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Terrestrial organisms have systems to escape from desiccation stresses. For example, tardigrades (also known as water bears) can survive severe dried and other extreme environments by anhydrobiosis. Although their extraordinary ability has enchanted people, little is known about the detailed molecular mechanisms of anhydrobiosis. Here, we focused on the tardigrade Ramazzottius varieornatus, one of the toughest animals on Earth. A transcriptome database of R. varieornatus shows that genes encoding a Ferritin‐like protein are upregulated during desiccation or ultraviolet radiation. This protein shows sequence similarity to enigmatic proteins in desiccation‐tolerant bacteria and plants, which are hypothesized to be desiccation‐related. However, because these proteins lack detailed biological information, their functions are relatively unknown. We determined an atomic (1.05 Å) resolution crystal structure of a Ferritin‐like protein from R. varieornatus. The structure revealed a dinuclear metal binding site, and we showed that this Ferritin‐like protein has phosphatase activity toward several metabolite compounds including unusual nucleotide phosphates produced by oxidative or radiation damage. We also found that a homologous protein from a desiccation‐ and ultraviolet‐tolerant bacterium Deinococcus radiodurans is a metabolite phosphatase. Our results indicate that through cleaning up damaged metabolites or regulation of metabolite levels, this phosphatase family can contribute to stress tolerances. This study provides a clue to one of the universal molecular bases of desiccation‐stress tolerance. [ABSTRACT FROM AUTHOR]

Bacteria regulate virulence by using two‐component systems (TCSs) composed of a histidine kinase (HK) and a response regulator (RR). TCSs respond to environmental signals and change gene‐expression levels. The HK QseE and the RR QseF regulate the virulence of Enterobacteriaceae bacteria such as enterohemorrhagic Escherichia coli. The operon encoding QseE/QseF also contains a gene encoding an outer membrane lipoprotein, qseG. The protein product QseG interacts with QseE in the periplasmic space to control the activity of QseE and constitutes a unique QseE/F/G three‐component system. However, the structural bases of their functions are unknown. Here, crystal structures of the periplasmic regions of QseE and QseG were determined with the help of AlphaFold models. The periplasmic region of QseE has a helix‐bundle structure as found in some HKs. The QseG structure is composed of an N‐terminal globular domain and a long C‐terminal helix forming a coiled‐coil‐like structure that contributes to dimerization. Comparison of QseG structures obtained from several crystallization conditions shows that QseG has structural polymorphisms at the C‐terminus of the coiled‐coil structure, indicating that the C‐terminus is flexible. The C‐terminal flexibility is derived from conserved hydrophilic residues that reduce the hydrophobic interaction at the coiled‐coil interface. Electrostatic surface analysis suggests that the C‐terminal coiled‐coil region can interact with QseE. The observed structural fluctuation of the C‐terminus of QseG is probably important for interaction with QseE. [ABSTRACT FROM AUTHOR]

Some tardigrades can survive extremely desiccated conditions through transition into a state called anhydrobiosis. Anhydrobiotic tardigrades have proteins unique to them and they are thought to be keys to the understanding of unusual desiccation resistance. In fact, previous transcriptome data show that several tardigrade‐specific proteins are significantly upregulated under desiccated conditions. However, their physiological roles and chemical properties have been ambiguous because they show low or no similarity of amino acid sequences to proteins found in other organisms. Here, we report a crystal structure of one of such proteins. This protein shows a β‐sandwich structure composed of 8 β‐strands, three Ca2+‐binding sites, and hydrophobic residues on Ca2+‐binding (CBD) loops, which resemble characteristics of C2 domain proteins. We therefore conveniently describe this protein as tardigrade C2 domain protein (TC2P). Because the C2 domain functions as a Ca2+‐mediated membrane docking module, which is related to signal transduction or membrane trafficking, TC2Ps may play a role in Ca2+‐triggered phenomenon under desiccated situations. Our finding provides not only structural insights into a newly discovered desiccation‐related protein family but also insights into the evolution and diversity of C2 domain proteins. PDB Code(s): 7DF2; [ABSTRACT FROM AUTHOR]

