Sequential resonance assignment and secondary structure determination of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors

The solution conformation of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors, has been investigated by nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments of the 1H NMR spectrum have been obtained by using a number of two-dimensional techniques for identifying through-bond and through-space (less than 5-A) connectivities. Elements of regular secondary structure have been identified on the basis of a qualitative interpretation of the nuclear Overhauser enhancement, coupling constant, and amide exchange data. These are two beta-sheet regions. One double-stranded antiparallel beta-sheet comprises residues 11-14 (strand 1) and 37-39 (strand 2). The other triple-stranded sheet is formed by two antiparallel strands comprising residues 45-49 (strand 4) and 53-57 (strand 5) connected by a turn (residues 50-52), and a small strand consisting of residues 20-22 (strand 3) that is parallel to strand 4.     

Automated assignment of NOESY NMR spectra using a knowledge based method (KNOWNOE)

Automated assignment of NOESY spectra is a prerequisite for automated structure determination of biological macromolecules. With the program KNOWNOE we present a novel, knowledge based approach to this problem. KNOWNOE is devised to work directly with the experimental spectra without interference of an expert. Besides making use of routines already implemented in AUREMOL, it contains as a central part a knowledge driven Bayesian algorithm for solving ambiguities in the NOE assignments. These ambiguities mainly arise from chemical shift degeneration which allows multiple assignments of cross peaks. Using a set of 326 protein NMR structures, statistical tables in the form of atom-pairwise volume probability distributions (VPDs) were derived. VPDs for all assignment possibilities relevant to the assignments of interproton NOEs were calculated. With these data for a given cross peak with N possible assignments A _i(i = 1,...,N) the conditional probabilities P(A _i, a|V ₀) can be calculated that the assignment A _idetermines essentially all (a-times) of the cross peak volume V ₀. An assignment A _kwith a probability P(A _k, a|V ₀) higher than 0.8 is transiently considered as unambiguously assigned. With a list of unambiguously assigned peaks a set of structures is calculated. These structures are used as input for a next cycle of iteration where a distance threshold D _maxis dynamically reduced. The program KNOWNOE was tested on NOESY spectra of a medium size protein, the cold shock protein (TmCsp) from Thermotoga maritima. The results show that a high quality structure of this protein can be obtained by automated assignment of NOESY spectra which is at least as good as the structure obtained from manual data evaluation.     

Primary structure of Dioclea glabra trypsin inhibitor, DgTI, a Bowman-Birk inhibitor.

A novel serine proteinase inhibitor, DgTI, was purified from Dioclea glabra seeds by acetone precipitation, and ion-exchange and reverse phase chromatography. The inhibitor belongs to the Bowman-Birk family, and its primary sequence, determined by Edman degradation and mass spectrometry, of 67 amino acids is: SSGPCCDRCRCTKSEPPQCQCQDVRLNSCHSACEACVCSHSMPGLCSCLDITHFCHEPCKSSGDDED++ +. Although two reactive sites were determined by susceptibility to trypsin (Lys(13) and His(40)), the inhibitory function was assigned only to the first site. The inhibitor forms a 1:1 complex with trypsin, and Ki is 0.5 x 10(-9) M. Elastase, chymotrypsin, kallikreins, factor Xa, thrombin, and plasmin were not inhibited. By its properties, DgTI is a Bowman-Birk inhibitor with structural and inhibitory properties between the class of Bowman-Birk type I (with a fully active second reactive site), and Bowman-Birk type II (devoid of second reactive site).     

Location of a gene conditioning seed coat colour in linseed (Linum usitatissimum L.) using chromosomal interchanges

The geneG, conditioning seed coat colour was found to be located at 15.37±3.05 map units from the breakage point of one of six newly induced interchanges in linseed. Since no information about the karyotype in linseed is available, the chromosome on whichG is located, cannot be specified.Part of a thesis submitted by the second author at the Indian Agricultural Research Institute, New Delhi, for a Ph.D. degree.     

