Human brain protein 14-3-3 beta isoform; human tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase

Target ID:

YWHABA

Aliases:

GW128HS1KCIP-1

Deposition Date:

Tuesday 26th April 2005

Families:

Signalling proteins

Related Diseases:

SignallingCancerNeurobiology

Structure type:

apo

Download Coordinates:

2BQ0

Authors:

X.YANG,J.M.ELKINS,O.FEDOROV,E.J.LONGMAN,F.SOBOTT,L.J.BALL,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,D.A.DOYLE

Site:

Oxford

ICM Datapack:

ICM Datapack

2BQ0

tabs

Structure Details

Phosphorylation of a target protein by a specific kinase
alters the characteristics of the protein in a number of ways.
One alteration is that the attached phosphate group presents a handle
to which other molecules can bind thus forming a complex.
This is generally how the 14-3-3 molecules interact with their proteins.

14-3-3 proteins are highly conserved eukaryotic proteins
that bind to phosphorylated Ser (pS) residues
via the two consensus sequences of RXXXpSXP and R(S/X)XpSXP.
The functional unit is a dimer and their interaction with the phosphorylated target
can result in stabilisation of the protein, alteration of enzyme activity,
localisation within the cell, prevention of dephosphorylation and either blockage
or mediation of protein interactions.

The symmetric dimer conformation as seen for the previous 14-3-3 structures.
The circles represent the locations of the peptide binding sites.

Previously the structure of the 14-3-3 proteins were believed to be rigid
as the conformation of both the bound and unbound forms were essentially identical.
This lead to the idea that the target proteins undergo a conformational change
upon binding and that the 14-3-3 molecule remains unchanged.
The 14-3-3 b structure presented here changes this view.
The structure is again a dimer but this time for one of the monomers three helices,
that form part of the phospho-peptide binding site,
have undergone a rigid body rotation as illustrated below left.

Overlapping of chain A (grey) with chain B (red) shows that the three helices
that normally form the top of the peptide binding site have rotated away
from the conserved structural base in chain B, opening up the peptide binding site.
This structural change implies that the 14-3-3 molecules
do not interact in a rigid way with their target proteins but actively
take part in the process changing their conformation as they go from the unbound to the bound state.
A schematic picture of the mechanism is illustrated to the left.

Materials and Methods

YWHAB

PDB:2BQ0

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:4507949
Entry Clone Source:MGC
SGC Clone Accession:
Tag:C-terminal hexahistidine tag + TEV cleavage site: egenlyfqslehhhhhh
Host:BL-21(DE3) R3 (phage resistant strain)

Construct

Prelude:
Sequence:
Vector:pET21-TvHR2

Growth

Medium:
Antibiotics:
Procedure:A frozen glycerol stock of transformed E. coli cells was used to inoculate 1 litre of TB plus 100 mg/ml ampicillin. When OD600 reached ~0.5 the temperature was shifted down from 37oC to 25oC for 1 hour before induction with the addition of 1 mM IPTG. Protein expression was allowed to carry on for a futher 4 hours before harvest.

Purification

Procedure
Column 1: Low pressure chromatography using Bio-Rad Econo column (2.5 cm x 13 cm).

Ni-NTA column purification. Buffers: Wash buffer I (WB1): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % Glycerol, 10 mM Imidazole pH 8.0. Wash Buffer II (WBII): 50 mM Hepes pH 8.0, 300mM NaCl, 30 mM Imidazole pH 8.0. Elution buffer (EB): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % Glycerol, 250 mM Imidazole pH 8.0.

Procedure: Total volume of Ni-NTA added to BioRad drip column: 4 mls (50%). Resin washed with 12.5 ml of WB1. The supernatant was applied to a column using 5 ml pipette and allowed to pass over the resin. The flow through was collected in a 50 ml falcon tube and applied once more to the column. Two wash steps followed. Wash with 12.5 ml of WBI. Wash with 12.5 ml column vols of WBII. Elute with 14 mls of EB into 7x2 ml fractions. At this stage the purity of the protein was greater than 95 % based on SDS-PAGE analysis. The C-terminal hexahistidine tag was removed by TEV protease treatment. The TEV protease, a hexa-histidine-tagged construct, was over-expressed and purified in-house to a final concentration of 2.5 mg/mL.

Extraction

Procedure
All extraction steps were carried out at 4 ºC.

Extraction buffer (EX): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % Glycerol, 10 mM Imidazole pH 8.0. 1 tablet protein inhibitor in 10ml EX buffer was added to the 1L growth pellet. Total vol: 45 mL (estimate). Cell breakage: 5 passes through the Emulsiflex C5 high pressure homogeniser. Total vol: 50 mls (estimate). Centrifuge for 30 mins at 16000 rpm and 4°C to remove cell debris. Discard pellet.
Concentration:
Ligand
MassSpec:
Crystallization:The TEV protease cleaved YWHAB was concentrated to 28 mg/mL and distributed into 18x50 mL aliquots before being frozen at -80°C. Crystals grew from a 1:2 ratio mix of protein-to-reservoir (0.05 M magnesium chloride, 0.1 M HEPES pH 7.5, 30 %v/v polyethylene glycol MME 550)
NMR Spectroscopy:
Data Collection:
Data Processing: