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- EMDB-4605: Human Phenylalanine Hydroxylase (hPAH) apo structure -

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Basic information

Entry
Database: EMDB / ID: EMD-4605
TitleHuman Phenylalanine Hydroxylase (hPAH) apo structure
Map data
Sample
  • Complex: apo-hPAH
    • Protein or peptide: Phenylalanine-4-hydroxylase
Function / homology
Function and homology information


Phenylketonuria / Phenylalanine metabolism / phenylalanine 4-monooxygenase / phenylalanine 4-monooxygenase activity / tyrosine biosynthetic process / catecholamine biosynthetic process / L-phenylalanine catabolic process / amino acid biosynthetic process / iron ion binding / cytosol
Similarity search - Function
Phenylalanine-4-hydroxylase, tetrameric form / Eukaryotic phenylalanine-4-hydroxylase, catalytic domain / Tyrosine 3-monooxygenase-like / ACT domain / Aromatic amino acid hydroxylase, iron/copper binding site / Biopterin-dependent aromatic amino acid hydroxylases signature. / Aromatic amino acid hydroxylase / Aromatic amino acid hydroxylase, C-terminal / Aromatic amino acid monoxygenase, C-terminal domain superfamily / Aromatic amino acid hydroxylase superfamily ...Phenylalanine-4-hydroxylase, tetrameric form / Eukaryotic phenylalanine-4-hydroxylase, catalytic domain / Tyrosine 3-monooxygenase-like / ACT domain / Aromatic amino acid hydroxylase, iron/copper binding site / Biopterin-dependent aromatic amino acid hydroxylases signature. / Aromatic amino acid hydroxylase / Aromatic amino acid hydroxylase, C-terminal / Aromatic amino acid monoxygenase, C-terminal domain superfamily / Aromatic amino acid hydroxylase superfamily / Biopterin-dependent aromatic amino acid hydroxylase / Biopterin-dependent aromatic amino acid hydroxylase family profile. / ACT domain profile. / ACT domain / ACT-like domain
Similarity search - Domain/homology
Phenylalanine-4-hydroxylase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsAlcorlo-Pages M / Flydal MI / Martinez-Caballero S / Martinez A / Hermoso JA / Fernandez-Leiro R
Funding support Spain, 1 items
OrganizationGrant numberCountry
Spanish Ministry of Economy and CompetitivenessBFU2017-87316-P (to RFL) Spain
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin.
Authors: Marte Innselset Flydal / Martín Alcorlo-Pagés / Fredrik Gullaksen Johannessen / Siseth Martínez-Caballero / Lars Skjærven / Rafael Fernandez-Leiro / Aurora Martinez / Juan A Hermoso /
Abstract: Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental ...Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH in the precatalytic state. Crystal structures, solved at 3.18-Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH BH forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic Fe Upon BH binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH Importantly, BH binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH from the precatalytic toward the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted molecular dynamics simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations.
History
DepositionFeb 11, 2019-
Header (metadata) releaseFeb 27, 2019-
Map releaseJun 5, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4605.png

Downloads & links

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Map

FileDownload / File: emd_4605.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.023875885 - 0.048971385
Average (Standard dev.)0.0000858175 (±0.003215155)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 174.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z174.400174.400174.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0240.0490.000

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Supplemental data

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Mask #1

Fileemd_4605_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Multibody refinement body 2

Fileemd_4605_additional_1.map
AnnotationMultibody refinement body 2
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_4605_additional_2.map
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Half map: #2

Fileemd_4605_half_map_1.map
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Half map: #1

Fileemd_4605_half_map_2.map
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Sample components

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Entire : apo-hPAH

EntireName: apo-hPAH
Components
  • Complex: apo-hPAH
    • Protein or peptide: Phenylalanine-4-hydroxylase

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Supramolecule #1: apo-hPAH

SupramoleculeName: apo-hPAH / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: homo-tetramer
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria)
Molecular weightTheoretical: 207 KDa

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Macromolecule #1: Phenylalanine-4-hydroxylase

MacromoleculeName: Phenylalanine-4-hydroxylase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: phenylalanine 4-monooxygenase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria)
SequenceString: MSTAVLENPG LGRKLSDFGQ ETSYIEDNCN QNGAISLIFS LKEEVGALAK VLRLFEENDV NLTHIESRPS RLKKDEYEFF THLDKRSLPA LTNIIKILRH DIGATVHELS RDKKKDTVPW FPRTIQELDR FANQILSYGA ELDADHPGFK DPVYRARRKQ FADIAYNYRH ...String:
MSTAVLENPG LGRKLSDFGQ ETSYIEDNCN QNGAISLIFS LKEEVGALAK VLRLFEENDV NLTHIESRPS RLKKDEYEFF THLDKRSLPA LTNIIKILRH DIGATVHELS RDKKKDTVPW FPRTIQELDR FANQILSYGA ELDADHPGFK DPVYRARRKQ FADIAYNYRH GQPIPRVEYM EEEKKTWGTV FKTLKSLYKT HACYEYNHIF PLLEKYCGFH EDNIPQLEDV SQFLQTCTGF RLRPVAGLLS SRDFLGGLAF RVFHCTQYIR HGSKPMYTPE PDICHELLGH VPLFSDRSFA QFSQEIGLAS LGAPDEYIEK LATIYWFTVE FGLCKQGDSI KAYGAGLLSS FGELQYCLSE KPKLLPLELE KTAIQNYTVT EFQPLYYVAE SFNDAKEKVR NFAATIPRPF SVRYDPYTQR IEVLDNTQQL KILADSINSE IGILCSALQK IK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMHepesHepes
200.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0001 kPa / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Details: 3microL, no incubation, 2 seconds blot, no wait before plunging.
DetailsThe thawed sample was run through SEC (S200I) prior to grid preparation

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-80 / Number grids imaged: 1 / Number real images: 1493 / Average exposure time: 12.0 sec. / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1000000
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: INSILICO MODEL / In silico model: ab initio model (RELION 3.0)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 160000
FSC plot (resolution estimation)

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