pyk_the_second_secret
TRANSCRIPT
Pyruvate Kinase and the Second Secret of Life
Pyruvate Kinase and the Second Secret of Pyruvate Kinase and the Second Secret of LifeLife
‘The second secret of life’
(Monod, 1963)
Allostery
the comparison of how one ligand
binds in the absence, versus the presence, of a second ligand
The first secret of life
‘The structure of DNA’
(Perutz)
Monod Wyman, Changeux
(MWC) model for allostery:proteins
adopt various conformations in thermal equilibrium
and allosteric
regulators stabilize selected conformations
Allostery: the basis of protein communication
Signal Transduction MetabolismTranscription
p53 CDK
pyruvate
kinaseMdm2
cyclin/phosphate
fructose bisphosphate
DNA
ATP/protein substrate
ADP/phosphenolpyruvate
PGAM
PYKG
LYC
OSO
ME
ATPADP
glucoseglucose
glc-6-P
fru-6-PATPADP
fru-1,6-BPPFK
pyruvate
G-3-P
GA-3-P
ADPATP
G-1,3-BP
ADPATP
G-3-P
G-2-P
PEP
Pi
1. Anti-parasitic Glycolysis
is essential for ATP
production in T. brucei
2.
AnticancerPYK (M2 isoform) is upregulatedIn all cancer cells
PYKs
as drug targets
LeishmaniaLeishmania
mexicanamexicana PYK PYK
regulated by F26BPregulated by F26BP
M2 PYK (embryonic M2 PYK (embryonic or cancer cells) or cancer cells) regulated by F16BPregulated by F16BP
M1 PYK (adult tissue) M1 PYK (adult tissue) constitutively activeconstitutively active
O
O
O
P
O
OO
O O
PO
OO
O
6
2
O
O
OP
O
P O
O
O
O
OO
O O
6 1
Phosphoenolpyruvate Pyruvate
ADP + + ATPO
O
O
PO
O
O O
O
O
T to R Conformational transition of Leishmania Pyruvate
Kinase
(LmPYK)
O
O
OP
O
O
O
Phosphoenolpyruvate Pyruvate
PyK
ADP
ATP
ActiveSite
EffectorSite
Pyruvate kinase monomer
Active site of ATP-bound LmPYK
Phosphoenolpyruvate Pyruvate
ADP
ATP
O
O
O
PO
O
O O
O
O
F-2,6-BP binding stabilises effector
loop and rigidifies tetramer
•
Forms 4 salt bridges (K484….E498 & R493….D482)•
effector
loop pushed towards adjacent chain in tetramer
cc_ribbon.png
T to R Conformational transition of Leishmania
Pyruvate
Kinase
(LmPYK)
consistent with Monod, Wyman, and Changeux
allostery
model.
The active conformer(R-state) of LmPYK
is
stabilised by the F26BP effector
molecule
Increased activity correlates with increased thermal stability
http://eduliss.bch.ed.ac.uk/
5 million compounds, 24 suppliers
>1,500 molecular descriptor values
web interface
Using EDULISS to find hits for the active site
Look for ligands
to mimic the γ-phosphate of ATP and up to 3 coordinating water molecules
Sample hits
Sulphonate
mimics of ATP are reminiscent of trypan
blue and suramin
SURAMIN binds to LmPYK
(IC50 = 7μM) and overlaps with the ATP binding pocket
NH
NH
O
NHO
NH
OS
SO
O
OSO
O
OONH
O
NH
S
SSO
O
O
OO
O
O
O
O
O
O
O
Used since 1920’s as antihelminthicand against trypanosomiasis
HTS screen for LmPYK
inhibitors using 300,000 compound library yields70 hits with IC 50
values between 1 to 50 μM
(NIH, Doug Auld)
M2 PYK (embryonic M2 PYK (embryonic or cancer cells) or cancer cells) regulated by F16BPregulated by F16BP
M1 PYK (adult tissue) M1 PYK (adult tissue) constitutively activeconstitutively active
