was issued to extensive replica exchange molecular dynamics simulations to obtain a representative three-dimensional structure of this fragment. A linear all-atom model of the fragment was generated using AmberTools, employing the Amber99SB force field and implicit solvent model using generalized born. The replica exchange simulations were run over 20 temperatures spanning from 270 to 509 K. Each replica was heated to its respective starting temperature during 200 ps. The replica exchange simulations consisted of 10,000 exchanges, each of 4 ps (2 × 107 steps for each replica). The simulations were carried out using multisander in the Amber package. The resulting conformations at 300 K were extracted and clustered using cpptraj. The representative structures from the most frequently populated cluster were extracted and included adjacent to the ACT domains in the model prior to the SAXS-based rigid body simulation. Independent of the replica exchange simulation, PSIPRED was used to predict the secondary structure elements in this fragment. The N-terminal residues Gly36-Asp44 of TH1 align well with the equivalent residues of PAH (see ), with high sequence identity, including the motif [SXIED]. Based on this alignment, the residues Gly36-Ala45 were modelled in TH1 corresponding to the PAH structure (PDB ID 5DEN). […]

Pipeline specifications

Software tools ATSAS, GASBOR, CRYSOL, AMBER, PSIPRED
Application Small-angle scattering
Organisms Homo sapiens, Dipturus trachyderma
Chemicals Catecholamines, Cobalt, Dopamine, Iron, Tyrosine