Evidence for Weakened Activity of Mycobacterial Chaperonins

Activity Restoration of a Naturally Inactive Chaperonin via Facilitated Oligomerization

C. M. Santosh Kumar, Garima Khare, C. V. Srikanth, Anil K Tyagi, Abhijit A. Sardesai and Shekhar C. Mande

Centre for DNA Fingerprinting and Diagnostics, Bldg. 7, Gruhakalpa, 5-4-399 / B,Nampally,  Hyderabad - 500 001 

GroEL-GroES Chaperonin Complex• Best studied molecular chaperones• Two proteins – GroEL & GroESGroEL - Tetradecamer of 58 kDa subunits• Two isologus heptameric rings• Hydrophobic cavities bind protein substrates• ATP dependent GroES - Heptamer of 10 kDa, acts as a lid GroES

GroEL

~ 10% of newly synthesized proteins

• Two copies cpn60s: Mtb groEL1 (cpn60.1) & Mtb groEL2 (cpn60.2)

• Share 70% homology with E. coli counterpart, GroEL• Dimeric form & ATP independent• Cannot help refolding but prevent aggregation

GroEL of M. tuberculosis

Qamra and Mande., J. Bact. (2004)Qamra, Srinivas and Mande., JMB (2004)

Unusual GroELs of M. tuberculosis

GroEL

Cpn60.2

Substrate Recognition or Oligomerization ?

Mtb groEL1 & Mtb groEL2 cloned along with cohort Mtb groES

E. coli SV2 (groEL Ts mutant) and LG6 (GroES/L depletion strain)

Mtb groELs are expressed but not able to complement

In vivo Complementation Studies

I. Mtb GroES + GroEL1

II. Mtb GroES + GroEL2

III. E. coli GroES + GroEL

IV. Vector

PgroERBS

E. coli groES E. coli groEL

PlacRBS

E. coli groES

MG1655 LG6

E. coli groEL

Gene Shuffling

E. coli SV2

0.2 % L-arabinose

Incubated at 30 ℃ and 42 ℃

Sequence Analysis of Gene Shuffled Mutants

Apical domain is plastic & equatorial domain is conserved

Apical Domain

Intermediate Domain

Equatorial Domain

Equatorial Domains Exchanged

Impaired Oligomerization

– Activity Determinant

Complementation Bacteriophage Plaquing

Bacteriophage

SV2 Lawn

0.2 % L-arabinose

Incubated at 30 ℃

Resident GroEL Contamination?

GroELMEF active in vivo – GroES dependent

Ptac based vector

PgroERBS

E. coli groES E. coli groEL

PBADRBS

E. coli groES

MG1655

MGM100

E. coli groEL

E. coli GroEL

GroELMEF

GroELMER

GroELSp24

GroELSp32

Expressed in E. coli BL21 (DE3)

Auto Induction System or L-arabinose

Ammonium Sulphate Extraction

Ion Exchange or Hydrophobic Interaction Chromatography

Gel Filtration

Protein Purification

GroELMEF exists as oligomer

ATPase Activity

Prevention of Aggregation

Biochemical CharacterizationSubstrate Protein Refolding

Summary• Mtb rGroEL - Inherently Inactive Chaperones

• GroELSp24 & GroELSp32

o Efficiency of Substrate Binding

• GroELMEF - Functional Chaperone

o Complementation

o Exists as Oligomer - Refolding & ATPase Activity

o Stability

• GroELMER - Inactive as Chaperone

o Prevents Substrate Protein Aggregation

Impaired Oligomerization of Mtb rGroELs – Reduced Activity

Factor Mediated Oligomerization?

• Active ATPase• Slow Growing Mtb

– 24 hrs doubling time

• Residues mutated to Serine or Threonine• Eukaryotic like STPKs• Heat regulated oligomerization in chaperones

Can Mtb Tolerate Robust GroEL?

Heat or Phosphorylation?

What Forms in Mtb?

Mtb lysates resolved by Gel filtration Superdex S200 16/60

Peaks corresponding to different Oligomeric forms of GroEL

Normallized fraction of each peak probed - α-GroEL1 antibody

Mtb lysates resolved on Native PAGE

Probed with α-GroEL1 antibody

37 ℃ 42 ℃

Native PAGE SDS PAGE

GroEL1 predominates in three forms

Is GroEL Phosphorylated?Co-immuno Precipitation

Tetradecameric form is Phosphorylated on Serine residue

Which Form of GroEL?

Peaks corresponding to different Oligomeric forms of GroEL

α-Ser-P antibody

Western Blotting

Acrylamide plugs(Oligomeric forms of GroEL)

Mass-Spec Analysis

MALDI-TOF

MS/MSPeptides with serine

ESVEDAVAAAK

P

S393 is Phosphorylated

Which Serine?

Summary

• Mycobacterial GroEL1 Exists in Multiple Forms

o Heptameric Form - Ambient Conditions

o Tetra-decameric Form - Elevated Temperatures

• Phosphorylation

o S393 is Phosphorylated – No Threonine

o Switch Between Heptameric & Tetra-decameric Form

• GroEL2 is not phosphorylated!

Oligomerization (Activity) is Phosphorylation Mediated

Mitochondrial & Choloroplast Hsp60 Levy et. al., Eur. J. Biochem. 2001Dickson et. al., J. Biol. Chem. 2000

The Model

ACKNOWLEDGEMENTS

• CDFD, Hyderabad • Department of Biotechnology, India• Council of Scientific and Industrial Research, India• Wellcome Trust, UK