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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...

The diffusible signal factor (DSF)-dependent quorum sensing (QS) system adopts a novel protein-protein interaction mechanism to autoregulate the production of signal DSF. Here, we present the crystal structures of DSF synthase RpfF and its complex with the REC domain of sensor protein RpfC. RpfF is structurally similarity to the members of the crotonase superfamily and contains an N-terminal α/β spiral core domain and a C-terminal α-helical region. Further structural and mutational analysis identified two catalytic glutamate residues, which is the conserved feature of the enoyl-CoA hydratases/dehydratases. A putative substrate-binding pocket was unveiled and the key roles of the residues implicated in substrate binding were verified by mutational analysis. The binding of the REC domain may lock RpfF in an inactive conformation by blocking the entrance of substrate binding pocket, thereby negatively regulating DSF production. These findings provide a structural model for the RpfC-RpfF interaction-mediated QS autoinduction mechanism.

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Abstract. The chaperonins, GroEL and GroES, are present ubiquitously and provide a paradigm in the understanding of assisted protein folding. Due to its essentiality of function, GroEL exhibits high sequence conservation across species. Complete genome sequencing has shown the occurrence of duplicate or multiple copies of groEL genes in bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. Monophyly of each bacterial clade in the phylogenetic tree generated for the GroEL protein suggests a lineage-specific duplication. The duplicated groEL gene in Actinobacteria is not accompanied by the operonic groES despite the presence of upstream regulatory elements. Our analysis suggests that in these bacteria the duplicated groEL genes have undergone rapid evolution and divergence to function in a GroES-independent manner. Evaluation of multiple sequence alignment demonstrates that the duplicated genes have acquired mutations at functionally significant positions including those involved in substrate binding, ATP binding, and GroES binding and those involved in inter-ring and intra-ring interactions. We propose that the duplicate groEL genes in different bacterial clades have evolved independently to meet specific requirements of each clade. We also propose that the groEL gene, although essential and conserved, accumulates nonconservative substitutions to exhibit structural and functional variations.