Cytochrome b5 is an essential electron transfer protein, which is ubiquitously found in living systems and involved in wide variety of biological processes. Tardigrades (also known as water bears), some of which are famous for desiccation resistance, have many proteins unique to them. Here, we report spectroscopic and structural characterization of a cytochrome b5 like protein from one of the desiccation‐tolerant tardigrades, Ramazzottius varieornatus strain YOKOZUNA‐1 (RvCytb5). A 1.4 Å resolution crystal structure revealed that RvCytb5 is a new cytochrome b5 protein specific to tardigrades. PDB Code(s): 7BWH; [ABSTRACT FROM AUTHOR]

Tardigrades, also known as water bears, can survive extreme conditions. For example, tardigrades have high tolerance to extreme desiccation because they can enter an anhydrobiotic state, in which they show no or nearly undetectable metabolic processes. Proteins from anhydrobiotic tardigrades with low homology to known proteins from other organisms are new potential targets for structural genomics. Here, we present spectroscopic and structural characterization of an unprecedented globin protein (Kumaglobin: Kgb) found in an anhydrobiotic tardigrade. Spectroscopy reveals that Kgb contains hexacoordinated low‐spin heme, which is not capable of binding to hydrogen sulfide (H2S) unlike other globin proteins, such as neuroglobin. Interestingly however, when distal histidine is replaced with alanine, H2S is capable of binding to heme, implying that the distal histidine of Kgb binds tightly to heme. The overall structure of Kgb at 1.5 Å resolution shows high resemblance to well‐characterized eukaryotic globin proteins, such as myoglobin and cytoglobin. However, the heme coordination geometry in Kgb is unique because the distal histidinyl ligand is located at the 11th position of helix E while it is found at 7th position on helix E in many known globin proteins. The unusual conformation of distal histidine in Kgb is stabilized by a hydrogen bond with the carbonyl O atom of A103. Furthermore, bulky residues exist around the heme cofactor, resulting in a ruffling conformation of the porphyrin ring. Based on our study, Kgb is thought to be involved in electron transfer or enzymatic reactions rather than transporting or storing ligands. Database: Structural data are available in the Protein Data Bank under the accession numbers 5ZIQ (Kgb4‐SR) and 5ZM9 (Kgb7‐house). Tardigrades are renowned for being able to survive extreme conditions that would be intolerable to other animals. To survive extreme desiccation, they enter an anhydrobiotic state in which they virtually stop all metabolic processes. In this study, Tsuyoshi Inoue and co‐authors present a detailed spectroscopic and structural characterization of a newly discovered heme protein that is unique to anhydrobiotic tardigrades. Exploration of this protein, Kumaglobin (Kgb, named after the Japanese name for tardigrades) expands our knowledge of globin protein biodiversity. [ABSTRACT FROM AUTHOR]

Copper‐containing nitrite reductases (CuNIRs) are multifunctional enzymes that catalyse the one‐electron reduction of nitrite (NO2−) to nitric oxide (NO) and the two‐electron reduction of dioxygen (O2) to hydrogen peroxide (H2O2). In contrast to the mechanism of nitrite reduction, that of dioxygen reduction is poorly understood. Here, results from anaerobic synchrotron‐radiation crystallography (SRX) and aerobic in‐house radiation crystallography (iHRX) with a CuNIR from the thermophile Geobacillus thermodenitrificans (GtNIR) support the hypothesis that the dioxygen present in an aerobically manipulated crystal can bind to the catalytic type 2 copper (T2Cu) site of GtNIR during SRX experiments. The anaerobic SRX structure showed a dual conformation of one water molecule as an axial ligand in the T2Cu site, while previous aerobic SRX GtNIR structures were refined as diatomic molecule‐bound states. Moreover, an SRX structure of the C135A mutant of GtNIR with peroxide bound to the T2Cu atom was determined. The peroxide molecule was mainly observed in a side‐on binding manner, with a possible minor end‐on conformation. The structures provide insights into dioxygen chemistry in CuNIRs and hence help to unmask the other face of CuNIRs. [ABSTRACT FROM AUTHOR]

