evidence for weakened activity of mycobacterial chaperonins
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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
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