Evolution of the grass leaf by primordium extension and petiole-lamina remodeling.

The sheathing leaf found in grasses and other monocots is an evolutionary innovation, yet its origin has been a subject of long-standing debate. Here, we revisit the problem in the light of developmental genetics and computational modeling. We show that the sheathing leaf likely arose through WOX-gene-dependent extension of a primordial zone straddling concentric domains around the shoot apex. Patterned growth within this zone, oriented by two polarity fields, accounts for wild-type, mutant and mosaic grass leaf development, whereas zone contraction and growth remodeling accounts for eudicot leaf development. In contrast to the prevailing view, our results suggest that the sheath derives from petiole, whereas the blade derives from the lamina of the eudicot leaf, consistent with homologies proposed in the 19th century.

Substituted 1-methyl-4-phenylpyrrolidin-2-ones - Fragment-based design of N-methylpyrrolidone-derived bromodomain inhibitors.

N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4.

Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment substitute in bromodomain inhibition.

N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.

Solution structure of BSTI: a new trypsin inhibitor from skin secretions of Bombina bombina.

The three-dimensional solution structure of BSTI, a trypsin inhibitor from the European frog Bombina bombina, has been solved using (1)H NMR spectroscopy. The 60 amino acid protein contains five disulfide bonds, which were unambiguously determined to be Cys (4--38), Cys (13--34), Cys (17--30), Cys (21--60), and Cys (40--54) by experimental restraints and subsequent structure calculations. The main elements of secondary structure are four beta-strands, arranged as two small antiparallel beta-sheets. The overall fold of BSTI is disk shaped and is characterized by the lack of a hydrophobic core. The presumed active site is located on a loop comprising residues 21--34, which is a relatively disordered region similar to that seen in many other protease inhibitors. However, the overall fold is different to other known protease inhibitors with the exception of a small family of inhibitors isolated from nematodes of the family Ascaris and recently also from the haemolymph of Apis mellifera. BSTI may thus be classified as a new member of this recently discovered family of protease inhibitors.

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata.

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do not coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular "bracelet" with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the "clasp". The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor.

NARROW SHEATH1 functions from two meristematic foci during founder-cell recruitment in maize leaf development.

The narrow sheath duplicate genes (ns1 and ns2) perform redundant functions during maize leaf development. Plants homozygous for mutations in both ns genes fail to develop wild-type leaf tissue in a lateral domain that includes the leaf margin. Previous studies indicated that the NS gene product(s) functions during recruitment of leaf founder-cells in a lateral, meristematic domain that contributes to leaf margin development. A mosaic analysis was performed in which the ns1-O mutation was exposed in hemizygous, clonal sectors in a genetic background already homozygous for ns2-O. Analyses of mutant, sectored plants demonstrate that NS1 function is required in L2-derived tissue layers for development of the narrow sheath leaf domain. NS1 function is not required for development of the central region of maize leaves. Furthermore, the presence of the non-mutant ns1 gene outside the narrow sheath domain cannot compensate for the absence of the non-mutant gene within the narrow sheath domain. NS1 acts non-cell autonomously within the narrow sheath-margin domain and directs recruitment of marginal, leaf founder cells from two discrete foci in the maize meristem. Loss of NS1 function during later stages of leaf development results in no phenotypic consequences. These data support our model for NS function during founder-cell recruitment in the maize meristem.

The narrow sheath duplicate genes: sectors of dual aneuploidy reveal ancestrally conserved gene functions during maize leaf development.

The narrow sheath mutant of maize displays a leaf and plant stature phenotype controlled by the duplicate factor mutations narrow sheath1 and narrow sheath2. Mutant leaves fail to develop a lateral domain that includes the leaf margins. Genetic data are presented to show that the narrow sheath mutations map to duplicated chromosomal regions, reflecting an ancestral duplication of the maize genome. Genetic and cytogenetic evidence indicates that the original mutation at narrow sheath2 is associated with a chromosomal inversion on the long arm of chromosome 4. Meristematic sectors of dual aneuploidy were generated, producing plants genetically mosaic for NARROW SHEATH function. These mosaic plants exhibited characteristic half-plant phenotypes, in which leaves from one side of the plant were of nonmutant morphology and leaves from the opposite side were of narrow sheath mutant phenotype. The data suggest that the narrow sheath duplicate genes may perform ancestrally conserved, redundant functions in the development of a lateral domain in the maize leaf.

Developmental complexities of simple leaves.

Recent papers have explored early events in the development of simple leaves. Functional compartmentalization of the shoot apical meristem correlates with distinct fields of cells connected by plasmodesmata. Molecules important in the initiation of phyllotactic pattern are described and the relationship between dorsoventral patterning and lateral leaf expansion is investigated.

