Functional diversity in archaeal Hsp60: a molecular mosaic of Group I and Group II chaperonin.

External stress disrupts the balance of protein homeostasis, necessitating the involvement of heat shock proteins (Hsps) in restoring equilibrium and ensuring cellular survival. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, lacks the conventional Hsp100, Hsp90, and Hsp70, relying solely on a single ATP-dependent Group II chaperonin, Hsp60, comprising three distinct subunits (α, ß, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αßγ, Hsp60αß, and Hsp60ß, at temperatures of 60 °C, 75 °C, and 90 °C, respectively. This study delves into the intricacies of Hsp60 complexes in S. acidocaldarius, uncovering their ability to form oligomeric structures in the presence of ATP. The recognition of substrates by Hsp60 involves hydrophobic interactions, and the subsequent refolding process occurs in an ATP-dependent manner through charge-driven interactions. Furthermore, the Hsp60ß homo-oligomeric complex can protect the archaeal and eukaryotic membrane from stress-induced damage. Hsp60 demonstrates nested cooperativity in ATP hydrolysis activity, where MWC-type cooperativity is nested within KNF-type cooperativity. Remarkably, during ATP hydrolysis, Hsp60ß, and Hsp60αß complexes exhibit a mosaic behavior, aligning with characteristics observed in both Group I and Group II chaperonins, adding a layer of complexity to their functionality.

Distinguishing between concerted, sequential and barrierless conformational changes: Folding versus allostery.

Characterization of transition and intermediate states of reactions provides insights into their mechanisms and is often achieved through analysis of linear free energy relationships. Such an approach has been used extensively in protein folding studies but less so for analyzing allosteric transitions. Here, we point out analogies in ways to characterize pathways and intermediates in folding and allosteric transitions. Achieving an understanding of the mechanisms by which proteins undergo allosteric switching is important in many cases for obtaining insights into how they function.

Reduced ADP off-rate by the yeast CCT2 double mutation T394P/R510H which causes Leber congenital amaurosis in humans.

The CCT/TRiC chaperonin is found in the cytosol of all eukaryotic cells and assists protein folding in an ATP-dependent manner. The heterozygous double mutation T400P and R516H in subunit CCT2 is known to cause Leber congenital amaurosis (LCA), a hereditary congenital retinopathy. This double mutation also renders the function of subunit CCT2, when it is outside of the CCT/TRiC complex, to be defective in promoting autophagy. Here, we show using steady-state and transient kinetic analysis that the corresponding double mutation in subunit CCT2 from Saccharomyces cerevisiae reduces the off-rate of ADP during ATP hydrolysis by CCT/TRiC. We also report that the ATPase activity of CCT/TRiC is stimulated by a non-folded substrate. Our results suggest that the closed state of CCT/TRiC is stabilized by the double mutation owing to the slower off-rate of ADP, thereby impeding the exit of CCT2 from the complex that is required for its function in autophagy.

Heat shock response in Sulfolobus acidocaldarius and first implications for cross-stress adaptation.

Sulfolobus acidocaldarius, a thermoacidophilic crenarchaeon, frequently encounters temperature fluctuations, oxidative stress, and nutrient limitations in its environment. Here, we employed a high-throughput transcriptomic analysis to examine how the gene expression of S. acidocaldarius changes when exposed to high temperatures (92 °C). The data obtained was subsequently validated using quantitative reverse transcription-PCR (qRT-PCR) analysis. Our particular focus was on genes that are involved in the heat shock response, type-II Toxin-Antitoxin systems, and putative transcription factors. To investigate how S. acidocaldarius adapts to multiple stressors, we assessed the expression of these selected genes under oxidative and nutrient stresses using qRT-PCR analysis. The results demonstrated that the gene thß encoding the ß subunit of the thermosome, as well as hsp14 and hsp20, play crucial roles in the majority of stress conditions. Furthermore, we observed overexpression of at least eight different TA pairs belonging to the type II TA systems under all stress conditions. Additionally, four common transcription factors: FadR, TFEß, CRISPR loci binding protein, and HTH family protein were consistently overexpressed across all stress conditions, indicating their significant role in managing stress. Overall, this work provides the first insight into molecular players involved in the cross-stress adaptation of S. acidocaldarius.

