The major heat shock protein, chaperonin 60, has been established to have 
intercellular signaling activity in addition to its established protein-folding 
function. Mycobacterium tuberculosis is one of a small proportion of bacteria to 
encode two chaperonin 60 proteins. We have demonstrated that chaperonin 60.1 
from this bacterium is a very active stimulator of human monocytes. To determine 
structure/function relationships of chaperonin 60.1 we have cloned and expressed 
the apical, equatorial, and intermediate domains of this protein. We have found 
that the signaling activity of M. tuberculosis chaperonin 60.1 resides in the 
equatorial domain. This activity of the recombinant equatorial domain was 
completely blocked by treating the protein with proteinase K, ruling out 
lipopolysaccharide contamination as the cause of the cell activation. Blockade 
of the activity of the equatorial domain by anti-CD14 monoclonal antibodies 
reveals that this domain activates monocytes by binding to CD14. Looking at the 
oligomeric state of the active proteins, using native gel electrophoresis and 
protein cross-linking we found that recombinant M. tuberculosis chaperonin 60.1 
fails to form the prototypic tetradecameric structure of chaperonin 60 proteins 
under the conditions tested and only forms dimers. It is therefore concluded 
that the monocyte-stimulating activity of M. tuberculosis Cpn60.1 resides in the 
monomeric subunit and within this subunit the biological activity is due to the 
equatorial domain.