Abstract P-45: Structure of the Bacteriophage AR9 Bacillus Subtilis Chaperonin According to Cryo-Electron Microscopy

Background: Chaperonins are a family of molecular chaperones Hsp60 (heat shock proteins 60). GroEL is a bacterial chaperonin. It ensures the correct folding of proteins, using the energy of ATP hydrolysis. Three-dimensional reconstructions of its predicted orthologs were obtained and biochemically characterized in free and nucleotide-bound states for bacteriophages EL Pseudomonas aeruginosa, OBP Pseudomonas fluorescens (Kurochkina, L. P. et al., Journal of virology, 2012; Semenyuk, P. I. et al., Biochemical Journal, 2016; Stanishneva-Konovalova, T. B. et al., Journal of Structural Biology, 2020). Physicochemical studies were carried out for the bacteriophage AR9 Bacillus Subtilis and confirmed that the protein has chaperone activity and does not require co-chaperonin to function (Semenyuk P. I. et al., International Journal of Biological Macromolecules, 2020). Methods: The recombinant chaperonin of the B. subtilis bacterial phage AR9 (gp228) was isolated and purified in a free state and vitrified in Vitrobot Mark IV. Data were collected using a Titan Krios cryo-TEM and processed in Warp, RELION and cryoSPARC software. Results: The final structures of the chaperonin were reconstructed with a C1 and C7 symmetry at the resolution of 4.5 Å and 4.0 Å respectively. Significant heterogeneity of the apical domains was addressed further using 3D classification and symmetry expansion in RELION resulting in a set of classes reflecting the conformational transition of the subunits between different states. At least four different conformational states of the subunit were clearly resolved. Conclusion: Gp228 structure show similarities between bacteriophage chaperonin and also bacterial chaperonin GroEL. It is formed by a single ring consisting of seven identical subunits, each has three domains: equatorial, intermediate, and apical. The subunits of the apo-form chaperonin Gp228 exhibit significant conformational flexibility in the apical and intermediate domains.

TEM and STEM-EDS evaluation of metal nanoparticle encapsulation in GroEL/GroES complexes according to the reaction mechanism of chaperonin

Escherichia coli chaperonin GroEL, which is a large cylindrical protein complex comprising two heptameric rings with cavities of 4.5 nm each in the center, assists in intracellular protein folding with the aid of GroES and adenosine triphosphate (ATP). Here, we investigated the possibility that GroEL can also encapsulate metal nanoparticles (NPs) up to ∼5 nm in diameter into the cavities with the aid of GroES and ATP. The slow ATP-hydrolyzing GroELD52A/D398A mutant, which forms extremely stable complexes with GroES (half-time of ∼6 days), made it possible to analyze GroEL/GroES complexes containing metal NPs. Scanning transmission electron microscopy–energy-dispersive X-ray spectroscopy analysis proved distinctly that FePt NPs and Au NPs were encapsulated in the GroEL/GroES complexes. Dynamic light scattering measurements showed that the NPs in the GroEL/GroES complex were able to maintain their dispersibility in solution. We previously described that the incubation of GroEL and GroES in the presence of ATP·BeFx and adenosine diphosphate·BeFx resulted in the formation of symmetric football-shaped and asymmetric bullet-shaped complexes, respectively. Based on this knowledge, we successfully constructed the football-shaped complex in which two compartments were occupied by Pt or Au NPs (first compartment) and FePt NPs (second compartment). This study showed that metal NPs were sequentially encapsulated according to the GroEL reaction in a step-by-step manner. In light of these results, chaperonin can be used as a tool for handling nanomaterials.

