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L-ergothioneine is widely distributed among various microbes to regulate their physiology and pathogenicity within complex environments. One of the key steps in the ergothioneinebiosynthesis pathway, the C-S bond cleavage reaction, uses the pyridoxal 50-phosphate dependent C-S lyase to produce the final product L-ergothioneine. Here, we present the crystallographic structure of the ergothioneine-biosynthesis C-S lyase EgtE from Mycobacterium smegmatis (MsEgtE) represents the first published structure of ergothioneine-biosynthesis C-S lyases in bacteria and shows the effects of active site residues on the enzymatic reaction. The MsEgtE and the previously reported ergothioneine-biosynthesis C-S lyase Egt2 from Neurospora crassa (NcEgt2) fold similarly. However, discrepancies arise in terms of substrate recognition, as observed through sequence and structure comparison of MsEgtE and NcEgt2. The structural-based sequence alignment of the ergothioneinebiosynthesis C-S lyase from fungi and bacteria shows clear distinctions among the recognized substrate residues, but Arg348 is critical and an extremely conserved residue for substrate recognition. The a14 helix is exclusively found in the bacteria EgtE, which represent the most significant difference between bacteria EgtE and fungi Egt2, possibly resulting from the convergent evolution of bacteria and fungi. [ABSTRACT FROM AUTHOR]

Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO2 oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO2 interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the α2ϵ2 component from the α8β8γ8δ8ϵ8 CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H2O/OH- bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the ϵ-subunit and FeS domain not present in nonmethanogenic Ni-CODHs. [ABSTRACT FROM AUTHOR]

Genes encoding methanogenic methylamine methyltransferases all contain an in-frame amber (UAG) codon that is read through during translation. We have identified the UAG-encoded residue in a 1.55 angstrom resolution structure of the Methanosarcina barkeri monomethylamine methyltransferase (MtmB). This structure reveals a homohexamer comprised of individual subunits with a TIM barrel fold. The electron density for the UAG-encoded residue is distinct from any of the 21 natural amino acids. Instead it appears consistent with a lysine in amide-linkage to (4R,5R)-4-substituted-pyrroline-5-carboxylate. We suggest that this amino acid be named L-pyrrolysine. [ABSTRACT FROM AUTHOR]

Investigates the catalytic activities of DNA beta polymerase. Elaboration of DNA polymerase structures; Re-evaluation on the evidence regarding the existence of a rate-limiting conformational change in beta polymerase reaction pathway.

Determines the structures of Bradyrhizobium japonicum FixL heme domain (BjFixLH) bound to dioxygen, imidazole and nitric oxide to gain further insight into the FixL sensing mechanism. Indication that the structure of BjFixLH bound to imidazole elucidates the structural requirements for accommodating sterically bulky ligands.

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decar- boxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and cata- lytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine. [ABSTRACT FROM AUTHOR]

Determines the structures of the protein-inhibitor complexes of both the cobalt and the zinc containing Escherichia coli peptide deformylase bound to the transition-state analogue, (s)-2-O-(H-phosphonyxy)-L-caproyl-L-leucyl-p-nitroanilide (PCLNA). Examination of peptide deformylase as a potential target for antibacterial chemotherapy; Extended conformation of the PCLNA.

Reports on the crystallization and the x-ray structure solution of zinc containing Escherichia coli peptide deformylase. Geometric similarity of zinc metal's environment with active sites of the thermolysin family; Origin of the enzyme's specificity for N-formulated over N-acetylated substrates; Significance of the study in understanding protein function.

Translation initiation in eubacteria involves a formylmethionine at the N-terminus of newly synthesized polypeptides. This N-formyl group is removed by peptide deformylase (PDF) during the post-translation process. Such a formylation/deformylation cycle is essential for the cell survival of eubacteria, but is not utilized in eukaryotic cytosolic protein biosynthesis. In view of the absence of deformylase activity in mammalian cells, this is an attractive target for the design of novel antibiotic drugs. Co-crystallization of peptide deformylase from Leptospira interrogans (LiPDF) with its natural inhibitor actinonin produced diffraction-quality crystals that belong to space group P2⊂, with unit-cell parameters a = 87.5, b = 119.1, c = 95.8 &Aring, β = 111.6 &degrees;. The 3.1 Å resolution data set collected in-house was used to obtain phases by molecular replacement. Three schemes for the correction of the preliminary solutions were proposed and proved successful in determining the structure of LiPDF with eight subunits in the asymmetric unit. [ABSTRACT FROM AUTHOR]

The article investigates the fluoride permeation mechanism in the Fluc-Ec1 channel within liposomes using solid-state NMR and molecular dynamics simulations. It uncovers new fluoride binding sites in the channel's aqueous vestibules and identifies one as a water molecule. It also highlights a dynamic region in the channel, loop 1, and proposes a "water-mediated knock-on" mechanism for fluoride conduction.