Affinity thermoprecipitation of trypsin using soybean trypsin inhibitor conjugated with a thermo-reactive polymer,poly(N-vinyl caprolactam)

The soybean trypsin inhibitor conjugate with a thermo-reactive water soluble polymer, poly(N-vinyl caprolactam), was successfully used for thermally induced affinity precipitation of trypsin. The validity of the developed procedure was proven by a model separation of trypsin from dilute solution containing a large excess of bovine serum albumin.     

Circular proteins in plants: solution structure of a novel macrocyclic trypsin inhibitor from Momordica cochinchinensis

Much interest has been generated by recent reports on the discovery of circular (i.e. head-to-tail cyclized) proteins in plants. Here we report the three-dimensional structure of one of the newest such circular proteins, MCoTI-II, a novel trypsin inhibitor from Momordica cochinchinensis, a member of the Cucurbitaceae plant family. The structure consists of a small beta-sheet, several turns, and a cystine knot arrangement of the three disulfide bonds. Interestingly, the molecular topology is similar to that of the plant cyclotides (Craik, D. J., Daly, N. L., Bond, T., and Waine, C. (1999) J. Mol. Biol. 294, 1327-1336), which derive from the Rubiaceae and Violaceae plant families, have antimicrobial activities, and exemplify the cyclic cystine knot structural motif as part of their circular backbone. The sequence, biological activity, and plant family of MCoTI-II are all different from known cyclotides. However, given the structural similarity, cyclic backbone, and plant origin of MCoTI-II, we propose that MCoTI-II can be classified as a new member of the cyclotide class of proteins. The expansion of the cyclotides to include trypsin inhibitory activity and a new plant family highlights the importance and functional variability of circular proteins and the fact that they are more common than has previously been believed. Insights into the possible roles of backbone cyclization have been gained by a comparison of the structure of MCoTI-II with the homologous acyclic trypsin inhibitors CMTI-I and EETI-II from the Cucurbitaceae plant family.     

High precision NMR structure of YhhP, a novel Escherichia coli protein implicated in cell division

YhhP, a small protein of 81 amino acid residues encoded by the yhhP gene in the Escherichia coli database, is implicated in cell division although the precise biological function of this protein has not been yet identified. A variety of microorganisms have similar proteins, all of which contain a common CPxP sequence motif in the N-terminal region. We have determined the three-dimensional solution structure of YhhP by NMR spectroscopy in order to obtain insight into its biological function. It folds into a two-layered alpha/beta-sandwich structure with a betaalphabetaalphabetabeta fold, comprising a mixed four-stranded beta-sheet stacked against two alpha-helices, both of which are nearly parallel to the strands of the beta-sheet. The CPxP motif plays a significant structural role in stabilizing the first helix as a part of the new type N-capping box where the Cys-Pro peptide bond adopts a cis configuration. The structure of YhhP displays a striking resemblance to the C-terminal ribosome-binding domain of translation initiation factor IF3 (IF3C). In addition, the surface charge distribution of the RNA-recognition helix of IF3C is nearly the same as that of the corresponding helix of YhhP. These results suggest a structure-based hypothesis in which binding to an RNA target plays an essential role in the function of this ubiquitous protein.     

SSR-based population structure, molecular diversity and linkage disequilibrium analysis of a collection of flax (Linum usitatissimum L.) varying for mucilage seed-coat content

Flax seed mucilage (SM) presents specific biological activities useful for the food and pharmaceutical industries. Understanding the population structure, genetic diversity and linkage disequilibrium (LD) of germplasm varying for mucilage content is pivotal for the identification of genes and quantitative trait loci underlying mucilage variation by association mapping (AM). In this study, 150 microsatellite loci were used to assess the population structure, genetic diversity and LD of a set of 60 flax cultivars/accessions capturing the breadth of SM variation in flax germplasm. STRUCTURE analysis and similarity-based methods revealed the presence of three populations reflecting mainly their geographic origins (South Asia, South America and North America), and the impact of germplasm exchange within and between North American flax breeding programs. Analysis of molecular variance showed that 78.32% of the genetic variation resided within populations and 21.68% among populations. The phi-statistic (??_st) value of 0.22 confirmed the presence of a strong population structure. A total of 408 alleles were detected, with the South American population capturing the highest overall diversity. However, the genetic diversity was narrow, as indicated by the small number of alleles per locus (2.72) and gene diversity (mean?=?0.34). LD was significant between 3.9% (r ²) and 36.2% (D??) of the loci pairs (FDR?<?0.05). The mean r ² and D?? were 0.26 and 0.53, respectively. The results suggest that the collection could be useful in AM studies aimed at the discovery of genes/alleles involved in SM; however a greater diversity may be required to improve the AM resolution.     