Abstract: Though anhydrobiotic tardigrades (micro‐animals also known as water bears) possess many genes of secretory abundant heat soluble (SAHS) proteins unique to Tardigrada, their functions are unknown. A previous crystallographic study revealed that a SAHS protein (RvSAHS1) from one of the toughest tardigrades, Ramazzottius varieornatus, has a β‐barrel architecture similar to fatty acid binding proteins (FABPs) and two putative ligand binding sites (LBS1 and LBS2) where fatty acids can bind. However, some SAHS proteins such as RvSAHS4 have different sets of amino acid residues at LBS1 and LBS2, implying that they prefer other ligands and have different functions. Here RvSAHS4 was crystallized and analyzed under a condition similar to that for RvSAHS1. There was no electron density corresponding to a fatty acid at LBS1 of RvSAHS4, where a putative fatty acid was observed in RvSAHS1. Instead, LBS2 of RvSAHS4, which was composed of uncharged residues, captured a putative polyethylene glycol molecule. These results suggest that RvSAHS4 mainly uses LBS2 for the binding of uncharged molecules. [ABSTRACT FROM AUTHOR]

Upon stopping metabolic processes, some tardigrades can undergo anhydrobiosis. Secretory abundant heat-soluble ( SAHS) proteins have been reported as candidates for anhydrobiosis-related proteins in tardigrades, which seem to protect extracellular components and/or secretory organelles. We determined structures of a SAHS protein from Ramazzottius varieornatus ( Rv SAHS1), which is one of the toughest tardigrades. Rv SAHS1 shows a β-barrel structure similar to fatty acid-binding proteins ( FABPs), in which hydrophilic residues form peculiar hydrogen bond networks, which would provide Rv SAHS1 with better tolerance against dehydration. We identified two putative ligand-binding sites: one that superimposes on those of some FABPs and the other, unique to and conserved in SAHS proteins. These results indicate that SAHS proteins constitute a new FABP family. [ABSTRACT FROM AUTHOR]

Pseudoazurin from the denitrifying bacterium Alcaligenes faecalis ( AfPAz) is a blue copper protein and functions as an electron donor to copper-containing nitrite reductase (CuNIR). Conventionally, AfPAz has been crystallized using highly concentrated ammonium sulfate as a precipitant. Here, a needle-like crystal of AfPAz grown in a solution containing a macromolecular precipitant, polyethylene glycol 8000 (PEG 8000), is reported. The crystal belonged to space group P61, with unit-cell parameters a = b = 68.7, c = 94.2 Å. The structure has been determined and refined at 2.6 Å resolution. The asymmetric unit contained two AfPAz molecules contacting each other on negatively charged surfaces. The molecular packing of the crystal showed a right-handed double-helical arrangement of AfPAz molecules and hence of blue copper sites. This structure provides insight into the excluded-volume effect of PEG and the manner of assembly of AfPAz. [ABSTRACT FROM AUTHOR]

In order to develop a structure-based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on N, N′-diacetylchitobiose deacetylases (Dacs) from Pyrococcus horikoshii (Ph-Dac) and Pyrococcus furiosus (Pf-Dac). Neither Ph-Dac nor Pf-Dac was expressed in the soluble fraction of Escherichia coli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E. coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were active and thermostable up to 85 °C. The crystal structures of Ph-Dac and Pf-Dac were determined at resolutions of 2.0 Å and 1.54 Å, respectively. The Pyrococcus Dac forms a hexamer composed of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph-Dac, catalytic Zn situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N-acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the Nε atom of His264 (from the adjacent polypeptide in the Ph-Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. Mutation analysis also indicated that His264 was essential to the catalysis. These factors give the archaeal Dacs an unprecedented active site architecture a Zn-dependent deacetylases. Database Structural data are available in the Protein Data Bank database under accession numbers , , and . Structured digital abstract and by () and by () [ABSTRACT FROM AUTHOR]

