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