Stress granules (SGs) are cytoplasmic biomolecular condensates containing proteins and RNAs in response to stress. Ras-GTPase-activating protein binding protein 1 (G3BP1) is a core SG protein. Caprin-1 and ubiquitin specific peptidase 10 (USP10) interact with G3BP1, facilitating and suppressing SG formation, respectively. The crystal structures of the nuclear transport factor 2-like (NTF2L) domain of G3BP1 in complex with the G3BP1 -interacting motif (GIM) of Caprin-1 and USP10 show that both GIMS bind to the same hydrophobic pocket of G3BP1. Moreover, both GIMs suppressed the liquid-liquid phase separation (LLPS) of G3BP1, suggesting that Caprin-1 likely facilitates SG formation via other mechanisms. Thus, we dissected various domains of Caprin-1 and investigated their role in LLPS in vitro and SG formation in cells. The C-terminal domain of Caprin-1 underwent spontaneous LLPS, whereas the N-terminal domain and GIM of Caprin-1 suppressed LLPS of G3BP1. The opposing effect of the N- and C-terminal domains of Caprin-1 on SG formation were demonstrated in cells with or without the endogenous Caprin-1. We propose that the N- and C-terminal domains of Caprin-1 regulate SG formation in a "yin and yang" fashion, mediating the dynamic and reversible assembly of SGs. [ABSTRACT FROM AUTHOR]

Peptide deformylase is an attractive target for developing novel antibiotics. Previous studies at pH 3.0 showed peptide deformylase from Leptospira interrogans (LiPDF) exists as a dimer in which one monomer is in a closed form and the other is in an open form, with different conformations of the CD-loop controlling the entrance to the active pocket. Here we present structures of LiPDF at its active pH range. LiPDF forms a similar dimer at pH values 6.5–8.0 as it does at pH 3.0. Interestingly, both of the monomers are almost in the same closed form as that observed at pH 3.0. However, when the enzyme is complexed with the natural inhibitor actinotin, the conformation of the CD-loop is half-open. Two pairs of Arg109-mediated cation-π interactions, as well as hydrogen bonds, have been identified to stabilize the different CD-loop conformations. These results indicate that LiPDF may be found in different structural states, a feature that has never before been observed in the peptide deformylase family. Based on our results, a novel substrate binding model, featured by an equilibrium between the closed and the open forms, is proposed. Our results present crystallographic evidence supporting population shift theory, which is distinguished from the conventional lock-and-key or induced-fit models. These results not only facilitate the development of peptide deformylase-targeted drugs but also provide structural insights into the mechanism of an unusual type of protein binding event. [ABSTRACT FROM AUTHOR]

Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by the occurrence of benign tumors in various vital organs and tissues. TSC1 and TSC2, the TSC1 and TSC2 gene products, form the TSC protein complex that senses specific cellular growth conditions to control mTORC1 signaling. TBC1D7 is the third subunit of the TSC complex, and helps to stabilize the TSC1-TSC2 complex through its direct interaction with TSC1. Homozygous inactivation of TBC1D7 causes intellectual disability and megaencephaly. Here we report the crystal structure of a TSC1- TBC1D7 complex and biochemical characterization of the TSC1-TBC1D7 interaction. TBC1D7 interacts with the C-terminal region of the predicted coiled-coil domain of TSC1. The TSC1-TBC1D7 interface is largely hydrophobic, involving the 4 helix of TBC1D7. Each TBC1D7 molecule interacts simultaneously with two parallel TSC1 helices from two TSC1 molecules, suggesting that TBC1D7 may stabilize the TSC complex by tethering the C-terminal ends of two TSC1 coiled-coils. [ABSTRACT FROM AUTHOR]

Electron transfer (ET) is widely used for driving the processes that underlie the chemistry of life. However, our abilities to probe electron transfer mechanisms in proteins and design redox enzymes are limited, due to the lack of methods to site-specifically insert electron acceptors into proteins in vivo. Here we describe the synthesis and genetic incorporation of 4- fluoro-3-nitrophenylalanine (FNO2Phe), which has similar reduction potentials to NAD(P)H and ferredoxin, the most important biological reductants. Through the genetic incorporation of FNO2Phe into green fluorescent protein (GFP) and femtosecond transient absorption measurement, we show that photoinduced electron transfer (PET) from the GFP chromophore to FNO2Phe occurs very fast (within 11 ps), which is comparable to that of the first electron transfer step in photosystem I, from P700* to A0. This genetically encoded, low-reduction potential unnatural amino acid (UAA) can significantly improve our ability to investigate electron transfer mechanisms in complex reductases and facilitate the design of miniature proteins that mimic their functions. [ABSTRACT FROM AUTHOR]

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNAwith high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site- and sequence-specific covalent labeling of RNAs in mammalian cells by using tRNAIle2-agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide-bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site-specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA. [ABSTRACT FROM AUTHOR]

Peptide deformylase (PDF) is a prokaryotic enzyme that catalyzes the deformylation of nascent peptides generated during protein synthesis and water molecules play a key role in these hydrolases. Using X-ray absorption near edge spectroscopy (XANES) and ab initio calculations we accurately probe the local atomic environment of the metal ion binding in the active site of PDF at different pHvalues and with different metal ions. This new approach is an effective way to monitor existing correlations among functions and structural changes. We show for the first time that the enzymatic activity depends on pH values and metal ions via the bond length of the nearest coordinating water (Wat1) to the metal ion. Combining experimental and theoretical data we may claim that PDF exhibits an enhanced enzymatic activity only when the distance of the Wat1 molecule with the metal ion falls in the limited range from 2.15 to 2.55Å. [ABSTRACT FROM AUTHOR]

Photo-induced electron transfer (PET) is ubiquitous for photosynthesis and fluorescent sensor design. However, genetically coded PET sensors are underdeveloped, due to the lack of methods to site-specifically install PET probes on proteins. Here we describe a family of acid and Mn(III) turn-on fluorescent protein (FP) sensors, named iLovU, based on PET and the genetic incorporation of superior PET quenchers in the fluorescent flavoprotein iLov. Using the iLovU PET sensors, we monitored the cytoplasmic acidification process, and achieved Mn(III) fluorescence sensing for the first time. The iLovU sensors should be applicable for studying pH changes in living cells, monitoring biogentic Mn(III) in the environment, and screening for efficient manganese peroxidase, which is highly desirable for lignin degradation and biomass conversion. Our work establishes a platform for many more protein PET sensors, facilitates the de novo design of metalloenzymes harboring redox active residues, and expands our ability to probe protein conformational dynamics. [ABSTRACT FROM AUTHOR]

Tetrapyrrole biosynthesis in plants, algae, and most bacteria starts from the NADPH-dependent reduction of glutamyl-tRNA by glutamyl-tRNA reductase (GluTR). The GluTR-catalyzed reaction is the rate-limiting step, and GluTR is the target of multiple posttranslational regulations, such as heme feedback inhibition, for the tetrapyrrole biosynthetic pathway. A recently identified GluTR regulator, GluTR binding protein (GluBP), has been shown to spatially organize tetrapyrrole synthesis by distributing GluTR into different suborganellar locations. Here we report the complex structure of GluTR-GluBP from Arabidopsis thaliana. The dimeric GluBP binds symmetrically to the catalytic domains of the V-shaped GluTR dimer via its C-terminal domain. A substantial conformational change of the GluTR NADPH-binding domain is observed, confirming the postulated rotation of the NADPH-binding domain for hydride transfer from NADPH to the substrate. Arg146, "guarding the door" for metabolic channeling, adopts alternative conformations, which may represent steps involved in substrate recognition and product release. A coupled enzyme assay shows that GluBP stimulates GluTR catalytic efficiency with an approximate threefold increase of the 5-aminolevulinic acid formation rate. In addition, the GluTR activity can be inhibited by heme in a concentration-dependent way regardless of the presence of GluBP. A structural alignment indicates that GluBP belongs to a heme-binding family involved in heme metabolism. We propose a catalytic mechanism model for GluTR, through which photosynthetic organisms can achieve precise regulation of tetrapyrrole biosynthesis. [ABSTRACT FROM AUTHOR]

An avian-origin human-infecting influenza (H7N9) virus was recently identified in China. We have evaluated the viral hemagglutinin (HA) receptor-binding properties of two human H7N9 isolates, A/Shanghai/1/2013 (SH-H7N9) (containing the avian-signature residue Gln226) and A/Anhui/1/2013 (AH-H7N9) (containing the mammalian-signature residue Leu226). We found that SH-H7N9 HA preferentially binds the avian receptor analog, whereas AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9 mutant HA (Leu226 → Gln) was found to exhibit dual receptor-binding property, indicating that other amino acid substitutions contribute to the receptor-binding switch. The structures of SH-H7N9 HA, AH-H7N9 HA, and its mutant in complex with either avian or human receptor analogs show how AH-H7N9 can bind human receptors while still retaining the avian receptor-binding property. [ABSTRACT FROM AUTHOR]

Aim: Human protein disulfide isomerase (hPDI) is a key enzyme and a redox-regulated chaperone responsible for oxidative protein folding in the endoplasmic reticulum. This work aims to reveal the molecular mechanism underlying the redox-regulated functions of hPDI by determining the crystal structures of hPDI in different redox states. Results: The structures of hPDI (abb'xa') in both the reduced and oxidized states showed that the four thioredoxin domains of a, b, b', and a' are arranged as a horseshoe shape with two CGHC active sites, respectively, in domains a and a' facing each other at the two ends. In reduced hPDI, domains a, b, and b' line up in the same plane, whereas domain a' twists ∼45° out. The two active sites are 27.6 Åapart. In oxidized hPDI, the four domains are differently organized to stay in the same plane, and the distance between the active sites increases to 40.3 Å. In contrast to the closed conformation of reduced hPDI, oxidized hPDI exists in an open state with more exposed hydrophobic areas and a larger cleft with potential for substrate binding. Innovation: This is the first report of the high-resolution structures of hPDI containing all four domains in both the reduced and the oxidized states. It reveals the redox-regulated structural dynamic properties of the protein. Conclusion: The redox-regulated open/closed conformational switch of hPDI endows the protein with versatile target-binding capacities for its enzymatic and chaperone functions. [ABSTRACT FROM AUTHOR]

Thioesterase superfamily member 2 (THEM2) is essential for cell proliferation of mammalian cells. It belongs to the hotdog-fold thioesterase superfamily and catalyzes the hydrolysis of the thioester bonds of acyl-CoA in vitro. In this study, THEM2 protein from zebrafish (fTHEM2) was expressed in Escherichia coli and purified by Ni-affinity and gel-filtration chromatography. fTHEM2 crystals were obtained using the sitting-drop vapour-diffusion method with PEG 10 000 as precipitant. X-ray diffraction data were collected to 1.80 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=77.1, b=74.4, c=96.6 Å, β=93.7°. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana vegetative storage proteins, VSP1 and VSP2, are acid phosphatases and belong to the haloacid dehalogenase (HAD) superfamily. In addition to their potential nutrient storage function, they were thought to be involved in plant defense and flower development. To gain insights into the architecture of the protein and obtain clues about its function, we have tested their substrate specificity and solved the structure of VSP1. The acid phosphatase activities of these two enzymes require divalent metal such as magnesium ion. Conversely, the activity of these two enzymes is inhibited by vanadate and molybdate, but is resistant to inorganic phosphate. Both VSP1 and VSP2 did not exhibit remarkable activities to any physiological substrates tested. In the current study, we presented the crystal structure of recombinant VSP1 at 1.8 A resolution via the selenomethionine single-wavelength anomalous diffraction (SAD). Specifically, an a-helical cap domain on the top of the a/b core domain is found to be involved in dimerization. In addition, despite of the low sequence similarity between VSP1 and other HAD enzymes, the core domain of VSP1 containing conserved active site and catalytic machinery displays a classic haloacid dehalogenase fold. Furthermore, we found that VSP1 is distinguished from bacterial class C acid phosphatase P4 by several structural features. To our knowledge, this is the first study to reveal the crystal structure of plant vegetative storage proteins. [ABSTRACT FROM AUTHOR]

Eukaryotic organelles have developed elaborate protein quality control systems to ensure their normal activity, among which Deg/HtrA proteases play an essential role. Plant Deg2 protease is a homologue of prokaryotic DegQ/DegP proteases and is located in the chloroplast stroma, where its proteolytic activity is required to maintain the efficiency of photosynthetic machinery during stress. Here, we demonstrate that Deg2 exhibits dual protease-chaperone activities, and we present the hexameric structure of Deg2 complexed with co-purified peptides. The structure shows that Deg2 contains a unique second PDZ domain (PDZ2) following a conventional PDZ domain (PDZ1), with PDZ2 orchestrating the cage assembly of Deg2. We discovered a conserved internal ligand for PDZ2 that mediates hexamer formation and thus locks the protease in the resting state. These findings provide insight into the diverse modes of PDZ domain-mediated regulation of Deg proteases. [ABSTRACT FROM AUTHOR]

The C-terminal nuclear localization sequence of FUsed in Sarcoma (FUS-NLS) is critical for its nuclear import mediated by transportin (Trn1). Familial amyotrophic lateral sclerosis (ALS) related mutations are clustered in FUS-NLS. We report here the structural, biochemical and cell biological characterization of the FUS-NLS and its clinical implications. The crystal structure of the FUS-NLS/Trn1 complex shows extensive contacts between the two proteins and a unique α-helical structure in the FUS-NLS. The binding affinity between Trn1 and FUS-NLS (wide-type and 12 ALS-associated mutants) was determined. As compared to the wide-type FUS-NLS (KD = 1.7 nM), each ALS-associated mutation caused a decreased affinity and the range of this reduction varied widely from 1.4-fold over 700-fold. The affinity of the mutants correlated with the extent of impaired nuclear localization, and more importantly, with the duration of disease progression in ALS patients. This study provides a comprehensive understanding of the nuclear targeting mechanism of FUS and illustrates the significance of FUS-NLS in ALS. [ABSTRACT FROM AUTHOR]

Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated. [ABSTRACT FROM AUTHOR]

Piwi proteins are modified by symmetric dimethylation of arginine (sDMA), and the methylarginine-dependent interaction with Tudor domain proteins is critical for their functions in germline development.Cocrystal structures of an extended Tudor domain (eTud) of Drosophila Tudor with methylated peptides of Aubergine, a Piwi family protein, reveal thatsDMAis recognized by an asparagine-gated aromatic cage. Furthermore, the unexpectedTudor-SN/p100 fold of eTud is important for sensing the position of sDMA. The structural information provides mechanistic insights into sDMA-dependent Piwi-Tudor interaction, and the recognition of sDMA by Tudor domains in general. [ABSTRACT FROM AUTHOR]

Previous results suggest that methylotrophic yeasts may contain factors that modulate prion stability. Alcohol oxidase (AOX), a key enzyme in methanol metabolism, is an abundant protein that is specific to methylotrophic yeasts. We examined the effect of Pichia pastoris AOX1 on prion phenotypes in Saccharomyces cerevisiae. The S. cerevisiae prion states [ PSI+] and [ URE3] arise from aggregation of the proteins Sup35p and Ure2p respectively, and correlate with the ability of Sup35p and Ure2p to form amyloid-like fibrils in vitro. We found that expression of P. pastoris AOX1 in S. cerevisiae had no effect on propagation of the [ PSI+] prion, but inhibited propagation of [ URE3]. Addition of AOX1 early in the time-course of fibril formation inhibits Ure2p fibril formation in vitro. AOX1 has not previously been identified as an ATPase. However, we discovered that in addition to its flavin adenine dinucleotide-dependent AOX activity, AOX1 possesses ATPase activity. This study identifies AOX1 as a novel prion inhibitory factor and a potential ATPase. [ABSTRACT FROM AUTHOR]

MitoNEET was identified as an outer mitochondrial membrane protein that can potentially bind the anti-diabetes drug pioglitazone. The crystal structure of the cytoplasmic mitoNEET (residues 33-108) is determined in this study. The structure presents a novel protein fold and contains a [2Fe-2S] cluster-binding domain. The [2Fe-2S] cluster is coordinated to the protein by Cys-72, Cys-74, Cys-83, and His-87 residues. This coordination is also novel compared with the traditional [2Fe-2S] cluster coordinated by four cysteines or two cysteines and two histidines. The cytoplasmic mitoNEET forms homodimers in solution and in crystal. The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface. His-87 residue, which plays an important role in the coordination of the [2Fe-2S] cluster, is exposed to the solvent on the dimer surface. It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the [2Fe-2S] cluster. [ABSTRACT FROM AUTHOR]

Covalent modification by Nedd8 (neddylation) stimulates the ubiquitin-protein isopeptide ligase (E3) activities of Cullins. DCN-1, an evolutionarily conserved protein, promotes neddylation of Cullins in vivo, binds directly to Nedd8, and associates with Cdc53 in the budding yeast Saccharomyces cerevisiae. The 1.9Å resolution structure of yeast DCN-1 shows that the region encompassing residues 66-269 has a rectangular parallelepiped-like all -helical structures, consisting of an EF-hand motif N-terminal domain and a closely juxtaposed C-terminal domain with six -helices. The EF-hand motif structure is highly similar to that of the c-Cbl ubiquitin E3 ligase. We also demonstrate that DCN-1 directly binds to Rbx-1, a factor important for protein neddylation. The structural and biochemical results are consistent with the role of DCN-1 as a scaffold protein in a multisubunit neddylation E3 ligase complex. [ABSTRACT FROM AUTHOR]

The disulfide-bridged hendecapeptide (CWTKSIPPKPC) loop, derived from an amphibian skin peptide, is found to have strong trypsin inhibitory capability. This loop, called the trypsin inhibitory loop (TIL), appears to be the smallest serine protease inhibitor known. A series of synthetic peptides derived from this loop also exhibits trypsin inhibitory activity; some peptides even exhibit both antimicrobial and trypsin inhibitory activities. Antimicrobial peptides are attractive candidates for producing novel antibiotics, but their sensitivity to trypsin-like proteases appreciably limits their application. Bifunctional peptides with both antimicrobial and trypsin inhibitory activities could be ideal candidates for clinical antibiotics, since these reported synthetic peptides have shown resistance against trypsin. The crystal structure of a complex of trypsin with one TIL derivative is solved. The concept of TIL is introduced in this paper. Novel trypsin inhibitors or antimicrobial peptides can be designed readily on the basis of the TIL. Furthermore, functional analysis and a precursor comparison suggest that serine protease inhibitors may have a common ancestor with antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme catalyzing a series of intermolecular phosphoryl group transfer reactions. Its main function is to synthesize 2,3- bisphosphoglycerate, the allosteric effector of hemoglobin. In this paper, we directly observed real-time motion of the enzyme active site and the substrate during phosphoryl transfer. A series of high resolution crystal structures of human bisphosphoglycerate mutase co-crystallized with 2,3-bisphosphoglycerate, representing different time points in the phosphoryl transfer reaction, were solved. These structures not only clarify the argument concerning the substrate binding mode for this enzyme family but also depict the entire process of the key histidine phosphorylation as a "slow movie". It was observed that the enzyme conformation continuously changed during the different states of the reaction. These results provide direct evidence for an "in line" phosphoryl transfer mechanism, and the roles of some key residues in the phosphoryl transfer process are identified. [ABSTRACT FROM AUTHOR]

5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme m the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C317 ↵ T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops. [ABSTRACT FROM AUTHOR]

Peptide deformylase (PDF, EC 3.5.1.27) is essential for the normal growth of eubacterium but not for mammalians. Recently, PDF has been studied as a target for new antibiotics. Its activity is strongly dependent on the bound metal ion. The crystallographic studies did not show any significant structural difference upon various bound metal ions. In this paper, X-ray absorption spectroscopy was employed to determine the local structure around the zinc ion of PDF from Leptospira interrogans in dry powder. XANES (X-ray absorption near-edge structure) calculations were performed and the local geometry of the active center was reconstructed successfully. By comparing with the crystal structure of an enzyme-product complex, the results from calculations show that a water molecule has moved towards the zinc ion and lies in the distance range to coordinate with the zinc ion weakly. [ABSTRACT FROM AUTHOR]

Bisphosphoglycerate mutase is a trifunctional enzyme of which the main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. The gene coding for bisphosphoglycerate mutase from the human cDNA library was cloned and expressed in Escherichia coli. The protein crystals were obtained and diffract to 2.5 Å and produced the first crystal structure of bisphosphoglycerate mutase. The model was refined to a crystallographic R-factor of 0.200 and Rfree of 0.266 with excellent stereochemistry. The enzyme remains a dimer in the crystal. The overall structure of the enzyme resembles that of the cofactor-dependent phosphoglycerate mutase except the regions of 13-21, 98-117, 127-151, and the C-terminal tail. The conformational changes in the backbone and the side chains of some residues reveal the structural basis for the different activities between phosphoglycerate mutase and bisphosphoglycerate mutase. The bisphosphoglycerate mutase-specific residue Gly-14 may cause the most important conformational changes, which makes the side chain of Glu-13 orient toward the active site. The positions of Glu-13 and Phe-22 prevent 2,3-bisphosphoglycerate from binding in the way proposed previously. In addition, the side chain of Glu-13 would affect the Glu-89 protonation ability responsible for the low mutase activity. Other structural variations, which could be connected with functional differences, are also discussed. [ABSTRACT FROM AUTHOR]

