Archaeal-like chaperonins in bacteria Stephen M. Techtmann1 and Frank T. Robb2 aInstitute of Marine and Environmental Technology, Program in the Biology of Model Systems, 701 East Pratt Street, Baltimore, MD 21202 and Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, MD 21201 Edited by Arthur L. Horwich, Yale University School of Medicine, New Haven, CT, and approved September 14, 2010 (received for review April 16, 2010) Chaperonins (CPN) are ubiquitous oligomeric protein machines that by preventing aggregation of the misfolded substrates (16–18). mediate the ATP-dependent folding of polypeptide chains. These Additionally, the active Iterative Annealing Model proposes that chaperones have not only been assigned stress response and nor- the misfolded protein is refolded by several rounds of binding, mal housekeeping functions but also have a role in certain human release and reengagement (19–21). disease states. A longstanding convention divides CPNs into two While both groups of chaperonins catalyze protein folding via groups that share many conserved sequence motifs but differ in encapsulation, there are key structural and mechanistic differ- both structure and distribution. Group I complexes are the well ences between the two groups. The Group II chaperonins (also known GroEL/ES heat-shock proteins in bacteria, that also occur known as the thermosome or TCP1) are structurally different in some species of mesophilic archaea and in the endosymbiotic from Group I, the GroEL/GroES complexes. Both Group I organelles of eukaryotes. Group II CPNs are found only in the and Group II complexes are composed of two stacked rings. cytosol of archaea and eukaryotes. Here we report a third, diver- The Group II CPNs are octo- or nonomeric rings (10), whereas gent group of CPNs found in several species of bacteria. We the GroEL is a homotetradecamer. Group II CPNs are often het- propose to name these Group III CPNs because of their distant erooligomers comprised of a combination of multiple subunits relatedness to both Group I and II CPNs as well as their unique (10). For example many Group II chaperonins from eukaryotes genomic context, within the hsp70 operon. The prototype Group III are heterohexadecamers composed of eight different subunits. CPN, Carboxydothermus hydrogenoformans chaperonin (Ch-CPN), Additionally, the function of the Group I cochaperone, GroES, is able to refold denatured proteins in an ATP-dependent manner is replaced in Group II CPNs by a helical protrusion in the apical and is structurally similar to the Group II CPNs, forming a 16-mer domain of each subunit. These flexible domains combine to form with each subunit contributing to a flexible lid domain. The Group a “built-in lid” that is essential for encapsulation of the denatured BIOCHEMISTRY III CPN represent a divergent group of bacterial CPNs distinct from protein (22–24). The opening and closing of this lid is ATP- the GroEL/ES CPN found in all bacteria. The Group III lineage may responsive and is allosterically regulated (24, 25). Recent work represent an ancient horizontal gene transfer from an archaeon has also indicated that, while certain CPN domains share striking into an early Firmicute lineage. An analysis of their functional similarity between Group I and Group II, the extent and types and structural characteristics may provide important insights into of structural rearrangements that occur during the CPN folding the early history of this ubiquitous family of proteins. cycle vary greatly (8, 10, 22, 25, 26). evolution ∣ protein-folding ∣ thermophilic Results Phylogeny of Group III Chaperonins. Recently, we annotated a diver- olecular chaperones have an essential role in ensuring gent class of chaperonin genes in the genomes of two carboxydo- Mproper folding of cellular proteins. Two primary protein- trophic bacteria, as encoding TCP1-like chaperonins. As shown in folding machines, Hsp70 (DnaK) and the chaperonin (CPN) are Fig. 1, these CPNs are conserved in the genomes of 11 bacteria present in the majority of living organisms (1–6). These two and are monophyletic and distant from all known Group I and systems constitute the ATP-dependent folding functions of both Group II chaperonins. Phylogenetic analysis using multiple algo- nascent polypeptides as well as the salvage of stress-denatured rithms revealed that these proteins form a deeply rooted clade, proteins (7–10). and are highly divergent from the other known chaperonins with Chaperonins are able to positively influence refolding through robust bootstrap support (Fig. 1; individual trees, Fig. S1). Trends sequestration of nonnative proteins. The general mechanism in the divergence of these proteins mirror those seen in the phy- whereby