The Covid-19 lockdown in 2020 unexpectedly provided an ideal setting to reflect and revisit some long-standing questions. With laboratory work largely unfeasible, we seized this opportunity to embark on a purely computational research project to explore our ongoing curiosity. Unexpectedly, the resultant findings aligned well with our genetic and structural observations, prompting us to bring up a coherent narrative. So, we put these findings together and shared our discoveries in the Communications Biology journal.¹

As a bit of backstory, we have been working on chaperonin proteins for several years. Chaperonins are remarkable proteins. They are essential and highly conserved across all forms of life. They form a double toroid architecture, consisting of two stacked rings of seven or eight subunits, each with a large central cavity (called the Anfinsen cage). Chaperonins facilitate correct folding of a large subset of proteins (about 10=12% proteins) within cells, particularly in response to stress conditions, such as the elevated temperatures, which can cause the proteins to unfold. Much of our understanding of chaperonins stems from research on the Escherichia coli chaperonin, GroEL. The focus on this single model has often obscured the unique properties of less typical chaperonins.

The chaperonins found in Actinobacteria illustrate this argument. Actinobacteria is one of the largest bacterial phyla and includes several pathogenic species. Unlike E. coli, Actinobacteria encode multiple chaperonin genes, which differ significantly from GroEL in several key aspects. Notably, one chaperonin gene is essential, whereas additional chaperonin paralogues possess unusual characteristics, with some implicated in pathogenic mechanisms. A major distinguishing feature of these chaperonin homolgoues lies in their C-terminal segments (CTS). The essential chaperonin paralogues, including GroEL, possess a Gly-Met-rich CTS, while other Actinobacterial chaperonin paralogues exhibit CTSs with varied sequences. These differences hint at a potential evolutionary significance and species-specific functional relevance. Although GroEL's mechanism of action is well characterised, the role of its CTS remains unclear. Structurally, the CTS is located within the central cavity, it is flexible and unresolved in the crystal structures. The CTSs were thought to be non-functional, leading to their classification as potentially vestigial.² However, the functional signatures observed in Actinobacterial CTSs prompted us to investigate their evolutionary significance.

During the Covid-19 lockdown, we worked with a fantastic project student, Ms. Aisha Mai. As the bench work almost impossible, we initiated a computational phylogenetic project with her to examine the diversification of CTSs. We observed that Actinobacterial chaperonins exhibit substantial variation in their CTSs and that paralogues containing Gly-Met-rich CTSs experience stronger selective pressure, indicating an ancient functional significance. These findings corroborated with the genetic and structural investigations conducted at the University of Birmingham, UK, and the National Centre for Cell Science, India. GroEL variants with mutated CTSs failed to functionally replace GroEL, and the CTSs adopted cavity-specific structural conformations, further substantiating the CTS’s functional relevance.

As the chaperonins with non-canonical CTSs have been implicated in pathogenic processes, we hope that our study will inspire further research into potential CTS-related therapeutic strategies (chaperonopathies) against diseases. This may involve developing inhibitors targeting unique CTSs and their conformations and conducting fundamental research to fully elucidate the specific roles of these diverse CTS regions in the infection and bacterial physiology. More broadly, studying these proteins may enhance our understanding of protein folding disorders beyond bacterial systems, with possible implications for neurodegenerative diseases and other protein misfolding conditions. Beyond direct therapeutic applications, our findings underscore the importance of considering the diversity within protein families and the potential for seemingly 'vestigial' regions to possess critical functional roles, which could have broader implications in understanding protein folding and regulation in various biological systems.

References:

1. Kumar, C. M. S., Mai, A. M., Mande, S. C. & Lund, P. A. Genetic and structural insights into the functional importance of the conserved gly-met-rich C-terminal tails in bacterial chaperonins. Communications Biology 8 doi: 10.1038/s42003-025-07927-x (2025).
2. Brocchieri, L. & Karlin, S. Conservation among HSP60 sequences in relation to structure, function, and evolution. Protein Sci 9, 476-486, doi:10.1110/ps.9.3.476 (2000).