Structural bases for aspartate recognition and polymerization efficiency of cyanobacterial cyanophycin synthetase.

Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin synthetase (CphA1), and accumulates as a nitrogen reservoir during N2 fixation by most cyanobacteria. A recent structural study showed that three constituent domains of CphA1 function as two distinct catalytic sites and an oligomerization interface in cyanophycin synthesis. However, it remains unclear how the ATP-dependent addition of aspartate to cyanophycin is initiated at the catalytic site of the glutathione synthetase-like domain. Here, we report the cryogenic electron microscopy structures of CphA1, including a complex with aspartate, cyanophycin primer peptide, and ATP analog. These structures reveal the aspartate binding mode and phosphate-binding loop movement to the active site required for the reaction. Furthermore, structural and mutational data show a potential role of protein dynamics in the catalytic efficiency of the arginine condensation reaction.

Isolation and characterization of oligopeptides with vascular disease suppression effects derived from wheat gluten.

Wheat gluten was hydrolyzed with both alkaline protease and neutral protease to produce high-protein and low-wheat-weight oligopeptides (WOP), which was subjected to a multistage purification. Then, high performance liquid chromatography was applied to separate WOP. In order to identify WOP sequences, six major fractions were gathered for mass spectrometry. A total of 15 peptides were synthesized for further in vitro analyses of their antithrombotic activity, vasorelaxation activity, and cholesterol reducing activity. Two antithrombotic peptides (ILPR and ILR), three vasorelaxant peptides (VN, FPQ, and FR), and four cholesterol-lowering peptides (QRQ, ILPR, FPQ, and ILR) were identified. These active peptides in WOP were also quantified. These peptides are novel candidate peptides with vascular disease suppressing effects. The results indicate WOP as good protein sources for multifunctional peptides.

Structural bases of IMiD selectivity that emerges by 5-hydroxythalidomide.

Thalidomide and its derivatives exert not only therapeutic effects as immunomodulatory drugs (IMiDs) but also adverse effects such as teratogenicity, which are due in part to different C2H2 zinc-finger (ZF) transcription factors, IKZF1 (or IKZF3) and SALL4, respectively. Here, we report the structural bases for the SALL4-specific proteasomal degradation induced by 5-hydroxythalidomide, a primary thalidomide metabolite generated by the enzymatic activity of cytochrome P450 isozymes, through the interaction with cereblon (CRBN). The crystal structure of the metabolite-mediated human SALL4-CRBN complex and mutagenesis studies elucidate the complex formation enhanced by the interaction between CRBN and an additional hydroxy group of (S)-5-hydroxythalidomide and the variation in the second residue of ß-hairpin structure that underlies the C2H2 ZF-type neo-morphic substrate (neosubstrate) selectivity of 5-hydroxythalidomide. These findings deepen our understanding of the pharmaceutical action of IMiDs and provide structural evidence that the glue-type E3 ligase modulators cause altered neosubstrate specificities through their metabolism.

Comprehensive NMR analysis of two kinds of post-fermented tea and their anti-glycation activities in vitro.

Post-fermented tea (dark tea) is produced from enzyme-inactivated fresh tea leaves by microbial fermentation. Batabata tea and Awaban tea are two major dark teas fermented under aerobic and anaerobic conditions, respectively. However, how their chemical compositions and functionalities change during different post-fermentation processes remains unclear. Nuclear magnetic resonance (NMR)-based analyses showed that (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) decreased in Batabata tea during post-fermentation with aerobic molds. In contrast, EGC and EC increased, and pyrogallol was produced in Awaban tea during post-fermentation with lactic acid bacteria (LAB). The anti-glycation activities of two dark teas were investigated using an in vitro assay system with human serum albumin (HSA). The anti-glycation activity decreased in Batabata tea, but it was retained in Awaban tea during post-fermentation. Our results showed that post-fermentation with LAB was an efficient way to enhance phenol content and that pyrogallol contributed to anti-glycation activity of Awaban tea.

Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6.

Toll-like receptors (TLRs) mediate immune responses upon recognition of a variety of ligands. To further elucidate the function of TLRs, it is important to identify novel ligands and their action mechanisms including polymer assembly. In this study, we propose an efficient method for preparation of the extracellular domain of human Toll-like receptor 6 (TLR6ED) in Escherichia coli using the bubbling cultivation method. Our preparation method improved the level of expression of TLR6ED into a soluble fraction as compared with typical cultivation using a rotary shaker. Circular dichroism (CD) experiments confirmed the structural formation of TLR6ED with secondary structure contents similar to leucine-rich repeat (LRR) modules. In addition, we also provided a procedure for preparing this recombinant protein using Sf9 insect cells, which ensures preservation of some key posttranslational modifications often lacking in bacteria-expressed proteins. These materials would be useful for analyzing novel molecules that bind directly to TLR6, complex formations with other regulators including TLR2 and TLR4, and the functional effects of N-linked glycosylation.