chaperonin affect refolding is through the encapsulation logenetic trees (Table S1 and Fig. S2). There are several deletions of denatured protein within a hydrophilic cavity. While the exact in Group III CPN relative to the other groups of CPNs (Fig. S3). mechanism of CPN-mediated protein folding is still unclear, These indels account for the some of the sequence divergence much is known about key steps in the CPN reaction cycle. The and may influence localized structural elements. This clade repre- most well studied chaperonin homolog is the Escherichia coli sents a previously unknown and unique group of CPNs whose GroEL (8). GroEL forms a barrel structure composed of two physiological role has yet to be determined and which we propose seven-membered rings, with each ring containing a folding cavity. to name as Group III CPNs. The significant divergence from Controversy still prevails over the exact mechanism by which the two known classes of CPNs leads us to suggest that this group GroEL catalyzes proteins folding. According to one model, is a monophyletic subfamily of the Group II CPNs, which may partially folded proteins bind to hydrophobic residues that line represent the remnant of an ancestral chaperonin group. the GroEL cavity (11). ATP binding causes large conformational changes that simultaneously draw the nonnative protein into the Author contributions: S.M.T. and F.T.R. designed research; S.M.T. performed research; cavity, increase the number of polar residues within the cavity, S.M.T. and F.T.R. analyzed data; and S.M.T. and F.T.R. wrote the paper. and trigger rapid binding of the Cochaperone GroES (12). This The authors declare no conflict of interest. sequestration in a hydrophilic cavity forces the protein to refold This article is a PNAS Direct Submission. through forcing the internalization of the hydrophobic residues of 1Present address: Department of Biochemistry and Molecular Biology, Uniformed Services the nonnative protein (13, 14). ATP hydrolysis in the GroES- University for the Health Science, 4301 Jones Bridge Road, Bethesda, MD 20814. bound ring, followed by ATP binding to the opposite ring triggers 2To whom correspondence should be addressed. E-mail: Frobb@som. the release of GroES followed by the encapsulated protein. There umaryland.edu. are several alternative models for GroEL activity (15), including This article contains supporting information online at www.pnas.org/lookup/suppl/ the passive model whereby GroEL assists in refolding simply doi:10.1073/pnas.1004783107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1004783107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Phylogenetic analysis of Group III chaperonin: Un- rooted minimum evolution phylogenetic tree representing Group I, Group II, and the new Group III chaperonins, indi- cating the great phylogenetic distance between the Group III clade and the conserved Group I and Group II clusters. The same alignment was used to construct trees using multiple algorithms (minimum evolution, maximum parsimony, and neighbor-joining). Nodes present in trees created by different alogorithms and with high bootstrap values are denoted as described. The clade representing Group III CPN contains the following organisms: Carboxydothermus hydrogenoformans, Geobacillus spY412MC, Syntrophomo- nas. wolfei wolfei Goettingen, Desulforudis audaxviator, Ammonifex degensii, Heliobacterium modesticaldum, Ther- mosinus carboxydivorans, Desulfotomaculum acetoxidans, Desulfotomaculum reducens, Pelotomaculum thermopro- pionicum, and Gloeobacter violaceus. Genomic Context of Group III Chaperonins. All of the currently unique group of chaperonins we cloned, expressed, and charac- identified Group III chaperonins, with one exception, are found terized a prototype Group III CPN from the extreme thermophile internal to the Hsp70 molecular chaperone operon (Fig. 2A). Carboxydothermus hydrogenoformans (Ch-CPN). C. hydrogenofor- These species all have conventional Group I CPNs encoded in mans is a member of the phylum Firmicutes and is known for its separate operons with their GroES cochaperone. The Group ability to grow on carbon monoxide as its sole carbon and energy III chaperonins are the only CPNs found near or in association source, linking the oxidation of carbon monoxide to hydrogen with the Hsp70 operon. This unique operon structure and the unprecedented association with Hsp70 provide evidence for a functional distinction between Group II and Group III CPNs. Group III CPNs are found between hrcA, the heat-shock responsive transcriptional repressor, and the Hsp70 operon (grpE, dnaK, dnaJ). The location of the Group III chaperonin downstream of the hrcA suggested that Group III CPNs could be HrcA