Chlamydia heat shock proteins GroEL and GroES Molecular biology

[Chlamydiae.com is delighted to feature this presentation, which was given by Karuna Karunakuran from the University of British Columbia, at the conference of the Chlamydia basic research society in Memphis, March 2003. Others authors included Yasuyuki Noguchi, Tim Read, Artem Cherkasov, Jeffrey Kwee, Caixia Shen, Colleen Nelson & Bob Brunham. See: Karunakuran et al., 2003 & Memphis report].


Fig 1. This presentation based on Karunakaran et al., (2003). J. Bacteriol. 185, 1958 - 1966. © The authors and reproduced here with permission.	Fig 2. The structure of the E. coli GroEL heat shock protein. There are 14 subunits of GroEL. Attached to the apical domain (circled, top) of GroEL is GroES. The apical region is also capable of polypeptide binding. The lower region, (circled, bottom) is concerned with ATP binding.	Fig 3. Three groEL genes encode the equivalent of hsp60 in chlamydiae. These genes are expressed constitutively throughout the developmental cycle, show subtle differences, and probably have different biological roles.	Fig 4. Table showing the relationship of the three chlamydial groEL genes with similar genes in other bacteria. Comparison of the deduced sequences showed high homology only for groEL1. Note that widely different bacteria have multiple copies of the groEL genes.

Fig 5. A comparison of the ribbon structure of E. coli GroEL (left) and of the three C. trachomatis GroELs (right). Note their close structural similarities. Note also the subtle differences in structure between GroEL1, 2 and 3 (right).	Fig 6. Twenty two amino acid residues known to be critical for the chaperonin function of E. coli GroEL were conserved and identical in C. trachomatis GroEL1 as shown by the labels. The label shows the amino acid (single letter code) and its position number.	Fig 7. In C. trachomatis GroEL2, 13 of these 22 critical amino acids were conserved. Among the 9 amino acids which were not conserved (green crosses) 6 were in the ATP binding region (circled). It was suggested that changes here may lead to an altered specificity of the GroEL molecule for binding and chaperoning damaged proteins.	Fig 8. In C. trachomatis GroEL3, 13 of the 22 critical amino acids were conserved. Again, among the 9 amino acids which were not conserved (green crosses) 6 were again in the ATP binding region (circled). However comparison of the circled region in Figs 7 and 8 shows that there are subtle differences in the amino acid composition of GreEL2 and GroEL3 which may affect target specificity.

Fig 9. Phylogenetic relationships of Chlamydial GroEL1, 2 & 3 to other bacteria. Note that GroEL1 has a phylogenetic position quite different to that of the related GroEL2 & 3. The analysis indicates that these genes are likely to have been present since the beginning of the chlamydial lineage.	Fig 10. Transcription (rtPCR) and translation (Western blot) of groEL1, 2 & 3. The gene encoding groEL1 is transcribed and translated at higher levels than groEL2 or groEL3. Note the slightly different molecular weights of the chlamydial-expressed proteins.	Fig 11. Quantitation of the transcription of various chlamydial heat shock protein genes by mini microarray following experimental heat shock. Note that groEL1 is the most abundant transcript followed by the associated groES and then dnaK. Transcription of groEL2 or 3 was barely detectable.	Fig 12. Plates showing the ability of an arabinose inducible construct of chlamydial groEL1, groEL2, or groEL3 to compensate for heat shock in an E. coli knock-out mutant lacking a functioning groEL1 gene. Only chlamydial groEL1 and groES together provided effective compensation for defective GroEL in a temperature-sensitive E. coli groEL mutant.

Fig 13. Table showing the ability of various arabinose- and ITPG-inducible constructs of chlamydial groEL genes to compensate for defective E. coli groEL gene function. Chlamydial GroEL1 + groES complements the E. coli groEL mutant whereas groEL2, but not the groEL2 frame shift mutation (next figure) interacts negatively with the C-terminal 29 amino acids playing a critical role. The function of each of the three chlamydial GroEL proteins in development and pathogenesis warrants further study.	Fig 14. Table showing a frameshift mutation in chlamydial groEL2 which dramatically affects the amino acid readout. Interestingly, groEL2 uses rare codons for arginine (AGG; 4 instances) and for isoleucine (AUA; 7 instances) which may be significant for regulation at the level of translation.	Fig 15. Summary of overall findings.

Molecular biology of chlamydial Gro-EL & -ES heat shock proteins in pictures

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