Conformational selection of inhibitors and substrates by proteolytic enzymes: implications for drug design and polypeptide processing.

Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata.

BACKGROUND: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (PIs) that are derived from a 43 kDa precursor protein, NaProPI. NaProPI contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native PIs. An unusual feature of NaProPI is that the structural domains lie across adjacent repeats and that the sixth PI domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPI repeat (aPI1) forms a basis for unravelling the mechanism by which this protein may have evolved. RESULTS: The three-dimensional structure of aPI1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native PIs. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPI, exists as a disordered loop in aPI1. The presence of this loop in aPI1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. CONCLUSIONS: A single repeat from NaProPI is capable of folding into a compact globular domain that displays native-like PI activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPI evolved.

A novel two-chain proteinase inhibitor generated by circularization of a multidomain precursor protein.

Female reproductive tissues of the ornamental tobacco amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens. These PIs are produced from a precursor protein composed of six repeats each with a protease reactive site. Here we show that proteolytic processing of the precursor generates five single-chain PIs and a remarkable two-chain inhibitor formed by disulfide-bond linkage of N- and C-terminal peptide fragments. Surprisingly, PI precursors adopt this circular structure regardless of the number of inhibitor domains, suggesting this bracelet-like conformation is characteristic of the widespread potato inhibitor II (Pot II) protein family.

Phenotypic analysis and molecular cloning of discolored-1 (dsc1), a maize gene required for early kernel development.

Recessive mutations in the maize dsc1 locus prevent normal kernel development. Solidification of the endosperm in homozygous dsc1- mutant kernels was undetectable 12 days after pollination, at which time the tissue was apparently completely solidified in wild-type kernels. At later times endosperm did solidify in homozygous dsc1- mutant kernels, but there was a marked reduction in the volume of the tissue. Embryo growth in homozygous dsc1- kernels was delayed compared to wild-type kernels, but proceeded to an apparently normal stage 1 in which the scutellum, coleoptile, and shoot apex were clearly defined. Embryo growth then ceased and the embryonic tissues degraded. Late in kernel development no tissue distinctions were obvious in dsc1- mutant embryos. Immature mutant embryos germinated when transplanted from kernels to tissue culture medium prior to embryonic degeneration, but only coleoptile proliferation was observed. The dsc1 gene was isolated by transposon tagging. Analysis of the two different dsc1- mutations confirmed that transposon insertion into the cloned genomic locus was responsible for the observed phenotype. Dsc1 mRNA was detected specifically in kernels 5-7 days after pollination. These data indicate Dsc1 function is required for progression of embryo development beyond a specific stage, and also is required for endosperm development.

Clonal sectors reveal that a specific meristematic domain is not utilized in the maize mutant narrow sheath.

The narrow leaf and shortened stem phenotypes of the maize mutant narrow sheath (ns) are postulated to result from the lack of founder cell initialization in a region of the meristem that gives rise to leaf and stem margins. To test this model, a lineage map of the maize meristem is presented which compares the development of leaf margins in the narrow leaf mutant, narrow sheath (ns), and wild-type sibling plants. X-irradiation of mature seeds produced aneuploid albino sectors in wild-type and ns mutant plants. Of particular interest are sectors occurring in more than one leaf, which reflect a meristematic albino cell lineage. Analyses of these sectors indicated that: (1) a region of the ns meristem does not contribute to the founder cell population of the incipient leaf; (2) the margins of ns mutant leaves are derived from a lateral region of the meristem different from those in wild-type siblings; (3) founder cells in wild-type, juvenile-staged vegetative meristems encircle the meristem to a greater extent than do founder cells in adult-staged meristems; and (4) meristematic leaf founder cells may be subdivided into specific lateral domains, such that the position of a sector on the meristem correlates with a particular cell lineage. These data support our model for ns gene function in a specific domain of the meristem.

Solution structure and proposed binding mechanism of a novel potassium channel toxin kappa-conotoxin PVIIA.

BACKGROUND: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins. PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. RESULTS: The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore. Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA. CONCLUSIONS: Although the structure of PVIIA is considerably different to that of the alphaK scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.

The maize mutant narrow sheath fails to establish leaf margin identity in a meristematic domain.

The maize mutant narrow sheath (ns) displays a leaf shape and plant stature phenotype that suggests the preprimordial deletion of a leaf domain. The ns mutant phenotype is inherited as a recessive, duplicate-factor trait, conditioned upon homozygosity for each of the two unlinked mutations narrow sheath-1 (ns1) and narrow sheath-2 (ns2). Mutant leaves are missing a large domain including the leaf margin, and mutant internodes are shortened on the marginal side of the stem. This domain deletion extends from the internode to beyond the longitudinal mid-length of the blade, and corresponds to an alteration in the organization of a specific region of the shoot apical meristem. The premargin region of mutant founder cells fail to down-regulate expression of Knox genes, markers of nonleaf meristematic identity. Our results indicate that leaf domains may acquire identity in the meristem itself, and that the subdivision of preprimordial developmental fields into differential domains is a common feature of both plant and animal organogenesis.

