Chaperonin 60s are a ubiquitous class of proteins that promote folding and assembly of other cellular polypeptides in an ATP-dependent manner. The oligomeric state of chaperonin 60s has been shown to be crucial to their role as molecular chaperones. Chaperonin 60s are also known to be important stimulators of the immune system. Mycobacterium tuberculosis possesses a duplicate set of chaperonin 60s, both of which have been shown to be potent cytokine stimulators. The M. tuberculosis chaperonin 60s are present in the extracellular milieu at concentrations that are extremely low for the formation of an oligomer. Here we present the crystal structure of one of the chaperonin 60s of M. tuberculosis, also called Hsp65 or chaperonin 60.2, at 3.2-Å resolution. We were able to crystallize the protein in its dimeric state. The unusual dimerization of the protein leads to exposure of certain hydrophobic patches on the surface of the protein, and we hypothesize that this might have relevance in binding to immunogenic peptides, as it does in the eukaryotic homologs.Chaperonin 60 (Cpn60), also commonly referred to as heat shock protein 60 (Hsp60), is one of the major molecular chaperones that are present ubiquitously in all forms of life. These molecular chaperones are known to assist the folding, assembly, and transport of several cellular proteins (16). Cpn60s have been shown to be overexpressed under a variety of unnatural conditions, such as thermal stress, hypoxia, nutrient deprivation, phagocytosis, etc. Invasion of a host is an apparent form of a stress, and induction of Cpn60s has also been observed in pathogenic organisms. The overexpressed pathogenderived Cpn60s act as major antigens that result in strong immune responses from the host (43).In Escherichia coli, the chaperonin GroEL has been shown to be essential for growth at all temperatures (13). The E. coli chaperonin has provided a paradigm for understanding protein folding mechanisms mediated by the chaperonins (41). GroEL promotes de novo folding of ϳ10 to 15% of all proteins in the bacterial cytosol in coordination with the heptameric cochaperonin GroES (12).X-ray studies combined with electron microscopy studies have provided valuable insights into the functional cycle of the GroEL chaperonin (41,4,8). The crystal structures of unliganded GroEL and the GroEL-GroES complex revealed a cylindrical complex with subunits of GroEL assembled into two heptameric rings stacked back to back to form the native 14-mer. The two rings enclose a large central channel that facilitates proper protein folding in an ATP-dependent manner (41,4,3). The crystal structure of Cpn60 from Paracoccus denitrificans also revealed a similar arrangement of Cpn60 subunits as a tetradecamer (15).GroEL is assisted in its function by a 10-kDa cochaperonin, GroES. The cochaperonin exists as a heptamer and adopts a dome-like structure that can bind to either GroEL ring to enclose the central cavity (17,21). GroES acts as a lid to seal off the folding chamber and helps displace bound substrate p...