Synthesis and properties of cholecystokinin-releasing peptide (monitor peptide), a 61-residue trypsin inhibitor

A 61-residue cholecystokinin-releasing peptide (monitor peptide), which was obtained from rat pancreatic juice and found to stimulate pancreatic enzyme secretion, was recently reported to inhibit bovine trypsin and to possess epidermal growth factor (EGF)-like activities, at a concentration of about 10 nM. However, monitor peptide is structurally different from the EGF family of growth factors. To investigate whether monitor peptide contains the supposed EGF-like activities, it has been synthesized together with its [Ala23, Ala47] analog. The purified peptides, which were fully characterized by a range of methods including Cf-252 ionization mass spectrometry and enzymatic digestion to establish the locations of disulfide linkages, were shown to belong to the pancreatic secretory trypsin inhibitor family and not to the EGF family. Neither synthetic monitor peptide nor its analog were able to compete with 125I-EGF in A-431 cells or to stimulate growth of Swiss 3T3 and NRK 49F cells, up to 1 microM concentration. However, synthetic monitor peptide was as effective as the native product in the inhibition of trypsin. Replacement of the essential Arg23 in the [Ala23, Ala47]-analog led to loss of trypsin inhibition activity.     

Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea.

Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C-terminal sequence analysis, and showed that LTI is a 57-residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase-high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.     

Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C

Calphostin C (UCN-1028C), a newly isolated compound from Cladosporium cladosporioides, is a potent and specific inhibitor of protein kinase C, because it was 1000 times more inhibitory to protein kinase C (IC50, 0.05 microM) than other protein kinases such as cAMP-dependent protein kinase and tyrosine-specific protein kinase (IC50, greater than 50 microM). Calphostin C did not inhibit calcium activated neutral protease (calpain)-digested protein kinase C, indicating that it interacts with the regulatory domain of protein kinase C. In addition this compound showed inhibitory effects on the binding of [3H]PDBu to protein kinase C. The potent cytotoxic activity and antitumor activity of calphostin C might be due to the inhibition of protein kinase C, and thus it may be potentially useful for the therapeutic application.     

Livin, a novel inhibitor of apoptosis protein family member

A novel human inhibitor of apoptosis protein (IAP) family member termed Livin was identified, containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. The mRNA for livin was not detectable by Northern blot in most normal adult tissues with the exception of the placenta, but was present in developmental tissues and in several cancer cell lines. Highest levels were observed in two melanoma-derived cell lines, G361 and SK-Mel29. Transfection of livin in HeLa cells resulted in protection from apoptosis induced by expression of FADD, Bax, RIP, RIP3, and DR6. Similar to other IAP family members, the anti-apoptotic activity of Livin was dependent on the BIR domain. Livin was also capable of inhibiting DEVD-like caspase activity triggered by tumor necrosis factor-alpha. In vitro binding studies demonstrated a direct interaction between Livin and the active form of the downstream caspases, caspase-3 and -7, that was dependent on the BIR domain of Livin. In addition, the unprocessed and cleaved forms of caspase-9 co-immunoprecipitated with Livin in vivo, and recombinant Livin could inhibit the activation of caspase-9 induced by Apaf-1, cytochrome c, and dATP. The subcellular distribution of the transfected Livin was analyzed by immunofluorescence. Both Livin and Survivin were expressed in the nucleus and in a filamentous pattern throughout the cytoplasm. In contrast to the apoptotic activity, the COOH-terminal RING domain mediated its subcellular localization patterning. Further studies found that transfection of an antisense construct against livin could trigger apoptosis specifically in cell lines expressing livin mRNA. This was associated with an increase in DNA fragmentation and in DEVD-like caspase activity. Thus, disruption of Livin may provide a strategy to induce apoptosis in certain cancer cells.     