pyruvate
glucose
mitochondrionpyruvate
glucose
X
Amino acid synthesiscell division
Pyruvate
Kinase
and Cancer
Warburg Effect: increased uptake of glucose but low oxidative phosphorylationcaused by replacing M1 PYK isoform
by allosterically
controlled M2 isoform
Case Study: Expression and Purification of the 4 isoforms
of hPYK
M2 PYK is a splice variant of M1 PYK and differs by 22 AA over a 45 AA stretch
M1 (constitutively active) forms tetramersM2 (allosterically
activated) forms dimers
Structure of human M1 PYK
The 45 amino acid splice variant defines the C-C interface
12 of the 22 residues that differ between M1 and M2 (black) are in a loop that clamps K142 and stabilises the dimer
interface
300 Å
M2 PYK negative staining EM images showtetramers (black circles) and dimers
(red circles)
Images from Crick Wang and Laura Spagnolo
buffer 6 new PYK pro001:10_UV f16 only001:10_UV buffer 6 new PYK pro001:10_Fractions
0
20
40
60
80
100
120
mAU
0.0 5.0 10.0 15.0 20.0 25.0 30.0 mlF2 1A2 1A4 1A6 1A8 1A10 1A12 1B2 1B4 1B6 1B8 1B10 1B12 1C2 1C4 1C6 1C8 1C10 1C12 1D2 1D4 1D6 1D8 1D10 1D12 1E2 1E4 1E6 1E8 1E10 1E12 1F2 1F4 1F6 1F8 1F10 Wast
Addition of effector
(F16BP) pushes M2 to the tetrameric
state
Gel filtration of M2 PYK (12 mg/ml)
Red: addition of F16BP (1mM)M2 is tetrameric
Blue: no F16BP addedM2 is a mixture of tetramer and dimer
tetramerdimer
M2 (apo) M2 + F16BP
M2 in 100mM KCl M2 in 10mM MgCl2
10nm 20nm
Dynamic Light Scattering results for M2 PYK are consistent with a dimer-
tetramer equilibrium.
Tetramers are favoured by addition of F16BP (and / or Mg ions)
10nm 20nm
Thermal Stability of M1 and M2 measured by dynamic light scattering
M2 PYK (apo)
Tm = 420CM2 PYK + F16BPTm = 520C
M1 PYK
Tm = 520CM1 PYK + F16BPTm = 520C
•
B-domain movement•
effector
loop movement
•
slight rotation of C domains away from the central cavity
M2: Inactive to active transition (binding F16BP)
•
T408M may repulse Q489 and reintroduce E409 interactions in M2.
Comparison of C-C interface between M1(blue) and M2 (green) PYK
•The addition of the effector
F16BP greatly reduces overall thermalmotion (B-factor) and increases TmM2 PYK (apo)
Tm = 420C
M2 PYK + F16BPTm = 520C
M2 Pyruvate
kinase
X-ray structures with and without effector
molecule
Red = hot, Blue = cold
M2 PYK is prevented from forming tetramers by:-
allosteric
inhibitors (small molecules, phosphorylated
proteins)
-
phosphorylation-
viral oncoproteins
-
lack of effector
(F16BP)
oncoprotein
Inactive M2 PYK Active M2 PYK
M2 PYK activation provides a potential cancer therapy
Evolution of Allosteric
Control in Pyruvate
Kinase
No effector
bound(F16BP or F26BP)
-Inactive T-state(flexible)
Effector
bound (F16BP or F26BP)
-Active R-state (rigid)
clamp rock and lock dissociation
SUPPORT MRC, Wellcome, BBSRC, EC, NIH
Glycolytic Enzymes Hugh Morgan Iain McNaeMatt NowickiLiam Worrall Lindsay TullochLinda GilmorePaul Michels
LIDAEUS/ EDULISSPaul TaylorKun Yi HsinSteven Shave
CTCB FacilitiesMartin WearLiz BlackburnJanice BramhamSandra BruceConny Ludwig