Measuring protein stability in the GroEL chaperonin cage reveals massive destabilization

The thermodynamics of protein folding in bulk solution have been thoroughly investigated for decades. By contrast, measurements of protein substrate stability inside the GroEL/ES chaperonin cage have not been reported. Such measurements require stable encapsulation, that is no escape of the substrate into bulk solution during experiments, and a way to perturb protein stability without affecting the chaperonin system itself. Here, by establishing such conditions, we show that protein stability in the chaperonin cage is reduced dramatically by more than 5 kcal mol−1 compared to that in bulk solution. Given that steric confinement alone is stabilizing, our results indicate that hydrophobic and/or electrostatic effects in the cavity are strongly destabilizing. Our findings are consistent with the iterative annealing mechanism of action proposed for the chaperonin GroEL.

Helicobacter hepaticus as disease driver in a novel CD40-mediated model of colitis

ABSTRACTGut microbiota and the immune system are in constant exchange, which shapes both, host immunity and microbial communities. Here, improper immune regulation can cause inflammatory bowel disease (IBD) and colitis. Antibody therapies blocking signaling through the CD40 – CD40L axis showed promising results as these molecules have been described to be deregulated in certain IBD patients. To better understand the mechanism, we used transgenic DC-LMP1/CD40 animals, which lack intestinal CD103+ dendritic cells (DCs) and therefore cannot induce regulatory T (iTreg) cells due to a constitutive CD40-signal in CD11c+ cells. These mice rapidly develop spontaneous fatal colitis with an increase of inflammatory IL-17+IFN-γ+ Th17/Th1 and IFN-γ+ Th1 cells. In the present study we analyzed the impact of the microbiota on disease development and detected elevated IgA- and IgG-levels in sera from DC-LMP1/CD40 animals. Their serum antibodies specifically bound intestinal bacteria and we identified a 60 kDa chaperonin GroEL (Hsp60) from Helicobacter hepaticus (Hh) as the main specific antigen targeted in absence of iTregs. When rederived to a different Hh-free SPF-microbiota, mice showed few signs of disease without fatalities, but upon recolonization of mice with Hh we found rapid disease onset and the generation of inflammatory Th17/Th1 and Th1 cells in the colon. Thus, the present work identifies a major bacterial antigen and highlights the impact of specific microorganisms on modulating the host immune response and its role on disease onset, progression and outcome in this colitis model.

Crystal Structure of Chaperonin GroEL from Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae (Xoo) is a plant pathogen that causes bacterial blight of rice, with outbreaks occurring in most rice-growing countries. Thus far, there is no effective pesticide against bacterial blight. Chaperones in bacterial pathogens are important for the stabilization and delivery of effectors into host cells to cause disease. In bacteria, GroEL/GroES complex mediates protein folding and protects proteins against misfolding and aggregation caused by environmental stress. We determined the crystal structure of GroEL from Xanthomonas oryzae pv. oryzae (XoGroEL) at 3.2 Å resolution, which showed the open form of two conserved homoheptameric rings stacked back-to-back. In the open form structure, the apical domain of XoGroEL had a higher B factor than the intermediate and equatorial domains, indicating that the apical domain had a flexible conformation before the binding of substrate unfolded protein and ATP. The XoGroEL structure will be helpful in understanding the function and catalytic mechanism of bacterial chaperonin GroELs.

Chaperonin overproduction and metabolic erosion caused by mutation accumulation in Escherichia coli

Bacterial cells adapting to a constant environment tend to accumulate mutations in portions of their genome that are not maintained by selection. This process has been observed in bacteria evolving under strong genetic drift, and especially in bacterial endosymbionts of insects. Here, we study this process in hypermutable Escherichia coli populations evolved through 250 single-cell bottlenecks on solid rich medium in a mutation accumulation experiment that emulates the evolution of bacterial endosymbionts. Using phenotype microarrays monitoring metabolic activity in 95 environments distinguished by their carbon sources, we observe how mutation accumulation has decreased the ability of cells to metabolize most carbon sources. We study if the chaperonin GroEL, which is naturally overproduced in bacterial endosymbionts, can ameliorate the process of metabolic erosion, because of its known ability to buffer destabilizing mutations in metabolic enzymes. Our results indicate that GroEL can slow down the negative phenotypic consequences of genome decay in some environments.