1. Pyruvate Kinase and the Second Secret of
Life
Pyruvate Kinase and the Second Secret ofPyruvate 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)
2. 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 kinase
Mdm2 cyclin/phosphate fructose bisphosphate
DNA ATP/protein substrate ADP/phosphenolpyruvate
4. LeishmaniaLeishmania mexicanamexicana
PYKPYK
regulated by F26BPregulated by F26BP
M2 PYK (embryonicM2 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
O
O
O O
P
O
O
O
O
6
2
O
O
O
P
O
P O
O
O
O
O
O
O O
6 1
Phosphoenolpyruvate Pyruvate
ADP + + ATP
O
O
O
P
O
O
O
O
O
O
5. T to R Conformational transition of Leishmania
Pyruvate Kinase (LmPYK)
O
O
O
P
O
O
O
Phosphoenolpyruvate Pyruvate
PyK
ADP
ATP
Active
Site
Effector
Site
Pyruvate kinase monomer
6. Active site of ATP-bound LmPYK
Phosphoenolpyruvate Pyruvate
ADP
ATP
O
O
O
P
O
O
O
O
O
O
7. 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
8. T to R Conformational transition of
Leishmania Pyruvate Kinase (LmPYK)
consistent with Monod, Wyman,
and Changeux allostery model.
9. The active conformer
(R-state) of LmPYK is
stabilised by the F26BP
effector molecule
Increased activity correlates with increased thermal stability
11. 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
12. SURAMIN binds to LmPYK (IC50 = 7μM) and overlaps
with the ATP binding pocket
N
H
N
H
O
NHO
N
H
O
S
S
O
O
O
S
O
O
O
ONH
O
N
H
S
SS
O
O
O
O
O
O
O
O
O
O
O
O
Used since 1920’s as antihelminthic
and against trypanosomiasis
13. HTS screen for LmPYK inhibitors using 300,000 compound library yields
70 hits with IC 50 values between 1 to 50 μM
(NIH, Doug Auld)
14. M2 PYK (embryonicM2 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 synthesis
cell division
Pyruvate Kinase and Cancer
Warburg Effect: increased uptake of glucose but low oxidative phosphorylation
caused by replacing M1 PYK isoform by allosterically controlled M2 isoform
16. M2 PYK is a splice variant of M1 PYK and differs by 22 AA over a 45 AA stretch
M1 (constitutively active) forms tetramers
M2 (allosterically activated) forms dimers
17. Structure of human M1 PYK
The 45 amino acid splice variant defines the C-C interface
18. 12 of the 22 residues that differ between M1 and M2 (black) are in
a loop that clamps K142 and stabilises the dimer interface
19. 300 Å
M2 PYK negative staining EM images show
tetramers (black circles) and dimers (red circles)
Images from Crick Wang and Laura Spagnolo
20. 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 ml
F2 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 added
M2 is a mixture of tetramer and dimer
tetramer
dimer
21. 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
23. • B-domain movement
• effector loop movement
• slight rotation of C domains
away from the central cavity
M2: Inactive to active transition (binding F16BP)
24. • T408M may repulse Q489 and
reintroduce E409 interactions in M2.
Comparison of C-C interface between M1(blue) and M2 (green) PYK
25. •The addition of the effector F16BP greatly reduces overall thermal
motion (B-factor) and increases Tm
M2 PYK (apo) Tm = 420C
M2 PYK + F16BPTm = 520C
M2 Pyruvate kinase X-ray structures with and without effector molecule
Red = hot, Blue = cold
26. 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
27. 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
28. SUPPORT
MRC, Wellcome, BBSRC, EC, NIH
Glycolytic Enzymes
Hugh Morgan
Iain McNae
Matt Nowicki
Liam Worrall
Lindsay Tulloch
Linda Gilmore
Paul Michels
LIDAEUS/ EDULISS
Paul Taylor
Kun Yi Hsin
Steven Shave
CTCB Facilities
Martin Wear
Liz Blackburn
Janice Bramham
Sandra Bruce
Conny Ludwig
