In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL SR . The GroEL SR -GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL SR -GroES interaction systematically by concatenating seven copies of groES to generate groES 7 . GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES 7 . GroES 7

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Chaperonin and cochaperonin, represented by E. coli GroEL and GroES, are essential molecular chaperones for protein folding. The double-ring assembly of GroEL is required to function with GroES, and a single-ring GroEL variant GroELSR forms a stable complex with GroES, arresting the chaperoning reaction cycle. GroES I25 interacts with GroEL; however, mutations of I25 abolish GroES-GroEL interaction due to the seven-fold mutational amplification in heptameric GroES. To weaken GroELSR-GroES interaction in a controlled manner, we used groES 7, a gene linking seven copies of groES, to incorporate I25 mutations in selected GroES modules in GroES7. We generated GroES7 variants with different numbers of GroESI25A or GroESI25D modules and different arrangements of the mutated modules, and biochemically characterized their interactions with GroELSR. GroES7 variants with two mutated modules participated in GroELSR–mediated protein folding in vitro. GroES7 variants with two or three mutated modules collaborated with GroELSR to perform chaperone function in vivo: three GroES7 variants functioned with GroELSR under both normal and heat-shock conditions. Our studies on functional single-ring bacterial chaperonin systems are informative to the single-ring human mitochondrial chaperonin mtHsp60-mtHsp10, and will provide insights into how the double-ring bacterial system has evolved to the single-ring mtHsp60-mtHsp10.

Chaperonins are essential for cellular growth under normal and stressful conditions and consequently represent one of the most conserved and ancient protein classes. The paradigm chaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence of and homologs, paralogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions. spp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain three genes, (), , and We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novel -specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried the essentiality of the three genes through targeted mutagenesis in ChGroEL hydrolyzed ATP at a rate 25% of that of EcGroEL and bound with high affinity to ChGroES, and the ChGroEL-ChGroES complex could refold malate dehydrogenase (MDH). The chlamydial ChGroEL was selective for its cognate cochaperonin, ChGroES, while EcGroEL could function with both EcGroES and ChGroES. A P35T ChGroES mutant (ChGroESP35T) reduced ChGroEL-ChGroES interactions and MDH folding activities but was tolerated by EcGroEL. Both ChGroEL-ChGroES and EcGroEL-ChGroESP35T could complement an EcGroEL-EcGroES mutant. Finally, we successfully inactivated both paralogs but not , leading to minor growth defects in cell culture that were not exacerbated by heat stress. Collectively, our results support novel functions for the paralogs and solidify ChGroEL as a bona fide chaperonin that is biochemically distinct from EcGroEL. is an important cause of human diseases, including pneumonia, sexually transmitted infections, and trachoma. The chlamydial chaperonin ChGroEL and chaperonin paralog ChGroEL2 have been associated with survival under stress conditions, and ChGroEL is linked with immunopathology elicited by chlamydial infections. However, their exact roles in bacterial survival and disease remain unclear. Our results further substantiate the hypotheses that ChGroEL is the primary chlamydial chaperonin and that the paralogs play specialized roles during infection. Furthermore, ChGroEL and the mitochondrial GroEL only functioned with their cochaperonin, in contrast to the promiscuous nature of GroEL from and, which might indicate a divergent evolution of GroEL during the transition from a free-living organism to an obligate intracellular lifestyle.

The interaction between HIP family proteins (HIP1 and HIP12/1R) and clathrin is fundamental to endocytosis. We used circular dichroism (CD) to study the stability of a HIP1 sub-fragment (aa468-530) that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83°C. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494). The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae), HIP1 from D. melanogaster and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.