Electron microscopic recording of myosin head power stroke in hydrated myosin filaments.

Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed.

A secreted protein with plant-specific cysteine-rich motif functions as a mannose-binding lectin that exhibits antifungal activity.

Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the α1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family.

Molecular mechanism of strigolactone perception by DWARF14.

Strigolactones (SLs) are phytohormones that inhibit shoot branching and function in the rhizospheric communication with symbiotic fungi and parasitic weeds. An α/ß-hydrolase protein, DWARF14 (D14), has been recognized to be an essential component of plant SL signalling, although its precise function remains unknown. Here we present the SL-dependent interaction of D14 with a gibberellin signalling repressor SLR1 and a possible mechanism of phytohormone perception in D14-mediated SL signalling. D14 functions as a cleavage enzyme of SLs, and the cleavage reaction induces the interaction with SLR1. The crystal structure of D14 shows that 5-hydroxy-3-methylbutenolide (D-OH), which is a reaction product of SLs, is trapped in the catalytic cavity of D14 to form an altered surface. The D14 residues recognizing D-OH are critical for the SL-dependent D14-SLR1 interaction. These results provide new insight into crosstalk between gibberellin and SL signalling pathways.

A thermoacidophile-specific protein family, DUF3211, functions as a fatty acid carrier with novel binding mode.

STK_08120 is a member of the thermoacidophile-specific DUF3211 protein family from Sulfolobus tokodaii strain 7. Its molecular function remains obscure, and sequence similarities for obtaining functional remarks are not available. In this study, the crystal structure of STK_08120 was determined at 1.79-Å resolution to predict its probable function using structure similarity searches. The structure adopts an α/ß structure of a helix-grip fold, which is found in the START domain proteins with cavities for hydrophobic substrates or ligands. The detailed structural features implied that fatty acids are the primary ligand candidates for STK_08120, and binding assays revealed that the protein bound long-chain saturated fatty acids (>C14) and their trans-unsaturated types with an affinity equal to that for major fatty acid binding proteins in mammals and plants. Moreover, the structure of an STK_08120-myristic acid complex revealed a unique binding mode among fatty acid binding proteins. These results suggest that the thermoacidophile-specific protein family DUF3211 functions as a fatty acid carrier with a novel binding mode.

Structure of the Ca(2+)-saturated C-terminal domain of scallop troponin C in complex with a troponin I fragment.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin that triggers the contraction of striated muscles. In scallops, the striated muscles consume little ATP energy in sustaining strong contractile forces. The N-terminal domain of TnC works as the Ca(2+) sensor in vertebrates, whereas scallop TnC uses the C-terminal domain as the Ca(2+) sensor, suggesting that there are differences in the mechanism of the Ca(2+)-dependent regulation of muscles between invertebrates and vertebrates. Here, we report the crystal structure of Akazara scallop (Chlamys nipponensis akazara) adductor muscle TnC C-terminal domain (asTnCC) complexed with a short troponin I fragment (asTnIS) and Ca(2+). The electron density of a Ca(2+) ion is observed in only one of the two EF-hands. The EF-hands of asTnCC can only be in the fully open conformation with the assistance of asTnIS. The number of hydrogen bonds between asTnCC and asTnIS is markedly lower than the number in the vertebrate counterparts. The Ca(2+) modulation on the binding between asTnCC and asTnIS is weaker, but structural change of the complex depending on Ca(2+) concentration was observed. Together, these findings provide a detailed description of the distinct molecular mechanism of contractile regulation in the scallop adductor muscle from that of vertebrates.

Different Ca²âº-sensitivities between the EF-hands of T- and L-plastins.

Plastins are Ca(2+)-regulated actin-bundling proteins, and essential for developing and stabilizing actin cytoskeletons. T-plastin is expressed in epithelial and mesenchymal cells of solid tissues, whereas L-plastin is expressed in mobile cells such as hemopoietic cell lineages and cancer cells. Using various spectroscopic methods, gel-filtration chromatography, and isothermal titration calorimetry, we here demonstrate that the EF-hand motifs of both T- and L-plastin change their structures in response to Ca(2+), but the sensitivity to Ca(2+) is lower in T-plastin than in L-plastin. These results suggest that T-plastin is suitable for maintaining static cytoskeletons, whereas L-plastin is suitable for dynamic rearrangement of cytoskeletons.

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber.

Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

Structural basis of abscisic acid signalling.

