Identification of the Myeloid-Cell-Activating Site in M. tuberculosis Chaperonin 60.1

Key to understanding the mechanism of action of moonlighting proteins is the identification of the active moonlighting sites. An early study generated the three recombinant domains of the M. tuberculosis chaperonin 60.1 protein and, using these proteins, showed that only the equatorial domain (which contains both N- and C-terminal residues) had the ability to stimulate monocyte cytokine synthesis (Tormay et al. 2005). To refine the search for the active site, a range of protein mutants was generated in which successive 30 residue segments were removed from the C-terminus of the protein. This generated a series of mutants all missing individual 30 residue segments progressing from the C-terminus. This identified residues 460-491 as the active site of this protein. This was confirmed by generating this peptide and a series of sub-peptides. Only the complete 30-residue peptide was active in inducing activation of monocyte cytokine synthesis (Hu et al. 2013). Structural modeling of this peptide segment showed that it was present on the surface of the protein and had significant a-helical structure (Hu et al. 2013). It is not clear if this particular moonlighting site is also responsible for the other biological actions of this protein.

 
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