Affinity purification of a correctly folded fragment of synthetic HIV-1 mRNA using a HIV-1 Rev peptide-ligand.

Formation of a macromolecular complex between the RNA binding protein HIV-1 Rev and HIV-1 mRNA is an essential prerequisite for nuclear export and subsequent expression of HIV-1 mRNA. The arginine rich peptide TRQARRNRRRRWRARQR, corresponding to residues 34-50 of HIV-1 Rev, contains the mRNA binding motif. We prepared a thioether linked Rev34-50-cellulose conjugate to affinity purify a fragment of synthetic mRNA corresponding to the high affinity binding site for Rev. The correctly folded fraction of mRNA (27.5%) was isolated from a crude synthetic mixture.

Solution structure of the cardiostimulant polypeptide anthopleurin-B and comparison with anthopleurin-A.

BACKGROUND: The polypeptide anthopleurin-B (AP-B) is one of a number of related toxins produced by sea anemones. AP-B delays inactivation of the voltage-gated sodium channel of excitable tissue. In the mammalian heart, this effect is manifest as an increase in the force of contraction. As a result, there is interest in exploiting the anthopleurins as lead compounds in the design of novel cardiac stimulants. Essential to this endeavour is a high-resolution solution structure of the molecule describing the positions of functionally important side chains. RESULTS: AP-B exists in multiple conformations in solution as a result of cis-trans isomerization about the Gly40-Pro41 peptide bond. The solution structure of the major conformer of AP-B has been determined by two-dimensional 1H NMR at pH 4.5 and 25 degrees C. The core structure is a four-stranded, antiparallel beta-sheet (residues 2-4, 20-23, 34-37 and 45-48) and includes several beta-turns (6-9, 25-28, 30-33). Three loops connect the beta-strands, the longest and least well defined being the first loop, extending from residues 8-17. These features are shared by other members of this family of sea anemone toxins. The locations of a number of side chains which are important for the cardiac stimulatory activity of AP-B are well defined in the structures. CONCLUSIONS: We have described the solution structure of AP-B and compared it with that of AP-A, from which it differs by substitutions at seven amino acid positions. It shares an essentially identical fold with AP-A yet is about 10-fold more active. Comparison of the structures, particularly in the region of residues essential for activity, gives a clearer indication of the location and extent of the cardioactive pharmacophore in these polypeptides.

NMR solution structure of the RNA-binding peptide from human immunodeficiency virus (type 1) Rev.

NMR spectroscopy has been used to solve the three-dimensional solution structure of a minimal RNA-binding domain of the Rev protein from the human immunodeficiency virus (type 1), an essential regulatory protein for viral replication. The presence of 10 arginine residues in the 17-residue peptide Rev34-50 caused significant problems in assignment of the NMR spectra. To improve spectral resolution, the peptide was synthesized with an alanine replacing a nonessential arginine and with selectively 15N-labeled residues. Contrary to Chou-Fasman modeling predictions an alpha-helix was detected in both water and 20% trifluoroethanol (TFE) and was found to span residues that constitute the RNA-binding and nuclear-localizing domains of Rev. The sequence-specific information provided by the NMR data gives a full description of the solution conformation of Rev34-50 which serves as a template for investigating binding of the peptide to RNA from the Rev response element (RRE). Preliminary modeling suggests that the helix can fit neatly into the expanded major groove of the RRE where interactions between the peptide side chains and the RNA can be identified. These data may aid the construction of a suitable pharmacophore model for the rational design of molecules that block Rev-RNA binding and inhibit HIV replication.

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

A simplified liquid chromatography assay for the quantitation of halofantrine and desbutylhalofantrine in plasma and identification of a degradation product of desbutylhalofantrine formed under alkaline conditions.

The development of a new and simplified, validated LC assay for the quantitation of halofantrine and desbutylhalofantrine in plasma is described. The methodology employs an inexpensive, rapid and simple liquid-liquid extraction procedure in combination with previously reported chromatographic conditions. The method has been employed to study aspects of the pharmacokinetics of orally administered halofantrine in beagle dogs and some preliminary data are presented. During development of the extraction procedure, degradation of desbutylhalofantrine was observed under non-acidic conditions in the extraction solvent (tert-butyl methyl ether) and we also report the structural elucidation of the breakdown product and the conditions required to avoid this degradation.