The phytohormone abscisic acid (ABA) mediates the adaptation of plants to environmental stresses such as drought and regulates developmental signals such as seed maturation. Within plants, the PYR/PYL/RCAR family of START proteins receives ABA to inhibit the phosphatase activity of the group-A protein phosphatases 2C (PP2Cs), which are major negative regulators in ABA signalling. Here we present the crystal structures of the ABA receptor PYL1 bound with (+)-ABA, and the complex formed by the further binding of (+)-ABA-bound PYL1 with the PP2C protein ABI1. PYL1 binds (+)-ABA using the START-protein-specific ligand-binding site, thereby forming a hydrophobic pocket on the surface of the closed lid. (+)-ABA-bound PYL1 tightly interacts with a PP2C domain of ABI1 by using the hydrophobic pocket to cover the active site of ABI1 like a plug. Our results reveal the structural basis of the mechanism of (+)-ABA-dependent inhibition of ABI1 by PYL1 in ABA signalling.

Direct demonstration of the cross-bridge recovery stroke in muscle thick filaments in aqueous solution by using the hydration chamber.

Despite >50 years of research work since the discovery of sliding filament mechanism in muscle contraction, structural details of the coupling of cyclic cross-bridge movement to ATP hydrolysis are not yet fully understood. An example would be whether lever arm tilting on the myosin filament backbone will occur in the absence of actin. The most direct way to elucidate such movement is to record ATP-induced cross-bridge movement in hydrated thick filaments. Using the hydration chamber, with which biological specimens can be kept in an aqueous environment in an electron microscope, we have succeeded in recording ATP-induced cross-bridge movement in hydrated thick filaments consisting of rabbit skeletal muscle myosin, with gold position markers attached to the cross-bridges. The position of individual cross-bridges did not change appreciably with time in the absence of ATP, indicating stability of time-averaged cross-bridge mean position. On application of ATP, individual cross-bridges moved nearly parallel to the filament long axis. The amplitude of the ATP-induced cross-bridge movement showed a peak at 5-7.5 nm. At both sides of the filament bare region, across which the cross-bridge polarity was reversed, the cross-bridges were found to move away from, but not toward, the bare region. Application of ADP produced no appreciable cross-bridge movement. Because ATP reacts rapidly with the cross-bridges (M) to form complex (M x ADP x Pi) with an average lifetime >10 s, the observed cross-bridge movement is associated with reaction, M + ATP --> M x ADP x Pi. The cross-bridges were observed to return to their initial position after exhaustion of ATP. These results constitute direct demonstration of the cross-bridge recovery stroke.

Proteinase inhibitor from ginkgo seeds is a member of the plant nonspecific lipid transfer protein gene family.

A 9-kD proteinase inhibitor was isolated from the seeds of ginkgo (Ginkgo biloba) and purified to homogeneity. This protein was revealed to partial-noncompetitively inhibit the aspartic acid proteinase pepsin and the cysteine proteinase papain (inhibition constant = 10(-5)-10(-4) m). The cDNA of the inhibitor was revealed to contain a 357-bp open reading frame encoding a 119-amino acid protein with a potential signal peptide (27 residues), indicating that this protein is synthesized as a preprotein and secreted outside the cells. Semiquantitative reverse transcription-polymerase chain reaction revealed that this gene expresses only in seeds, not in stems, leaves, and roots, suggesting that the protein is involved in seed development and/or germination. The inhibitor showed about 40% sequence homology with type-I nonspecific lipid transfer protein (nsLTP1) from other plant species. Actually, this inhibitor exerted both lipid transfer activity and lipid-binding activity, while the protein did not show any antifungal and antibacterial activities. Furthermore, the site-directed mutagenesis study using a recombinant ginkgo nsLTP1 revealed that proline (Pro)-79 and phenylalanine-80 are important on phospholipid transfer activity and that Pro-79 and isoleucine-82 are essential for the binding activity toward cis-unsaturated fatty acids. On the other hand, the alpha-helical content of P79A and F80A mutants was significantly lower than that of the wild-type protein. It was noteworthy that the papain-inhibitory activity of P79A and F80A mutants was elevated twice as much as that of the wild-type protein. In summary, we concluded that Pro-79 plays a critical role in both the lipid transfer and binding activities of ginkgo nsLTP1.

Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle.

Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca(2+) ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca(2+)-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca(2+)-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca(2+)-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca(2+)-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca(2+)-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca(2+) signal to TnI in Tn ternary complex.

Crystallization and preliminary X-ray analysis of the Ca2+-bound C-terminal lobe of troponin C in complex with a troponin I-derived peptide fragment from Akazara scallop.

Troponin C (TnC) is the Ca(2+)-binding component of troponin and triggers muscle contraction. TnC of the invertebrate Akazara scallop can bind only one Ca(2+) at the C-terminal EF-hand motif. Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. The crystals diffracted X-rays to 1.80 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.1, b = 42.2, c = 60.0 A. The asymmetric unit was assumed to contain one molecular complex of the Akazara scallop TnC C-lobe and TnI fragment, with a Matthews coefficient of 1.83 A(3) Da(-1) and a solvent content of 33.0%.