GroEL array systemPaul Voziyan Bryan TiemanMary JohnstonGreg BomhoffAngela Chao

Cryo-EM Chaperonin-GS structuresScott Falke, graduate student (KUMC)

Florence Tama Charles Brooks Scripps

Edward Gogol collaborator - UMKC

KUMC Lied Research Fund

Designing a Chaperonin/Osmolyte Folding Array System

The need for a broad folding array system

Expressed proteins membrane proteins,truncated proteins, mutant proteins, affinity tagged proteins

Soluble and properly folded

Proper products or Crystallized proteins (low hanging fruit)

purification

large, smallAggregates orinclusion bodiesare usually shelved

Soluble butmisfolded,kineticallytrapped

+

ribosome

Active protein

misfoldingpathway IX

aggregation (mass action)

aggregate growth

aggregate growth

Cellular Folding

unfolding

folding

MOLECULARCHAPERONES

ATP

ADP

Chaperonins

142 Ao

155 Ao

33 Ao

GroEL (14 subunits)no nucleotide

GroES (7 subunits)

140Ao

140Ao

14 ATP binding sites

Chaperonins from E. coli (homologous to Hsp60 in mitochondria)

GroEL-GroES-ADP

Diagram courtesy ofHelen Saibil, Birbeck College

The chaperonin cycle

Step 3

ATP ATP

180o

ATP ATP ADP ADP

ATP ATP

ADP

ADP ADP

ATPase

ATP +

ATP ATP

ATP

Step 1 Step 2

Step 4

folded

GroEL

GS monomer51 kDa

Glutamine synthetasedodecamer (Class II(?))

Domains fit inside 10 A EM density map

GroEL no nucleotides

highest affinityfor folding polypeptides

polypeptidebinding sites

Ranson et al., 2001 Cell, 107, 869-879.

o

GroEL + ATP

low affinityfor folding polypeptides

polypeptidebinding sitesbecome lesssolvent exposed in ATP boundform.

Domains fit inside 10 A Cryo-EM density map

Ranson et al., 2001 Cell, 107, 869-879.

o

flipped180o

GroEL cryo images 13 A collected by S. Falke

o

Data collected at Scripps in collaboration with R. Milligan

flipped180o

substrate GS

GroEL cryo images 13 A collected by S. Falke

o

Data collected at Scripps in collaboration with R. Milligan

cis

Flexible fitting of crystal structure to EM mapNormal mode analysis fitting – F. Tama, C. Brooks

cis

Trans ring

3 mer fit fromNormal modeanalysisview from inside cavity

Substrate free

cis (Protein bound ring)

Trans ring

3 mer fit fromNormal modeanalysisview from inside cavity

GS bound

spontaneous foldingof Glutamine synthetase

Stable GroEL-GS complex

addition of GroESand ATP

50

% o

rigi

nal

act

ivit

y

Time (min)30 60 90 120 150 180

100

75

25

Typical protein folding experiment with chaperonins Fisher, 1992, Biochemistry

Up-down dimer contacts of Glutamine synthetase consist of swapped secondary elements.

Valine 468(c-terminal amino acid)

Truncated GS does not fold + GroE

spontaneousNative

Native

V468

V468

+ GroE

Fra

ctio

nal a

ctiv

ity

reco

vere

d

GroEL/ES-GS 468 complex

0 1 2 5 10 30 120 min

12-mer of native wt GS

misfolded aggregate

NB

Reassembled wt GS 12-mer

A0 1 2 5 10 30 120 min NGroEL/ES - wt GS complex

12-mer ofnative GS 468

Truncated GS does not assemble from GroE

Combination of other chaperone systemsand lower temperatures did not facilitate GS 468 assembly and reactivation

•DnaK (hsp 70), DnaJ (hsp40), GrpE

•small hsps

•GroE chaperonins + all of the above

•All of the above at 20C.

•All of the above at 15C.

NO ACTIVITY OBSERVED

Additive Activity recovered after 20 h (fraction of native) Additive alone with GroEL-ATP with GroEL-GroES-ATP

1 M betaine << 0.130.01 0.130.01

1 M sarcosine << 0.040.01 0.200.06

1 M sucrose 0.050.02 0.360.07 0.300.07

0.5 M KGlu << 0.090.01 0.350.06

1 M TMAO << 0.220.05 0.450.09

4M glycerol 0.180.04 0.480.08 0.470.09

<<, activity was below the detection limit of the assay.

