Crystal structure of a Ca2+-dependent regulator of flagellar motility reveals the open-closed structural transition

Sperm chemotaxis toward a chemoattractant is very important for the success of fertilization. Calaxin, a member of the neuronal calcium sensor protein family, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis of ascidian, Ciona intestinalis. Here, we present the crystal structures of calaxin both in the open and closed states upon Ca2+ and Mg2+ binding. The crystal structures revealed that three of the four EF-hands of a calaxin molecule bound Ca2+ ions and that EF2 and EF3 played a critical role in the conformational transition between the open and closed states. The rotation of α7 and α8 helices induces a significant conformational change of a part of the α10 helix into the loop. The structural differences between the Ca2+- and Mg2+-bound forms indicates that EF3 in the closed state has a lower affinity for Mg2+, suggesting that calaxin tends to adopt the open state in Mg2+-bound form. SAXS data supports that Ca2+-binding causes the structural transition toward the closed state. The changes in the structural transition of the C-terminal domain may be required to bind outer-arm dynein. These results provide a novel mechanism for recognizing a target protein using a calcium sensor protein

A new target region for changing the substrate specificity of amine transaminases

(R)-stereospecific amine transaminases (R-ATAs) are important biocatalysts for the production of (R)-amine compounds in a strict stereospecific manner. An improved R-ATA, ATA-117-Rd11, was successfully engineered for the manufacture of sitagliptin, a widely used therapeutic agent for type-2 diabetes. The effects of the individual mutations, however, have not yet been demonstrated due to the lack of experimentally determined structural information. Here we describe three crystal structures of the first isolated R-ATA, its G136F mutant and engineered ATA-117-Rd11, which indicated that the mutation introduced into the 136th residue altered the conformation of a loop next to the active site, resulting in a substrate-binding site with drastically modified volume, shape, and surface properties, to accommodate the large pro-sitagliptin ketone. Our findings provide a detailed explanation of the previously reported molecular engineering of ATA-117-Rd11 and propose that the loop near the active site is a new target for the rational design to change the substrate specificity of ATAs.UTokyo Research掲載「さまざまな薬の合成に重要な酵素が作用する相手を認識する仕組み」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/how-an-important-enzyme-used-in-drug-production-recognizes-its-substrate.htmlUTokyo Research "How an important enzyme used in drug production recognizes its substrate" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/how-an-important-enzyme-used-in-drug-production-recognizes-its-substrate.htm

Primary Structure and Conformation of a Tetrodotoxin-Binding Protein in the Hemolymph of Non-Toxic Shore Crab <i>Hemigrapsus sanguineus</i>

Tetrodotoxin (TTX)-binding proteins are present in toxic TTX-bearing animals, such as pufferfish and gastropods. These may prevent autotoxicity. However, TTX-binding proteins are also found in the nontoxic marine shore crab, Hemigrapsus sanguineus. Here, we isolated the TTX-binding protein, HSTBP (Hemigrapsus sanguineus TTX-binding protein), from the hemolymph of H. sanguineus and elucidated its primary structure using cDNA cloning. HSTBP, a 400 kDa acidic glycoprotein by gel filtration high-performance liquid chromatography, comprises 3 subunits, 88 kDa (subunit-1), 65 kDa (subunit-2), and 26 kDa (subunit-3) via sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced conditions. The open reading frame of the cDNA comprises 5049 base pairs encoding 1683 amino acid residues, and the mature protein contains 1650 amino acid residues from Arg34 to Ser1683. The three subunits are arranged in tandem in the following order: subunit-3 (Arg34-Gln261), subunit-1 (Asp262-Phe1138), and subunit-2 (Val1139-Ser1683). A BLAST homology search showed weak similarity of HSTBP to clotting proteins of crustaceans (29–40%). SMART analysis revealed a von Willebrand factor (vWF)-type (⇒delete hyphen) D domain at Phe1387-Gly1544. We confirmed that the recombinant protein of HSTBP subunit-2 containing the vWF-type (⇒delete hyphen) D domain bound to TTX at a molecular ratio of 1:1

Crystallization and preliminary X-ray analysis of γ-­hexachlorocyclohexane dehydrochlorinase LinA from Sphingobium japonicum UT26

Purification and crystallization of LinA allowed the collection of data to 2.25 Å resolution

Association between uncooperativeness and the glucose metabolism of patients with chronic behavioral disorders after severe traumatic brain injury: a cross-sectional retrospective study

Abstract Bakground Patients with behavioral disorders following severe traumatic brain injury (sTBI) often have disorders of consciousness that make expressing their emotional distress difficult. However, no standard method for assessing the unsettled and unforeseen responses that are associated with behavioral disorders has yet to be established. Because the thalamus is known to play a role in maintaining consciousness and cognition, we used 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) to examine the association between brain glucose metabolism in the thalamus and behavioral disorders. Methods We retrospectively analyzed 70 consecutive patients with sTBI who had been involved in motor vehicle accidents. To assess behavioral disorders, we evaluated 18 symptoms using the Brief Psychiatric Rating Scale (BPRS): Emotional Withdrawal, Conceptual Disorganization, Tension, Mannerisms and Posturing, Motor Retardation, Uncooperativeness, Blunted Affect, Excitement, Somatic Concern, Anxiety, Feeling of Guilt, Grandiosity, Depressive Mood, Hostility, Suspiciousness, Hallucinatory Behavior, Unusual Thought Content, and Disorientation. First, we identified clinical characteristics of sTBI patients with behavioral disorders. Next, we retrospectively analyzed 18F-FDG-PET/CT data to assess how thalamic activity was related with abnormal behaviors. Results Twenty-six patients possessed the minimum communicatory ability required for psychiatric interview. Among them, 15 patients (57.7%) were diagnosed with behavioral disorder, 14 of whom had reached a stable psychiatric state after about 426.6 days of treatment. Excitement (13 patients) and uncooperativeness (10 patients) were the most frequently observed symptoms. Available 18F-FDG-PET/CT data indicated that thalamic glucose metabolism was imbalanced and lateralized (p = 0.04) in 6 patients who exhibited uncooperativeness. Conclusions Behavioral symptoms of excitement and uncooperativeness were common in patients with sTBI, although most symptoms improved as the chronic stage continued. Our data support the idea that imbalanced laterality of glucose metabolism in the thalamus might be related to behavioral disorders characterized by uncooperativeness. Trial registration UMIN 000029531. Registered 27 March 2017, retrospectively registered

Additional file 1: of Association between uncooperativeness and the glucose metabolism of patients with chronic behavioral disorders after severe traumatic brain injury: a cross-sectional retrospective study

Figure S1. Representative images of a three-dimensional volume of interest measurement (a) and color mapped image (b) of glucose metabolism measured via 18F-fluorodeoxyglucose positron emission tomography/computed tomography. (PPTX 380 kb