Two-faced Fcab prevents polymerization with VEGF and reveals thermodynamics and the 2.15 Å crystal structure of the complex

Fcabs (Fc domain with antigen-binding sites) are promising novel therapeutics. By engineering of the C-terminal loops of the CH3 domains, 2 antigen binding sites can be inserted in close proximity. To elucidate the binding mode(s) between homodimeric Fcabs and small homodimeric antigens, the interaction between the Fcabs 448 and CT6 (having the AB, CD and EF loops and the C-termini engineered) with homodimeric VEGF was investigated. The crystal structures of these Fcabs, which form polymers with the antigen VEGF in solution, were determined. However, construction of heterodimeric Fcabs (JanusFcabs: one chain Fc-wt, one chain VEGF-binding) results in formation of distinct JanusFcab–VEGF complexes (2:1), which allowed elucidation of the crystal structure of the JanusCT6–VEGF complex at 2.15 Å resolution. VEGF binding to Janus448 and JanusCT6 is shown to be entropically unfavorable, but enthalpically favorable. Structure-function relationships are discussed with respect to Fcab design and engineering strategies.     

The 1.8 Å cholix toxin crystal structure in complex with NAD+ and evidence for a new kinetic model

Certain Vibrio cholerae strains produce cholix, a potent protein toxin that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 1.8 Å crystal structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD⁺). We also substituted hallmark catalytic residues by site-directed mutagenesis and analyzed both NAD⁺ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These data are the basis for a new kinetic model of cholix toxin activity. Further, the new structural data serve as a reference for continuing inhibitor development for this toxin class.     

Refined 2.5 Å X-ray crystal structure of the complex formed by porcine kallikrein A and the bovine pancreatic trypsin inhibitor: Crystallization,patterson search,structure determination,refinement, structure and comparison with its components and with the bovine trypsin-pancreatic trypsin inhibitor complex

The complex formed by porcine pancreatic kallikrein A with the bovine pancreatic trypsin inhibitor (PTI) has been crystallized at pH 4 in tetragonal crystals of space group P4₁2₁2 with one molecule per asymmetric unit. Its crystal structure has been solved applying Patterson search methods and using a model derived from the bovine trypsin-PTI complex (Huber et al., 1974) and the structure of porcine pancreatic kallikrein A (Bode et al., 1983). The kallikrein-PTI model has been crystallographically refined to an R-value of 0·23 including X-ray data to 2·5 Å.The root-mean-square deviation, including all main-chain atoms, is 0·45 Å and 0·65 Å for the PTI and for the kallikrein component, respectively, compared with the refined models of the free components. The largest differences are observed in external loops of the kallikrein molecule surrounding the binding site, particularly in the C-terminal part of the intermediate helix around His172. Overall, PTI binding to kallikrein is similar to that of the trypsin complex. In particular, the conformation of the groups at the active site is identical within experimental error (in spite of the different pH values of the two structures). Ser195 OG is about 2·5 Å away from the susceptible inhibitor bond Lys15 C and forms an optimal 2·5 Å hydrogen bond with His57 NE.The PTI residues Thr11 to Ile18 and Val34 to Arg39 are in direct contact with kallikrein residues and form nine intermolecular hydrogen bonds. The reactive site Lys15 protrudes into the specificity pocket of kallikrein as in the trypsin complex, but its distal ammonium group is positioned differently to accommodate the side-chain of Ser226. Ser226 OG mediates the ionic interaction between the ammonium group and the carboxylate group of Asp189. Model-building studies indicate that an arginine side-chain could be accommodated in this pocket. The PTI disulfide bridge 14–38 forces the kallikrein residue Tyr99 to swing out of its normal position. Model-building experiments show that large hydrophobic residues such as phenylalanine can be accommodated at this (S2) site in a wedge-shaped hydrophobic cavity, which is formed by the indole ring of Trp215 and by the phenolic side-chain of Tyr99, and which opens towards the bound inhibitor/substrate chain. Arg17 in PTI forms a favorable hydrogen bond and van der Waals' contacts with kallikrein residues, whereas the additional hydrogen bond formed in the trypsin-PTI complex between Tvr39 OEH and Ile19 N is not possible The kallikrein binding site offers a qualitative explanation of the unusual binding and cleavage at the N-terminal Met-Lys site of kininogen. Model-building experiments suggest that the generally restricted capacity of kallikrein to bind protein inhibitors with more extended binding segments might be explained by steric hindrance with some extruding external loops surrounding the kallikrein binding site (Bode et al., 1983).     