Numerous osmolytes can facilitate folding of 468 GSfrom GroE (Voziyan et al., 2000, J. Pharm. Sci. Voziyan and Fisher, 2000, Protein. Sci.

Additive Activity recovered after 20 h (fraction of native) Additive alone with GroEL-ATP with GroEL-GroES-ATP

1 M betaine << 0.130.01 0.130.01

1 M sarcosine << 0.040.01 0.200.06

1 M sucrose 0.050.02 0.360.07 0.300.07

0.5 M KGlu << 0.090.01 0.350.06

1 M TMAO << 0.220.05 0.450.09

4M glycerol 0.180.04 0.480.08 0.470.09

<<, activity was below the detection limit of the assay.

Numerous osmolytes can facilitate folding of 468 GSfrom GroE (Voziyan et al., 2000, J. Pharm. Sci. Voziyan and Fisher, 2000, Protein. Sci.

What is the highest protein concentration we can fold with the chaperonin system?

Concentrate complex

+GroE

•With this method, V468 GS and GS can be refolded with >75% activity at 2 mg/ml (no limit found yet)

• Rhodanese - 5 mg/ml (no limit found as of yet).

Protein concentration

Act

ivit

y re

gain

GroEL-protein complexes can be concentrated(Fisher, J. Biol. Chem., 1993, Smith and Fisher, J. Biol. Chem. 1995

Voziyan et al., J. Pharm. Sci. 2000)

Activity regain no longerconcentration dependent

Protein concentration

Optimal GroEL-substrate formation

Assembly and reactivation of dodecameric GSoptimal vs. concentrated (37oC)

active dodecamer

0.25 mg/ml

0.025 mg/ml

20

40

60

80

10 20 30

Time (minutes)

Rea

ctiv

atio

n (

% o

f or

igin

al)

Another Advantage of this system - test for conditions that remove the GroES and ATP requirement from refolding assay. Typical chaperonin substrates...

•Mitochondrial malate dehydrogenase - folds with ATP/and all osmolytes tested at 20-37 C, ADP alone works in place of ATP

•Rhodanese - folds with ATP/glycerol even at 37 C

•Citrate synthase- ATP and polyols (no GroES needed), ADP alone works in place of ATP

•Alcohol dehydrogenase - ATP and most osmolytes

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60

Porcine Mitochondrial Malate Dehydrogenase Chaperonin Requirements

Spontaneous

GroEL Alone

GroEL and ATP

GroEL, GroES, and ATP

PmMDH

Time (minutes)

37°C

PmMDH Renaturation with GroE Chaperonins in 35% Glycerol

Time (minutes)

0 20 40 60 80 100 120

Fra

ctio

n of

Rec

over

ed P

mM

DH

Act

ivity

0.0

0.2

0.4

0.6

0.8

1.0

Spontaneous

GroEL Alone

GroEL and ATP

GroEL, GroES, and ATP

In the Presence of 35% Glycerol, the Chaperonin Requirements for folding of PmMDH become less stringent

Tieman et al., 2001, J. Biol. Chem.

35% glycerol alone

Glutamine synthetase can fold from the chaperonin with glycerol (or sucrose) alone. No longer requires ATPVoziyan and Fisher, 2002, Arch. Biochem. Biophys.

Fra

ctio

n R

ecov

ered

Act

ivit

y

Time (hr)

0 1 2 3

0.8

0.6

0.4

0.2

Reused beads

+ATP

Renaturation of Glutamine synthetase from immobilized EL-Beads(attached through succinimide linkage)

Glycerol 2M (no ATP)

Beads alone (no EL attached)

MDH +Beads alone

MDH+EL beads

MDH +EL-beadsGlycerolATP

MDH +EL-beadsGlycerolATP2nd trial-same beads

Fra

cti

on

al acti

vit

y

reg

ain

aft

er

two

hou

rs

0.15

0.30

0.45

Minimal requirements with osmolytes preserved using the immobilized system –with a more stringentGroE substrate - Malate dehydrogenase.

