Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90.

The molecular chaperone and heat shock protein Hsp90 is part of many protein complexes in eukaryotic cells. Together with its cochaperones, Hsp90 is responsible for the maturation of hundreds of clients. Although having been investigated for decades, it still is largely unknown which components are necessary for a functional complex and how the energy of ATP hydrolysis is used to enable cyclic operation. Here we use single-molecule FRET to show how cochaperones introduce directionality into Hsp90's conformational changes during its interaction with the client kinase Ste11. Three cochaperones are needed to couple ATP turnover to these conformational changes. All three are therefore essential for a functional cyclic operation, which requires coupling to an energy source. Finally, our findings show how the formation of sub-complexes in equilibrium followed by a directed selection of the functional complex can be the most energy efficient pathway for kinase maturation.

Aha1 regulates Hsp90's conformation and function in a stoichiometry-dependent way.

The heat shock protein 90 (Hsp90) is a molecular chaperone, which plays a key role in eukaryotic protein homeostasis. Co-chaperones assist Hsp90 in client maturation and in regulating essential cellular processes such as cell survival, signal transduction, gene regulation, hormone signaling, and neurodegeneration. Aha1 (activator of Hsp90 ATPase) is a unique co-chaperone known to stimulate the ATP hydrolysis of Hsp90, but the mechanism of their interaction is still unclear. In this report, we show that one or two Aha1 molecules can bind to one Hsp90 dimer and that the binding stoichiometry affects Hsp90's conformation, kinetics, ATPase activity, and stability. In particular, a coordination of two Aha1 molecules can be seen in stimulating the ATPase activity of Hsp90 and the unfolding of the middle domain, whereas the conformational equilibrium and kinetics are hardly affected by the stoichiometry of bound Aha1. Altogether, we show a regulation mechanism through the stoichiometry of Aha1 going far beyond a regulation of Hsp90's conformation.

A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories.

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We test them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerization and interaction with ArlI, the motor ATPase of the archaellum.

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.

Overwhelming Postsplenectomy Infection: A Prospective Multicenter Cohort Study.

BACKGROUND: Recent population-based cohort studies have questioned the role of pneumococci as the most frequent pathogen causing severe infection in patients after splenectomy. The aim of the study was to define the causative pathogens and clinical presentation of patients with overwhelming postsplenectomy infection (OPSI). METHODS: In a prospective cohort study in 173 German intensive care units, we searched for patients with and without asplenia and community-acquired severe sepsis/septic shock. Clinical and laboratory variables and survival of patients were assessed. RESULTS: Fifty-two patients with severe sepsis or septic shock with asplenia and 52 without asplenia were included. OPSI patients more often had a history of malignancy (38% vs 17%; P = .016) and had a lower body mass index (24 kg/m(2) vs 28 kg/m(2); P = .004). Streptococcus pneumoniae was detected more frequently in OPSI patients (42% vs 12% without asplenia; P < .001) and more frequently manifested as bloodstream infection (31% vs 6%; P = .002). Gram-negative infection was similar in both groups (12% vs 19%; P = .157). Pneumococcal vaccine coverage of OPSI patients was low overall (42% vs 8% among patients without asplenia; P < .001). Purpura fulminans was a frequent complication, developing in 19% of OPSI patients vs 5% of patients without asplenia (P = .038). The interval between splenectomy and OPSI was 6 years (range, 1 month-50 years). On multivariable Poisson regression, asplenia was the only predictive variable independently associated with pneumococcal sepsis (adjusted relative risk, 2.53 [95% confidence interval, 1.06-6.08]). CONCLUSIONS: Pneumococcal infections remain the most important cause of severe sepsis and septic shock following splenectomy.