Mutations of human BEST1, encoding a Ca2+-activated Cl− channel (hBest1), cause macular degenerative disorders. Best1 homolog structures reveal an evolutionarily conserved channel architecture highlighted by two landmark restrictions (named the "neck" and "aperture", respectively) in the ion conducting pathway, suggesting a unique dual-switch gating mechanism, which, however, has not been characterized well. Using patch clamp and crystallography, we demonstrate that both the neck and aperture in hBest1 are Ca2+-dependent gates essential for preventing channel leakage resulting from Ca2+-independent, spontaneous gate opening. Importantly, three patient-derived mutations (D203A, I205T and Y236C) lead to Ca2+-independent leakage and elevated Ca2+-dependent anion currents due to enhanced opening of the gates. Moreover, we identify a network of residues critically involved in gate operation. Together, our results suggest an indispensable role of the neck and aperture of hBest1 for channel gating, and uncover disease-causing mechanisms of hBest1 gain-of-function mutations. Ji and Kittredge et al. demonstrate that the neck and aperture of human Bestrophin1, a Ca2+-activated Cl- channel, are both Ca2+-dependent gates. By showing hyper-activity of Bestrophin1, which is associated with its enhanced gate opening in the presence of three patient-derived mutations, this study provides mechanistic insights into the gain-of-function mutations that cause macular degenerative disease. [ABSTRACT FROM AUTHOR]

Some tardigrades can survive extremely desiccated conditions through transition into a state called anhydrobiosis. Anhydrobiotic tardigrades have proteins unique to them and they are thought to be keys to the understanding of unusual desiccation resistance. In fact, previous transcriptome data show that several tardigrade-specific proteins are significantly upregulated under desiccated conditions. However, their physiological roles and chemical properties have been ambiguous because they show low or no similarity of amino acid sequences to proteins found in other organisms. Here, we report a crystal structure of one of such proteins. This protein shows a β-sandwich structure composed of 8 β-strands, three Ca 2+ -binding sites, and hydrophobic residues on Ca 2+ -binding (CBD) loops, which resemble characteristics of C2 domain proteins. We therefore conveniently describe this protein as tardigrade C2 domain protein (TC2P). Because the C2 domain functions as a Ca 2+ -mediated membrane docking module, which is related to signal transduction or membrane trafficking, TC2Ps may play a role in Ca 2+ -triggered phenomenon under desiccated situations. Our finding provides not only structural insights into a newly discovered desiccation-related protein family but also insights into the evolution and diversity of C2 domain proteins., (© 2020 The Authors. Protein Science published by Wiley Periodicals LLC. on behalf of The Protein Society.)

The glycosylation of small hydrophobic compounds is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Because glycosylation is an invaluable tool for improving the stability and water solubility of hydrophobic compounds, UGTs have attracted attention for their application in the food, cosmetics, and pharmaceutical industries. However, the ability of UGTs to accept and glycosylate a wide range of substrates is not clearly understood due to the existence of a large number of UGTs. Pa GT2, a UGT from Phytolacca americana , can regioselectively glycosylate piceatannol but has low activity toward other stilbenoids. To elucidate the substrate specificity and catalytic mechanism, we determined the crystal structures of Pa GT2 with and without substrates and performed molecular docking studies. The structures have revealed key residues involved in substrate recognition and suggest the presence of a nonconserved catalytic residue (His81) in addition to the highly conserved catalytic histidine in UGTs (His18). The role of the identified residues in substrate recognition and catalysis is elucidated with the mutational assay. Additionally, the structure-guided mutation of Cys142 to other residues, Ala, Phe, and Gln, allows Pa GT2 to glycosylate resveratrol with high regioselectivity, which is negligibly glycosylated by the wild-type enzyme. These results provide a basis for tailoring an efficient glycosyltransferase.

The soluble region (residues 32-354) of GK0767, a copper-containing nitrite reductase from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426, has been cloned and overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 1.3 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 115.1, c = 87.5 Å. Preliminary studies and molecular-replacement calculations reveal the presence of one subunit of the homotrimeric structure in the asymmetric unit; this corresponds to a V(M) value of 3.14 Å(3) Da(-1).