Folding from Inclusion bodies

isolate inclusion body - sucrose cushion

wash and unfold (GnHCl)

refold with chaperonin-osmolyte array

test for correct folding

Phosphatidylinositol transfer protein (PITP) Folding from Inclusion bodies

% N

ativ

e ac

tivi

ty

Collaboration with G. Helmkamp KUMC

Folding from inclusion bodies –Protein X in vitro (Arginine best osmolyte)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 218M urea +Buffer+BSA +Buffer +GroEL + + + + + + + + + + + + + +GroES + + + + + + +ARG + + + + + + + + + +GS/GSSG + + + + + + + +PDI + + + + + + + + + +

28.4 kDa

21.7 kDa

Standard,10 ng

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

68.8 kDa

52.5 kDa

Western Blot of soluble fractions with GP-antibody detection

Additives

IX

inactive protein

GroELGroEL-substratefolding platform

concentrate

multiple wellfolding array

additive 1

additive 2

additive 3

additive i

ATP(ADP)

0%

10%

90%

0%

BEST FOLDING

CONDITIONS

Dilute into wells

Free or immobilizedGroEL

(patent pending)

More applications:

GroE-osmolyte system– testing for ligands or osmolytes to correct a structural defect using an array!

Known –some Osmolyte systems can reverse assembly or folding defects

•If the protein binds a ligand (L)

release I

I N N+L N-L

excess ligand shifts equilibrium to foldedform.

GroEL array system may allow us to screen for small molecules to prevent aggregation.

Advantage of GroEL – all intermediates are initially bound.

Aggregate

GroEL-I

Failures can be reversed with the correct ligand

collaboration with R. Middaugh at KU Lawrence

Acid Fibroblast growth factoraggregates easily

CHAPERONINS enhancedaggregation!!! `

•The ligand Heparin shifts the foldingequilibrium to the folded soluble state.

Folding mutant Protein

Disease Osmolyte or ligand

Reference

Cystic Fibrosis transmembrane conductance regulator protein (chloride transport)

Cystic Fibrosis Glycerol, CPX ? ?(SciClone)

Brown et al., 1996 Cell stress Chaperones, 1: 117-125

aquaporin Nephrogenic Diabetes Insipidus

Glycerol Tamarappoo et al., 1999, JBC 274: 34825

Alpha 1 trypsin inhibitor Emphysema, liverinjury

Glycerol Burrows et al., (2000) PNAS 97: 1796

Beta glucosidase Gaucher Disease N-n-nonyl)deoxynojirimycin – NN-DNJ

Sawkar et al., 2002, PNAS, 99: 15428

Transthyretin Familial AmyloidCardiomyophathy

L-thyroxine, Flurbiprofien, resveratrol etc.

Klabunde et al., 2000, Nat. Struc. Biol. 7:312.

V2 vasopression receptor

Nephrogenic Diabetes Insipidus

Nonpeptidic V2R antagonists

Morello et al., 2000, J. Clin. Invest. 105: 887

Jiu-Li Song and David Chuang ( 2001, J. Biol. Chem., 276, 40241-46 )

Branched chain - ketoacid decarboxylase normally a tetramer, - mutant (Maple syrup urine disease) an inactive soluble dimer.

With complete GroE system –in combination with TMAO corrects defect. Once identified, TMAO ALONE corrects assembly defects in branched chain - ketoacid decarboxylase (not as well as GroE-TMAO combination)

FOLDED ACTIVE Tetramer STATE REMAINED ACTIVE WHEN THE OSMOLYTE IS REMOVED!!

Using the GroEL-ES-osmolyte system

•Potential to rapidly identify the best folding solution

•When used as a folding platform, the chaperonins capture folding intermediates and can maintain thesemetastable states in aggregation free states.

•Substrates released from GroEL at very highconcentrations.

•Requirement for GroES (and ATP) can be eliminated.

•Works from an immobilized support and is reusable.

•May be used to search for therapeutic ligands to preventaggregation

Implications of this research:

2.5 A atomic model of E coli Glutamine synthetase dodecamer

Top view(one hexamer) Side view

Almassy et al., 1987

Mg ions

Time (min)

Truncated GS folds in the presence of Glycerol(20-35%) and GroE

V468 GS

Native GS

+ ATP

Fra

ctio

nal

Act

ivit

y re

cove

red