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Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments., (© 2021. The Author(s).)

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

To understand a biological system, it is important to observe structures of biomolecules in the solution where the system is functionalized. Small-Angle X-ray Scattering coupled with Size Exclusion Chromatography (SEC-SAXS) is one of techniques to selectively observe the target molecules in the multi-component system. However, this technique is believed to be available only with a synchrotron-based SAXS instrument due to requirement of high beam intensity and, therefore, the limitation of the beam time was obstacle to satisfy demands from many bio-researchers. We newly developed Laboratory-based Size exclusion chromatography SAXS System (La-SSS) by utilizing a latest laboratory-based SAXS instrument and finely optimization of the balance between flow rate, cell volume, irradiation time and so on. La-SSS succeeded not only decoupling of target protein(s) from non-specific aggregates but also measurement of each concerned component in a multi-component system. In addition, an option: "stopping mode", which is designed for improving statistics of SAXS profile, realized a high S/N data acquisition for the most interesting protein in a multi-component system. Furthermore, by utilizing a column having small bed volume, the small-scale SEC-SAXS study makes available. Through optimization of instrumental parameters and environments, La-SSS is highly applicable for experimental requirements from various biological samples. It is strongly expected that a La-SSS concept must be a normal option for laboratory-based SAXS in the near future.

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine. (1)H-(13)C heteronuclear single-quantum coherence (HSQC) spectral data of HIV-1 RT, in which the methionine methyl groups of the p66 subunit were selectively labeled with (13)C, were collected in the presence and absence of these NNRTIs. We found that the methyl (13)C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs.

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b' and a' domains provide substrate binding sites and undergo an open-closed domain rearrangement depending on the redox states of the a' domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37-Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b'-a' domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b' domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a' domain partially masks the binding surface of the b' domain, causing steric hindrance against the peptide in the reduced form of the b'-a' domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI.