Thermus parvatiensis RLT sp. nov., Isolated from a Hot Water Spring,Located Atop the Himalayan Ranges at Manikaran,India

A Gram negative, yellow pigmented, rod shaped bacterium designated as RL^T was isolated from a hot water spring (90–98 °C) located at Manikaran in Northern India. The isolate grows at 60–80 °C (optimum, 70 °C) and at pH 7.0–9.0 (optimum pH 7.2). Phylogenetic analysis of 16S rRNA gene sequences and levels of DNA–DNA relatedness together indicate that the new isolate represents a novel species of the genus Thermus with closest affinity to Thermus thermophilus HB8^T (99.5 %) followed by Thermus arciformis (96.4 %). A comparative analysis of partial sequences of housekeeping genes (HKG) further revealed that strain RL^T is a novel species belonging to the genus Thermus. The melting G+C content of strain RL^T was calculated as 68.7 mol%. The DNA–DNA relatedness value of strain RL^T with its nearest neighbours (>97 %) was found to be less than 70 % indicating that strain RL^T represents a novel species of the genus Thermus. MK-8 was the predominant respiratory quinone. The presence of characteristic phospholipid and glycolipid further confirmed that strain RL^T belongs to the genus Thermus. The predominant fatty acids of strain RL^T were iso-C_17:0 (23.67 %) and iso-C_15:0 (24.50 %). The results obtained after DNA–DNA hybridization, biochemical and physiological tests clearly distinguished strain RL^T from its closely related species. Thus, strain RL^T represents a novel species of the genus Thermus for which the name Thermus parvatiensis is proposed (=DSM 21745^T= MTCC 8932^T).

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0538-4) contains supplementary material, which is available to authorized users.     

Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca²⁺ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N₃ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC₅₀ = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.     

Full Implementation of the Genetic Code by Tryptophanyl-tRNA Synthetase Requires Intermodular Coupling

Tryptophanyl-tRNA Synthetase (TrpRS) Urzyme (fragments A and C), a 130-residue construct containing only secondary structures positioning the HIGH and KMSKS active site signatures and the specificity helix, accelerates tRNA^Trp aminoacylation with ∼10-fold specificity toward tryptophan, relative to structurally related tyrosine. We proposed that including the 76-residue connecting peptide 1 insertion (Fragment B) might enhance tryptophan affinity and hence amino acid specificity, because that subdomain constrains the orientation of the specificity helix. We test that hypothesis by characterizing two new constructs: the catalytic domain (fragments A–C) and the Urzyme supplemented with the anticodon-binding domain (fragments A, C, and D). The three constructs, together with the full-length enzyme (fragments A–D), comprise a factorial experiment from which we deduce individual and combined contributions of the two modules to the steady-state kinetics parameters for tryptophan-dependent ³²PP_i exchange, specificity for tryptophan versus tyrosine, and aminoacylation of tRNA^Trp. Factorial design directly measures the energetic coupling between the two more recent modules in the contemporary enzyme and demonstrates its functionality. Combining the TrpRS Urzyme individually in cis with each module affords an analysis of long term evolution of amino acid specificity and tRNA aminoacylation, both essential for expanding the genetic code. Either module significantly enhances tryptophan activation but unexpectedly eliminates amino acid specificity for tryptophan, relative to tyrosine, and significantly reduces tRNA aminoacylation. Exclusive dependence of both enhanced functionalities of full-length TrpRS on interdomain coupling energies between the two new modules argues that independent recruitment of connecting peptide 1 and the anticodon-binding domain during evolutionary development of Urzymes would have entailed significant losses of fitness.     

Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207)

Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived α_s2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the α_s2-casein f(183–207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of α_s2-casein f(193–203) and α_s2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid α_s2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.     

The amino- and carboxyl-terminal sequence of bovine rhodopsin

The amino terminus of bovine rhodopsin is blocked and has the sequence x-Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys-Thr-Gly-Val-Val-Arg, where CHO represents sites of carbohydrate attachment. The carboxyl-terminal sequence of rhodopsin is Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala. Upon short-term digestion of rod outer segment (ROS) membranes with thermolysin, opsin (～ 35,000 daltons) is converted to a membrane-bound fragment O′ (～ 30,500 daltons) and 2 peptides containing 12 amino acids are released from the carboxyl terminus of rhodopsin into the supernatant. Upon long-term digestion of ROS with thermolysin, opsin and O′ are replaced by the membrane-bound fragments F₁ (～25,000 daltons), and F₂ (～9,500 daltons). When ³²P-ROS are digested, F₂ carries the ³²P. Both O′ and F₁ contain the amino-terminal glycopeptide.     

Amino Acid Composition of Cucumber Green Mottle Mosaic Virus Coat Protein and Sequence Determination of Its Tryptic Peptides

Amino acid composition of the CGMMV* coat protein was determined to be as follows: Asp₂₀, Thr₁₀, Ser₂₄, Glu₁₀. Pro₆, Gly₉, Ala₂₁, Val₇, Ile₇, Leu₁₈, Tyr₄, Phe₉, Lys₄, His₁, Arg₈, Trp₂. No terminal α-amino group was detected by dinitrophenylation method. The carboxyl-terminus was found to be serine by hydrazinolysis of the protein and digestion with carboxypeptidase A.

For sequence analysis of the coat protein, tryptic digestion was accomplished at pH 8.0 resulting in ten soluble and several insoluble peptides at pH 4.5. The amino acids contained in soluble peptides accounted for 91 out of 160 residues in the whole protein. The amino acid sequences of ten soluble peptides were determined.

From the similarities of amino acid sequence of the peptides to those of TMV* protein, CGMMV was assumed to be a strain of TMV group.     

Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG3(T2AG3)3 and (T2AG3)4 human telomere sequences in Na+ and K+ solutions

The quadruplex structures of the human telomere sequences AG₃(T₂AG₃)₃ I and (T₂AG₃)₄ II were investigated in the presence of Na⁺ and K⁺ ions, through the cross-linking of adenines and guanines by the cis- and trans-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ complexes 1 and 2. The bases involved in chelation of the cis- and trans-Pt(NH₃)₂ moieties were identified by chemical and 3′-exonuclease digestions of the products isolated after denaturing gel electrophoresis. These are the four adenines of each sequence and four out of the 12 guanines. Two largely different structures have been reported for I: A from NMR data in Na⁺ solution and B from X-ray data of a K⁺-containing crystal. Structure A alone agrees with our conclusions about the formation of the A1–G10, A13–G22, A1–A13 platinum chelates at the top of the quadruplex and A7–A19, G4–A19 and A7–G20 at the bottom, whether the Na⁺ or K⁺ ion is present. At variance with a recent proposal that structures A and B could be the major species in Na⁺ and K⁺ solutions, respectively, our results suggest that structure A exists predominantly in the presence of both ions. They also suggest that covalent platinum cross-linking of a human telomere sequence could be used to inhibit telomerase.     

Whole-Genome Optical Mapping and Finished Genome Sequence of Sphingobacterium deserti sp. nov., a New Species Isolated from the Western Desert of China

A novel Gram-negative bacterium, designated ZW^T, was isolated from a soil sample of the Western Desert of China, and its phenotypic properties and phylogenetic position were investigated using a polyphasic approach. Growth occurred on TGY medium at 5–42°C with an optimum of 30°C, and at pH 7.0–11.0 with an optimum of pH 9.0. The predominant cellular fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c or C_16:1 ω6c/C_16:1 ω7c) (39.22%), iso-C15:0 (27.91%), iso-C_17:0 3OH (15.21%), C_16:0 (4.98%), iso-C_15:0 3OH (3.03%), C16:0 3OH (5.39%) and C_14:0 (1.74%). The major polar lipid of strain ZW^T is phosphatidylethanolamine. The only menaquinone observed was MK-7. The GC content of the DNA of strain ZWT is 44.9 mol%. rDNA phylogeny, genome relatedness and chemotaxonomic characteristics all indicate that strain ZW^T represents a novel species of the genus Sphingobacterium. We propose the name S. deserti sp. nov., with ZW^T (= KCTC 32092T = ACCC 05744T) as the type strain. Whole genome optical mapping and next-generation sequencing was used to derive a finished genome sequence for strain ZW^T, consisting of a circular chromosome of 4,615,818 bp in size. The genome of strain ZW^T features 3,391 protein-encoding and 48 tRNA-encoding genes. Comparison of the predicted proteome of ZW^T with those of other sphingobacteria identified 925 species-unique proteins that may contribute to the adaptation of ZW^T to its native, extremely arid and inhospitable environment. As the first finished genome sequence for any Sphingobacterium, our work will serve as a useful reference for subsequent sequencing and mapping efforts for additional strains and species within this genus.     

Amino acid sequences of proteins from Leptospira serovar pomona

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Characterization of the specific DNA-binding properties of Tnp26, the transposase of insertion sequence IS26

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1–P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix–turn–helix domain (amino acids I13–R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1–D12) did not bind. The N-terminal M1–P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1–M234) and Tnp26 M1–P56 fragment. These findings indicate that the helix–turn–helix and disordered domains of Tnp26 play a role in Tnp26–TIR complex formation. Both domains are conserved in all members of the IS26 family.     

Role of ATP hydrolysis in the DNA translocase activity of the bovine papillomavirus (BPV-1) E1 helicase

The E1 protein of bovine papillomavirus type-1 is the viral replication initiator protein and replicative helicase. Here we show that the C-terminal ~300 amino acids of E1, that share homology with members of helicase superfamily 3 (SF3), can act as an autonomous helicase. E1 is monomeric in the absence of ATP but assembles into hexamers in the presence of ATP, single-stranded DNA (ssDNA) or both. A 16 base sequence is the minimum for efficient hexamerization, although the complex protects ~30 bases from nuclease digestion, supporting the notion that the DNA is bound within the protein complex. In the absence of ATP, or in the presence of ADP or the non–hydrolysable ATP analogue AMP–PNP, the interaction with short ssDNA oligonucleotides is exceptionally tight (T_1/2 > 6 h). However, in the presence of ATP, the interaction with DNA is destabilized (T_1/2 ~60 s). These results suggest that during the ATP hydrolysis cycle an internal DNA-binding site oscillates from a high to a low-affinity state, while protein–protein interactions switch from low to high affinity. This reciprocal change in protein–protein and protein–DNA affinities could be part of a mechanism for tethering the protein to its substrate while unidirectional movement along DNA proceeds.     

Sequence-function relationships provide new insight into the cleavage site selectivity of the 8-17 RNA-cleaving deoxyribozyme

Many sequence variations of the 8–17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8–17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5′ rN₁₈-N_1.1 3′) cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3′ position (N_1.1) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5′ position (rN₁₈) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8–17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000-fold over background. Three optimal 8–17 variants, identified from ~75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of ≥0.1 min⁻¹, and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.     

Purification and structure of the host-specific toxin from Helminthosporium carbonum race 1

The host-specific toxin from Helminthosporiumcarbonum race 1 was purified from culture filtrates by solvent extraction, gel filtration, and high pressure liquid chromatography. High resolution mass spectrometry of the purified toxin gave a MW of 436.2318 and an elemental composition C₂₁H₃₂N₄O₆. Amino acid analysis and proton and¹³C-NMR indicated a peptide containing four amino acids. Their sequence was determined by gas chromatography mass spectrometry. Finally, digestion of the amino acids with D- and L-amino acid oxidases gave the complete structure cyclo[(L-2-amino-9, 10-epoxy-8-oxodecanoyl)-D-prolyl-L-alanyl-L-alanyl].     

Clostridium botulinum types A, B, C1, and E produce proteins with or without hemagglutinating activity: Do they share common amino acid sequences and genes?

Clostridium botulinum produce the antigenically distinct 150 kD neurotoxin serotypes (e.g., A, B, C₁, and E) and simultaneously proteins, A Hn⁺, B Hn⁺, C Hn⁺, and E Hn^–, that have high, low, and no hemagglutinating activity. A Hn⁺ and B Hn⁺ are serologically cross-reactive. A Hn⁺, B Hn⁺, and C Hn⁺ found as large aggregates (900–220 kD) can be dissociated on SDS-PAGE into multiple subunits, the smallest for A Hn⁺, B Hn⁺ is 17 kD and 27 kD for C Hn⁺. The 116 kD E Hn^– does not aggregate. We determined the sequences of 10–33 amino terminal residues of the 17, 21.5, 35, and 57 kD subunits of A Hn⁺ and B Hn⁺. Each of these subunits have unique sequences, indicating that the larger units studied are not homomers or heteromers of smaller units. The subunits of A Hn⁺ and B Hn⁺ of comparable size have striking sequence identity (e.g., 21.5 kD subunits from the two are identical and 57 kD subunits have 80% identity).In vitro proteolysis of 116 kD E Hn^– with different proteases did not impart hemagglutinating activity to the fragments. The 116 kD E Hn^– and one of its proteolytic fragments (87 kD) were partially sequenced. Sixty-two base pairs downstream from the termination codon of the cloned 33 kD subunit of C Hn⁺, there is an initiation codon followed by an open reading frame for at least 34 amino acid residues (Tsuzukiet al., 1990). The derived amino acid sequence of this open reading frame, we found, has 73–84% sequence identity with those of the 17 kD subunits of A Hn⁺ and B Hn⁺ and significant identity with the N-terminal of E Hn^–. These highly conserved sequences show existence of genetic linkage among the Hn⁺ and Hn^– proteins.     

Flavobacterium plurextorum sp. nov. Isolated from Farmed Rainbow Trout (Oncorhynchus mykiss)

Five strains (1126-1H-08^T, 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 µm wide and 3 µm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25°C. They grew in a temperature range between 15°C to 30°C, but they did not grow at 37°Cor 42°C. Based on 16S rRNA gene sequence analysis, the isolates belonged to the genus Flavobacterium. Strain 1126-1H-08^T exhibited the highest levels of similarity with Flavobacterium oncorhynchi CECT 7678^T and Flavobacterium pectinovorum DSM 6368^T (98.5% and 97.9% sequence similarity, respectively). DNA–DNA hybridization values were 87 to 99% among the five isolates and ranged from 21 to 48% between strain 1126-1H-08^T, selected as a representative isolate, and the type strains of Flavobacterium oncorhynchi CECT 7678^T and other phylogenetic related Flavobacterium species. The DNA G+C content of strain 1126-1H-08^T was 33.2 mol%. The predominant respiratory quinone was MK-6 and the major fatty acids were iso-C_15∶0 and C_15∶0. These data were similar to those reported for Flavobacterium species. Several physiological and biochemical tests differentiated the novel bacterial strains from related Flavobacterium species. Phylogenetic, genetic and phenotypic data indicate that these strains represent a new species of the genus Flavobacterium, for which the name Flavobacterium plurextorum sp. nov. was proposed. The type strain is 1126-1H-08^T ( = CECT 7844^T = CCUG 60112^T).     

The Octarepeat Region of the Prion Protein Is Conformationally Altered in PrPSc

Background

Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP^C. Little is known about the details of the structural rearrangement of physiological PrP^C into a still-elusive disease-associated conformation termed PrP^Sc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.

Methodology/Principal Findings

Here, we report that trypsin digestion of PrP^Sc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP^Sc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP^Sc are exposed in PrP^C.

Conclusions/Significance

We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP^Sc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrP^C participates in PrP^Sc conversion, and may also be exploited for diagnostic purposes.     

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A₂ (PLA₂), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA₂ protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA₂ protein, suggesting the presence of multiple forms in the starfish PLA₂. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA₂. In phylogenetic tree, the starfish PLA₂ should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA₂, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA₂ from porcine pancreas.     

Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins

Telomeres are protein–DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins, they play an essential role in genome stability. We determined the structure of the DNA-binding domain of NgTRF1, a double-stranded telomere-binding protein of tobacco, using multidimensional NMR spectroscopy and X-ray crystallography. The DNA-binding domain of NgTRF1 contained the Myb-like domain and C-terminal Myb-extension that is characteristic of plant double-stranded telomere-binding proteins. It encompassed amino acids 561–681 (NgTRF1^561–681), and was composed of 4 α-helices. We also determined the structure of NgTRF1^561–681 bound to plant telomeric DNA. We identified several amino acid residues that interacted directly with DNA, and confirmed their role in the binding of NgTRF1 to telomere using site-directed mutagenesis. Based on a structural comparison of the DNA-binding domains of NgTRF1 and human TRF1 (hTRF1), NgTRF1 has both common and unique DNA-binding properties. Interaction of Myb-like domain with telomeric sequences is almost identical in NgTRF1^561–681 with the DNA-binding domain of hTRF1. The interaction of Arg-638 with the telomeric DNA, which is unique in NgTRF1^561–681, may provide the structural explanation for the specificity of NgTRF1 to the plant telomere sequences, (TTTAGGG)_n.