eIF3k, the smallest subunit of eukaryotic initiation factor 3 (eIF3), interacts with several other subunits of eIF3 and the 40 S ribosomal subunit, eIF3k is conserved among high eukaryotes, including mammals, insects, and plants, and it is ubiquitously expressed in human t tissues. Interestingly, eIF3k does not exist in some species of yeast. Thus, eIF3k may play a unique regulatory role in higher organisms. Here we report the crystal structure of human eIF3k, the first high-resolution c structure of an eIF3 component. This novel structure (contains two distinct domains, a HEAT (named for Huntington, elongation factor 3, A subunit of protein phosphatase 2A, target of rapamycin) repeat-like HAM (HEAT analogous motif) domain and a winged-helix-like WH domain. Through structural comparison and sequence conservation analysis, we show that eIF3k has three putative protein-binding surfaces and has potential RNA binding activity. The structure provides key information for understanding the structure and function of the eIF3 complex. [ABSTRACT FROM AUTHOR]

Defensins are small cysteine-rich antimicrobial peptides that are widely distributed in plants, insects and mammals. As potent defenders in protecting plants from pathogenic fungal attack, plant defensins are presumed to play an important role in the innate immunity of plants and are expected to find applications in the production of transgenic crops. A novel plant defensin protein SPE10 from Pachyrrhizus erosus seeds was purified and partially sequenced. Crystallization screening using the hanging-drop vapour-diffusion method resulted in two crystal forms, from one of which a diffraction data set was collected to 0.98 Å resolution. The crystal belongs to space group P21, with unit-cell parameters a = 32.71, b = 28.11, c = 54.85 Å, β = 103.78°. Preliminary crystallographic studies revealed two subunits in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Coactosin-like protein (CLP) is an actin-binding protein as well as a 5-lipoxygenase binding partner. Human coactosin-like protein has been expressed in high yield and the His-tagged protein was purified by affinity chromatography. Several different crystal forms were obtained by the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.0 Å resolution were collected from the best crystal. The space group was determined to be P212121, with unit-cell parameters a = 38.4, b = 48.7, c = 72.6 Å. [ABSTRACT FROM AUTHOR]

A 16 kDa protein SPE16 was purified from the seeds of Pachyrrhizus erosus. Its N-terminal amino-acid sequence showed significant sequence homology to pathogenesis-related proteins from the PR-10 family. An activity assay indicated that SPE16 possesses ribonuclease activity as do some other PR-10 proteins. SPE16 crystals were obtained by the hanging-drop vapour-diffusion method. The space group is P2[sub 1]2[sub 1]2[sub 1], with unit-cell parameters a = 53.36, b = 63.70, o c = 72.96 Å. [ABSTRACT FROM AUTHOR]

2-Dehydro-3-deoxygalactarate (DDG) aldolase is a member of the class II aldolase family and plays an important role in the pyruvate-metabolism pathway, catalyzing the reversible aldol cleavage of DDG to pyruvate and tartronic semialdehyde. As it is a potential novel antibiotic target, it is necessary to elucidate the catalytic mechanism of DDG aldolase. To determine the crystal structure, crystals of DDG aldolase from Leptospira interrogans were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution using a Cu Kα rotating-anode X-ray source. The crystal belonged to space group C2, with unit-cell parameters a = 293.5, b = 125.6, c = 87.6 Å, β = 100.9°. The VM is calculated to be 2.4 Å3 Da−1, assuming there to be 12 protein molecules in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Human B-type 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (dPGM-B) has been cloned, overexpressed and purified, with a yield of 30% of the total protein. Crystals of human dPGM-B were obtained using the hanging-drop vapour-diffusion technique. X-ray diffraction data were collected to 2.8 Å resolution. The human dPGM-B crystals belong to space group P21, with unit-cell parameters a = 130.5, b = 75.9, c = 187.0 Å, α β= 94.4°. There could be between 9 and 18 monomers per asymmetric unit, with 12 molecules being the most likely. [ABSTRACT FROM AUTHOR]