Structural basis for the affinity of four insolubilized lectins,with a specificity for α-D-mannose,towards various glycopeptides with the N-glycosylamine linkage and related oligosaccharides

Precisions are given on the fine specificity and on the usefulness of immobilized concanavalin A,Lens culinaris, Vicia faba andPisum sativum agglutinins for fractionation of glycopeptides with the N-glycosylamine linkage.While insolubilized concanavalin A represents a very useful tool for the fractionation of both N-acetyllactosaminic and oligomannosidic type glycopeptides or related oligo-saccharides, immobilizedLens culinaris, as well asVicia faba orPisum sativum agglutinins allow the subfractionation of some N-acetyllactosaminic glycopeptide populations on the basis of the presence of an α-L-fucose residue substituting in C-6 position the N-acetylglucosamine residue involved in the N-glycosylamine bond.     

The surface structure of a complex substrate revealed by enzyme kinetics and Freundlich constants for α-amylase interaction with the surface of starch

Background

Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates.

Methods

For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution–solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR).

Results

Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and Δ_gelH was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid–liquid interface and slowest to granules possessing ordered crystalline surfaces.

Conclusions

Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods.

General Significance

Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.     

Secondary structure of β-hydroxydecanoyl thiol ester dehydrase, a 39-kDa protein, derived from Hα, Cα, Cβ and CO signal assignments and the Chemical Shift Index: Comparison with the crystal structure

Summary Nearly complete backbone ¹H, ¹⁵N and ¹³C signal assignments are reported for -hydroxydecanoyl thiol ester dehydrase, a 39-kDa homodimer containing 342 amino acids. Although ¹⁵N relaxation data show that the protein has a rotational correlation time of 18 ns, assignments were derived from triple-resonance experiments recorded at 500 MHz and pH 6.8, without deuteration. The Chemical Shift Index, CSI, identified two long helices and numerous -strands in dehydrase. The CSI predictions are in close agreement with the secondary structure identified in the recently derived crystal structure, particularly when one takes account of the numerous bulges in the -strands. The assignment of dehydrase and a large deuterated protein [Yamazaki et al. (1994) J. Am. Chem. Soc., 116, 11655–11666] suggest that assignment of 40–60 kDa proteins is feasible. Hence, further progress in understanding the chemical shift/structure relationship could open the way to determine the structures of such large proteins. Supplementary Material is available on request, comprising Table S1 listing the spectral parameters; Table S2 listing the assignments; Fig. S1 showing the 2D ¹H–¹⁵N HSQC spectrum; Fig. S2 showing sequential NOEs, secondary shifts, H-exchange and ³J_HN data; and Fig. S3 showing plots of the H, C, CO and C Chemical Shift Indexes.To whom correspondence should be addressed.     

Colocalization of GH,TSH and prolactin,but not ACTH,with βLH-immunoreactivity: evidence for pluripotential cells in the ovine pituitary

Increasing evidence suggests that multihormonal cells in the pituitary gland may be more commonplace than previously thought. This has forced us to reconsider our classical view of cell populations in the pituitary gland. Studies so far have focused almost exclusively on the rat, and there is a dearth of information on other species. Our first objective was to determine whether a subpopulation of gonadotropes also express somatotropin in the ewe, as reported in the rat. In addition, we sought to determine whether gonadotropes express any of the other known pituitary hormones. Finally, we investigated whether the stage of the estrous cycle influenced the occurrence of these pluripotential gonadotropes. We found that a small population of LH-immunoreactive cells also expresses immunoreactive GH, prolactin and TSH. No gonadotropes colocalized with ACTH. Significantly (P<0.001) more gonadotropes expressed GH during the luteal (10.7±0.4%) than the late follicular (5.4±0.3%) phase but there was no difference between the luteal and follicular phases in the proportion of gonadotropes expressing prolactin (follicular: 5.7±0.7%; luteal: 5.5±0.6%) or TSH (follicular: 3.1±0.7%; luteal: 4.2±0.5%). Similarly, there was a significant (P<0.05) difference in the proportion of GH-immunoreactive cells expressing LH immunoreactivity in the luteal (5.9±0.3%) and follicular (3.4±0.5%) phases but no difference in the proportion of prolactin- (follicular: 2.2±0.7%; luteal: 2.0±0.8%) or TSH-immunoreactive cells (follicular: 9.6±3.7%; luteal: 10.8±2.9%) expressing LH. The specific function of these multihormonal gonadotropes in sheep remains to be determined. This research was supported by NIH grant (5 P20 RR15553).     