Analysis of inter-alpha-trypsin inhibitor and a novel trypsin inhibitor, pre-alpha-trypsin inhibitor, from human plasma. Polypeptide chain stoichiometry and assembly by glycan

The polypeptide chain composition of protein material referred to in the literature as "inter-alpha-trypsin inhibitor" was investigated. The material was found to consist of distinct proteins of 125,000 and 225,000 Da, each of which contained more than one polypeptide chain. The links that assemble each protein were found to be stable to various strong denaturants, but susceptible to treatment with trifluoromethanesulfonic acid or hyaluronidase, indicating a glycan nature. The 225,000-Da protein migrated with inter-alpha mobility on agarose gel electrophoresis and is designated inter-alpha-trypsin inhibitor, whereas the 125,000-Da protein migrated with pre-alpha mobility, and we designate it pre-alpha-trypsin inhibitor. Analysis of the proteins, the separated chains, and proteolytic derivatives thereof revealed that each protein contained a single, identical, trypsin-inhibitory chain of 30,000 Da. Inter-alpha-trypsin inhibitor contains noninhibitory heavy chains of 65,000 and 70,000 Da, whereas pre-alpha-trypsin inhibitor contains a heavy chain of 90,000 Da. Our data allow identification of several recently reported cDNA clones and clarify the confusion surrounding the composition of plasma proteins referred to as inter-alpha-trypsin inhibitor.     

Three-dimensional structure of a recombinant variant of human pancreatic secretory trypsin inhibitor (Kazal type).

A modified version of the human pancreatic trypsin inhibitor (PSTI), generated in a protein-design project, has been crystallized in spacegroup P4(3) with lattice constants a = 40.15 A, c = 33.91 A. The structure has been solved by molecular replacement. Refinement of the structure by simulated annealing and conventional restrained least-squares yielded for 8.0 to 2.3 A data a final R-value of 19.1%. Differences to the known structures of porcine PSTI complexed with trypsinogen and modified human PSTI complexed with chymotrypsinogen occur at the flexible N-terminal part of the molecule. These differences are influenced by crystal packing, as are low temperature factors for the binding loop. The geometry of the binding loop is similar to the complexed structures.     

SABA (secondary structure assignment program based on only alpha carbons): a novel pseudo center geometrical criterion for accurate assignment of protein secondary structures

Most widely used secondary structure assignment methods such as DSSP identify structural elements based on N-H and C=O hydrogen bonding patterns from X-ray or NMR-determined coordinates. Secondary structure assignment algorithms using limited Cα information have been under development as well, but their accuracy is only ~80% compared to DSSP. We have hereby developed SABA (Secondary Structure Assignment Program Based on only Alpha Carbons) with~90% accuracy. SABA defines a novel geometrical parameter, termed a pseudo center, which is the midpoint of two continuous Cαs. SABA is capable of identifying α-helices, 3(10)-helices, and β-strands with high accuracy by using cut-off criteria on distances and dihedral angles between two or more pseudo centers. In addition to assigning secondary structures to Cα-only structures, algorithms using limited Cα information with high accuracy have the potential to enhance the speed of calculations for high capacity structure comparison.     

The X-ray structure of Escherichia coli RraA (MenG), A protein inhibitor of RNA processing

The Escherichia coli protein regulator of RNase E activity A (RraA) has recently been shown to act as a trans-acting modulator of RNA turnover in bacteria; it binds to the essential endonuclease RNase E and inhibits RNA processing in vivo and in vitro. Here, we report the 2.0A X-ray structure of RraA. The structure reveals a ring-like trimer with a central cavity of approximately 12A in diameter. Based on earlier sequence analysis, RraA had been identified as a putative S-adenosylmethionine:2-demethylmenaquinone and was annotated as MenG. However, an analysis of the RraA structure shows that the protein lacks the structural motifs usually required for methylases. Comparison of the observed fold with that of other proteins (and domains) suggests that the RraA fold is an ancient platform that has been adapted for a wide range of functions. An analysis of the amino acid sequence shows that the E.coli RraA exhibits an ancient relationship to a family of aldolases.     

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.