Mycobacterial chaperonins in cellular proteostasis: Evidence for chaperone function of Cpn60.1 and Cpn60.2-mediated protein folding.

Mycobacterium tuberculosis encodes two chaperonin proteins, MtbCpn60.1 and MtbCpn60.2, that share substantial sequence similarity with the Escherichia coli chaperonin, GroEL. However, unlike GroEL, MtbCpn60.1 and MtbCpn60.2 purify as lower-order oligomers. Previous studies have shown that MtbCpn60.2 can functionally replace GroEL in E. coli, while the function of MtbCpn60.1 remained an enigma. Here, we demonstrate the molecular chaperone function of MtbCpn60.1 and MtbCpn60.2, by probing their ability to assist the folding of obligate chaperonin clients, DapA, FtsE and MetK, in an E. coli strain depleted of endogenous GroEL. We show that both MtbCpn60.1 and MtbCpn60.2 support cell survival and cell division by assisting the folding of DapA and FtsE, but only MtbCpn60.2 completely rescues GroEL-depleted E. coli cells. We also show that, unlike MtbCpn60.2, MtbCpn60.1 has limited ability to support cell growth and proliferation and assist the folding of MetK. Our findings suggest that the client pools of GroEL and MtbCpn60.2 overlap substantially, while MtbCpn60.1 folds only a small subset of GroEL clients. We conclude that the differences between MtbCpn60.1 and MtbCpn60.2 may be a consequence of their intrinsic sequence features, which affect their thermostability, efficiency, clientomes and modes of action.

GroEL2 of Mycobacterium tuberculosis Reveals the Importance of Structural Pliability in Chaperonin Function.

UNLABELLED: Intracellular protein folding is mediated by molecular chaperones, the best studied among which are the chaperonins GroEL and GroES. Conformational changes and allosteric transitions between different metastable states are hallmarks of the chaperonin mechanism. These conformational transitions between three structural domains of GroEL are anchored at two hinges. Although hinges are known to be critical for mediating the communication between different domains of GroEL, the relative importance of hinges on GroEL oligomeric assembly, ATPase activity, conformational changes, and functional activity is not fully characterized. We have exploited the inability of Mycobacterium tuberculosis GroEL2 to functionally complement an Escherichia coli groEL mutant to address the importance of hinge residues in the GroEL mechanism. Various chimeras of M. tuberculosis GroEL2 and E. coli GroEL allowed us to understand the role of hinges and dissect the consequences of oligomerization and substrate binding capability on conformational transitions. The present study explains the concomitant conformational changes observed with GroEL hinge variants and is best supported by the normal mode analysis. IMPORTANCE: Conformational changes and allosteric transitions are hallmarks of the chaperonin mechanism. We have exploited the inability of M. tuberculosis GroEL2 to functionally complement a strain of E. coli in which groEL expression is repressed to address the importance of hinges. The significance of conservation at the hinge regions stands out as a prominent feature of the GroEL mechanism in binding to GroES and substrate polypeptides. The hinge residues play a significant role in the chaperonin activity in vivo and in vitro.

Elucidating the role of multivalency, shape, size and functional group density on antibacterial activity of diversified supramolecular nanostructures enabled by templated assembly.

With the increased prevalence of antibiotic-resistant infections, there is an urgent need to develop novel antibacterial materials. In addition, gaining a complete understanding of the structural features that impart activity toward target microorganisms is essential to enable materials optimisation. Here we have reported a rational design to fabricate antibacterial supramolecular nanoparticles with variable shape, size and cationic group density, by exploiting noncovalent interactions between a shape determining template amphiphile and a cationic amphiphile to introduce charge on the nanoparticle surface. We have shown that the monomeric cationic amphiphile alone showed poor antibacterial activity, whereas nanostructures formed by co-assembling the complementary units showed significantly enhanced antibacterial efficiency. Further, the systematic variation of several structural parameters such as shape, spacing between the cationic groups and size of these nanostructures allowed us to elicit the role of each parameter on the overall antibacterial properties. Finally, we investigated the origin of the differing antibacterial activity of these nanoparticles having different shape and size but with the same molecular composition, by comparing the thermodynamic parameters of their binding interactions with a bacterial membrane mimic.

Correction: Elucidating the role of multivalency, shape, size and functional group density on antibacterial activity of diversified supramolecular nanostructures enabled by templated assembly.

Correction for 'Elucidating the role of multivalency, shape, size and functional group density on antibacterial activity of diversified supramolecular nanostructures enabled by templated assembly' by Amrita Sikder et al., Mater. Horiz., 2023, 10, 171-178, https://doi.org/10.1039/D2MH01117D.

Chaperonin Abundance Enhances Bacterial Fitness.

The ability of chaperonins to buffer mutations that affect protein folding pathways suggests that their abundance should be evolutionarily advantageous. Here, we investigate the effect of chaperonin overproduction on cellular fitness in Escherichia coli. We demonstrate that chaperonin abundance confers 1) an ability to tolerate higher temperatures, 2) improved cellular fitness, and 3) enhanced folding of metabolic enzymes, which is expected to lead to enhanced energy harvesting potential.

Facilitated oligomerization of mycobacterial GroEL: evidence for phosphorylation-mediated oligomerization.

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.

Ser/Thr protein kinase PrkC-mediated regulation of GroEL is critical for biofilm formation in Bacillus anthracis.

PrkC is a conserved Ser/Thr protein kinase encoded in Bacillus anthracis genome. PrkC is shown to be important for B. anthracis pathogenesis, but little is known about its other functions and phosphorylated substrates. Systemic analyses indicate the compelling role of PrkC in phosphorylating multiple substrates, including the essential chaperone GroEL. Through mass spectrometry, we identified that PrkC phosphorylates GroEL on six threonine residues that are distributed in three canonical regions. Phosphorylation facilitates the oligomerization of GroEL to the physiologically active tetradecameric state and increases its affinity toward the co-chaperone GroES. Deletion of prkC in B. anthracis abrogates its ability to form biofilm. Overexpression of native GroEL recovers the biofilm-forming ability of prkC deletion strain. Similar overexpression of GroEL phosphorylation site mutants (Thr to Ala) does not augment biofilm formation. Further analyses indicate the phosphorylation of GroEL in diverse bacterial species. Thus, our results suggest that PrkC regulates biofilm formation by modulating the GroEL activity in a phosphorylation-dependent manner. The study deciphers the molecular signaling events that are important for biofilm formation in B. anthracis.

Multiple chaperonins in bacteria--novel functions and non-canonical behaviors.

Chaperonins are a class of molecular chaperones that assemble into a large double ring architecture with each ring constituting seven to nine subunits and enclosing a cavity for substrate encapsulation. The well-studied Escherichia coli chaperonin GroEL binds non-native substrates and encapsulates them in the cavity thereby sequestering the substrates from unfavorable conditions and allowing the substrates to fold. Using this mechanism, GroEL assists folding of about 10-15 % of cellular proteins. Surprisingly, about 30 % of the bacteria express multiple chaperonin genes. The presence of multiple chaperonins raises questions on whether they increase general chaperoning ability in the cell or have developed specific novel cellular roles. Although the latter view is widely supported, evidence for the former is beginning to appear. Some of these chaperonins can functionally replace GroEL in E. coli and are generally indispensable, while others are ineffective and likewise are dispensable. Additionally, moonlighting functions for several chaperonins have been demonstrated, indicating a functional diversity among the chaperonins. Furthermore, proteomic studies have identified diverse substrate pools for multiple chaperonins. We review the current perception on multiple chaperonins and their physiological and functional specificities.