Mössbauer spectra of the heme peptide (HP) 1–50 and the heme peptide:non-heme peptide (NHP) non-covalent complex 1–50:51–104 derived from cytochrome c: evidence for cytochrome c iron site solvation in aqueous solution

Mössbauer spectroscopic studies on a heme peptide (HP) derived from cytochrome c and on the HP recombined non-covalently with the remaining cleaved section are reported. The results suggest that the environment of the heme site in the known crystal structure of cytochrome c may differ in detail from the environment of the heme in the working protein.     

Distinct Structural Requirements for Interaction of the Integrins α5β1, αvβ5, and αvβ6 with the Central Cell Binding Domain in Fibronectin

At least 10 different members of the integrin family have been reported to bind to fibronectin, and eight of these interact with the arginine-glycine-aspartic acid (RGD) site in the tenth type III repeat. However, studies utilizing recombinant fibronectin fragments have shown that for three of these, α5β1, αIIbβ3, and αvβ3, the structural requirements for binding to fibronectin differ. In the present study. we report that two additional integrins, αvβ6. and αvβ5 also demonstrate unique requirements for interaction with recombinant fibronectin fragments. αvβ5, like αvβ3, can support cell adhesion to the RGD-containing tenth repeat alone, and does not require the presence of a synergy site in the adjacent ninth repeat. In the cells used in this study. αvβ5 only minimally supported adhesion to intact fibronectin. but did support adhesion to fragments composed of the eighth, ninth and tenth repeats or the tenth repeat. alone. Mutant fragments in which the eighth and tenth repeats were adjacent to one another enhanced adhesion mediated by αvβ5, as well as adhesion mediated by αvβ6. αvβ5 and αvβ6-mediated adhesion to all fibronectin fragments required interaction with the RGD site, as inferred by inhibition of adhesion with an RGD-containing peptide. These data suggest that each integrin that interacts with the RGD site in fibronectin has unique structural requirements for this interaction.     

Disulfide bonds Cys9–Cys57, Cys34–Cys88 and Cys38–Cys90 of the β-subunit of human chorionic gonadotropin are crucial for heterodimer formation with the α-subunit: experimental evidence for the conclusions from the crystal structure of hCG

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α- and β-subunits of hCG are highly cross-linked internally by disulfide bonds that seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This paper describes the results of our studies on the role of the disulfide bonds of hCG-β in heterodimer formation with the α-subunit. Six disulfide peptides incorporating each of the six disulfide bonds of hCG-β were screened, along with their linear counterparts, for their ability to competitively inhibit the recombination of α- and β-subunits. The disulfide peptides Cys (9–57), Cys (34–88) and Cys (38–90) were found to inhibit the α/β recombination whereas the remaining three disulfide peptides viz. Cys (23–72), Cys (26–110) and Cys (93–100) did not exhibit any inhibition activity. Interestingly, none of the linear peptides could inhibit the α/β recombination. Results clearly demonstrate that the disulfide bonds Cys⁹–Cys⁵⁷, Cys³⁴–Cys⁸⁸ and Cys³⁸–Cys⁹⁰ of the β-subunit of hCG are crucial for heterodimer formation with the α-subunit thus providing experimental confirmation of the conclusions from the crystal structure of the hormone.     

Genetic structure and variability of the endemic and vulnerable <Emphasis Type="Italic">Vellozia gigantea</Emphasis> (Velloziaceae) associated with the landscape in the Espinhaço Range,in southeastern Brazil: implications for conservation

The Espinhaço Range, in eastern Brazil, has a peculiar landscape that has influenced the vegetation pattern of the region because of its valleys, canyons, ranges and disjunct rock outcrops found at high elevations. In this region, the vegetation type known as campos rupestres (rupestrian fields), which occurs in the disjunct outcrops, has high levels of species richness and endemism. Vellozia gigantea, a 6-m tall dracenoid monocot, is a vulnerable species endemic to this vegetation and has a narrow distribution that extends approximately 27 km. This region is located in a disturbed area, where populations are divided into three geographical groups, separated by a canyon and a valley. For this study, we used ISSR markers to measure the genetic diversity of the species and test the hypothesis that the canyon and the valley constitute geographical barriers to gene flow in V. gigantea. Nine populations and 173 individuals were analyzed using nine ISSR primers, which produced 89 fragments. In spite of being a vulnerable species with a narrow distribution, the populations of V. gigantea have high genetic diversity (mean percentage of polymorphic loci = 56.6%; mean Shannon’s index of diversity = 0.278; mean expected heterozygosity = 0.183). Genetic divergence among populations was high (Φ_ST = 0.28), and principal coordinate, neighborjoining and Bayesian analyses showed that only the canyon may constitute a partial barrier to gene flow in this species. Groups of populations separated by the canyon should be managed separately because they contain different gene pools.     