A diminished hydrophobic effect inside the GroEL/ES cavity contributes to protein substrate destabilization.

Confining compartments are ubiquitous in biology, but there have been few experimental studies on the thermodynamics of protein folding in such environments. Recently, we reported that the stability of a model protein substrate in the GroEL/ES chaperonin cage is reduced dramatically by more than 5 kcal mol-1 compared to that in bulk solution, but the origin of this effect remained unclear. Here, we show that this destabilization is caused, at least in part, by a diminished hydrophobic effect in the GroEL/ES cavity. This reduced hydrophobic effect is probably caused by water ordering due to the small number of hydration shells between the cavity and protein substrate surfaces. Hence, encapsulated protein substrates can undergo a process similar to cold denaturation in which unfolding is promoted by ordered water molecules. Our findings are likely to be relevant to encapsulated substrates in chaperonin systems, in general, and are consistent with the iterative annealing mechanism of action proposed for GroEL/ES.

Minimal Yet Powerful: The Role of Archaeal Small Heat Shock Proteins in Maintaining Protein Homeostasis.

Small heat shock proteins (sHsp) are a ubiquitous group of ATP-independent chaperones found in all three domains of life. Although sHsps in bacteria and eukaryotes have been studied extensively, little information was available on their archaeal homologs until recently. Interestingly, archaeal heat shock machinery is strikingly simplified, offering a minimal repertoire of heat shock proteins to mitigate heat stress. sHsps play a crucial role in preventing protein aggregation and holding unfolded protein substrates in a folding-competent form. Besides protein aggregation protection, archaeal sHsps have been shown recently to stabilize membranes and contribute to transferring captured substrate proteins to chaperonin for refolding. Furthermore, recent studies on archaeal sHsps have shown that environment-induced oligomeric plasticity plays a crucial role in maintaining their functional form. Despite being prokaryotes, the archaeal heat shock protein repository shares several features with its highly sophisticated eukaryotic counterpart. The minimal nature of the archaeal heat shock protein repository offers ample scope to explore the function and regulation of heat shock protein(s) to shed light on their evolution. Moreover, similar structural dynamics of archaeal and human sHsps have made the former an excellent system to study different chaperonopathies since archaeal sHsps are more stable under in vitro experiments.

Partitioning the Hill coefficient into contributions from ligand-promoted conformational changes and subunit heterogeneity.

Heterooligomers that undergo ligand-promoted conformational changes are ubiquitous in nature and involved in many essential processes. Conformational switching often leads to positive cooperativity in ligand binding that is reflected in a Hill coefficient with a value greater than one. The subunits comprising heterooligomers can differ, however, in their affinity for the ligand. Such so-called site heterogeneity results in apparent negative cooperativity that is reflected by a Hill coefficient with a value less than one. Consequently, positive cooperativity due to the ligand-promoted allosteric switch can be masked, in cases of such heterooligomers, by apparent negative cooperativity owing to site heterogeneity. Here, we derived expressions for the Hill coefficient, in the case of a heterodimer, in which the contributions from the ligand-promoted allosteric switch and site heterogeneity are separated. Using these equations and simulations for higher order oligomers, we show under which conditions site heterogeneity can significantly mask the extent of observed positive cooperativity.

Archaeal Hsp14 drives substrate shuttling between small heat shock proteins and thermosome: insights into a novel substrate transfer pathway.

Heat shock proteins maintain protein homeostasis and facilitate the survival of an organism under stress. Archaeal heat shock machinery usually consists of only sHsps, Hsp70, and Hsp60. Moreover, Hsp70 is absent in thermophilic and hyperthermophilic archaea. In the absence of Hsp70, how aggregating protein substrates are transferred to Hsp60 for refolding remains elusive. Here, we investigated the crosstalk in the heat shock response pathway of thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In the present study, we biophysically and biochemically characterized one of the small heat shock proteins, Hsp14, of S. acidocaldarius. Moreover, we investigated its ability to interact with Hsp20 and Hsp60 to facilitate the substrate proteins' folding under stress conditions. Like Hsp20, we demonstrated that the dimer is the active form of Hsp14, and it forms an oligomeric storage form at a higher temperature. More importantly, the dynamics of the Hsp14 oligomer are maintained by rapid subunit exchange between the dimeric states, and the rate of subunit exchange increases with increasing temperature. We also tested the ability of Hsp14 to form hetero-oligomers via subunit exchange with Hsp20. We observed hetero-oligomer formation only at higher temperatures (50 °C-70 °C). Furthermore, experiments were performed to investigate the interaction between small heat shock proteins and Hsp60. We demonstrated an enthalpy-driven direct physical interaction between Hsp14 and Hsp60. Our results revealed that Hsp14 could transfer sHsp-captured substrate proteins to Hsp60, which then refolds them back to their active form.

Archaeal SRP RNA and SRP19 facilitate the assembly of SRP54-FtsY targeting complex.

The signal recognition particle (SRP) plays an essential role in protein translocation across biological membranes. Stable complexation of two GTPases in the signal recognition particle (SRP) and its receptor (SR) control the delivery of nascent polypeptide to the membrane translocon. In archaea, protein targeting is mediated by the SRP54/SRP19/7S RNA ribonucleoprotein complex (SRP) and the FtsY protein (SR). In the present study, using fluorescence resonance energy transfer (FRET), we demonstrate that archaeal 7S RNA stabilizes the SRP54·FtsY targeting complex (TC). Moreover, we show that archaeal SRP19 further assists 7S RNA in stabilizing the targeting complex (TC). These results suggest that archaeal 7S RNA and SRP19 modulate the conformation of the targeting complex and thereby reinforce TC to execute protein translocation via concomitant GTP hydrolysis.

The oligomeric plasticity of Hsp20 of Sulfolobus acidocaldarius protects environment-induced protein aggregation and membrane destabilization.

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that rescue misfolded proteins from irreversible aggregation during cellular stress. Many such sHsps exist as large polydisperse species in solution, and a rapid dynamic subunit exchange between oligomeric and dissociated forms modulates their function under a variety of stress conditions. Here, we investigated the structural and functional properties of Hsp20 from thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. To provide a framework for investigating the structure-function relationship of Hsp20 and understanding its dynamic nature, we employed several biophysical and biochemical techniques. Our data suggested the existence of a ~24-mer of Hsp20 at room temperature (25 °C) and a higher oligomeric form at higher temperature (50 °C-70 °C) and lower pH (3.0-5.0). To our surprise, we identified a dimeric form of protein as the functional conformation in the presence of aggregating substrate proteins. The hydrophobic microenvironment mainly regulates the oligomeric plasticity of Hsp20, and it plays a key role in the protection of stress-induced protein aggregation. In Sulfolobus sp., Hsp20, despite being a non-secreted protein, has been reported to be present in secretory vesicles and it is still unclear whether it stabilizes substrate proteins or membrane lipids within the secreted vesicles. To address such an issue, we tested the ability of Hsp20 to interact with membrane lipids along with its ability to modulate membrane fluidity. Our data revealed that Hsp20 interacts with membrane lipids via a hydrophobic interaction and it lowers the propensity of in vitro phase transition of bacterial and archaeal lipids.

The Archaeal Signal Recognition Particle: Present Understanding and Future Perspective.

The signal recognition particle (SRP) and its receptor constitute universally conserved and essential cellular machinery that controls the proper membrane localization of nascent polypeptides with the transmembrane domain. In the past decade, there has been an immense advancement in our understanding of this targeting machine in all three domains of life. A significant portion of such progress came from the structural analysis of archaeal SRP components. Despite the availability of structural insights from different archaeal SRP components, little is known about protein translocation in this domain of life compared to either bacteria or eukaryotes. One of the primary reasons being limited availability of the genetic and cell biological tools in archaea. In the present review, an attempt has been made to explore the structural information available for archaeal SRP components to gain insights into the protein translocation mechanism of this group of organisms. Besides, many exciting avenues of archaeal research possible using the recently developed genetic and cell biological tools for some species have been identified.