<Emphasis Type="Italic">Syndesmis</Emphasis> François, 1886 (Rhabdocoela: Umagillidae): a revisitation,with a synopsis and an identification key to species,and new molecular evidence for ascertaining the phylogeny of the group

Syndesmis François, 1886 is a genus of umagillid turbellarians comprising species which are typically endosymbionts of echinoids, i.e. sea urchins. This group is likely key in addressing the issue of transition between a free-living and a parasitic mode of life in the Platyhelminthes. Accordingly, its phylogeny should be considered for detailed analysis, namely by addressing molecular evidence for its different species. At the present time, a revisitation of Syndesmis is required and fully justified by the following lines of argument: (i) the body of knowledge on Syndesmis is large, but the information is scattered through many different works in the literature; (ii) for about 60 years, it was a common practice to assign the umagillids isolated from sea urchins as a single species, i.e. the type-species, Syndesmis echinorum François, 1886, which was later split into several species on morphological grounds; and (iii) the type-species - for which no molecular information is available - was redescribed and new species were described in recent years but the generic diagnosis of Syndesmis was not emended accordingly. The present state of art additionally justifies the necessity of (i) an updated synopsis of species and (ii) an identification key to the 26 species described from different hosts and geographical locations. All these aspects define the aims of the present study. It is proposed that S. antillarum is attributed to Stunkard & Corliss (1951) and not to Powers (1936).     

The molecular structure and magnetic properties of the dimeric N,N′-ethylenebis(salicylamine)Fe(II)-μ-methoxo-N,N′-ethylene(o-hydroxylphenylglycine)salicylamine Fe(III): A complex with a μ-monodentate acetato bridge

We have prepared and characterized a dimeric complex of the formula Fe₂L(OCH₃)L′·solvent where L = N,N′-ethylenebis(salicylamine) and L′ = N,N′-ethylene(o-hydroxylphenylglycine)salicylamine. Crystals of the methanol solvate were found to be orthorhombic, space group P2nb, with a = 13.748 (5), b = 22.426(9), c = 11.762(6) Å and d_calc = 1.41 g/cm³. Least squares refinement of 966 reflections gave a final R factor of 0.066. The structure shows that the two iron atoms are coordinated to two different ligands and are bridged by a methoxide ion and a μ-monodentate acetate oxygen from the pendant arm of L′. The magnetic susceptibility was measured over the range 2–300 K and gave a spin coupling constant J = −8.3 cm⁻¹. The significance of acetate bridges to the exchange coupling pathway is discussed.     

Genetic evidence for the recognition of two fiddler crabs, Uca iranica and U.?albimana (Crustacea: Brachyura: Ocypodidae), from the northwestern Indian Ocean, with notes on the U.?lactea species-complex

The status of two poorly known fiddler crabs, Uca iranica Pretzmann, 1971, from the Persian Gulf, and U. albimana (Kossmann, 1877), from the Red Sea, was studied using two mitochondrial genes: the large subunit (16S) ribosomal (r)RNA and cytochrome oxidase subunit I (COI). A molecular phylogeny shows both U. iranica and U. albimana to be members of a monophyletic U. lactea species-complex containing six taxa, with three highly supported internal clades. Uca iranica and U. albimana are the closest genetically, but are different enough to be considered valid species (16S rRNA nucleotide divergence > 7.0%, and COI > 11.9%), and form a highly supported “western” clade with U. annulipes (in line with the original morphological concept). A West Pacific “eastern” clade includes U. lactea in the north and the more widely ranging U. perplexa. An Australian endemic species, U. mjoebergi, forms a third monotypic clade.     

Synthesis, X-ray crystal structure and magnetic study of the 1D {[Cu2(N,N,N′,N′-tetramethyl-ethylenediamine)2(μ-1,5-dca)2(dca)2]}n complex

Reaction of Cu(NO₃)₂ · 3H₂O, N,N,N′,N′-tetramethyl-ethylenediamine (L) and sodium dicyanamide (Nadca) in aqueous medium yields a complex the {[Cu₂L₂(μ-1,5-dca)₂(dca)₂]}_n complex, 1. Single crystal X-ray analysis reveals that complex 1 has a 1D infinite chain structure in which copper(II) ions are bridged by single dicyanamide anions in an end-to-end fashion. The coordination environment around copper(II) is distorted square pyramidal. Two among the four coordination sites of the basal plane are occupied by the nitrogen atoms of the diamine and two remaining sites are occupied by the terminal nitrogen atom of a bridging and of a monodentate dca anions. The fifth coordination site (apical) is occupied by a nitrogen atom from a bridging dca anion of an adjacent CuL(dca)₂ moiety, yielding the [Cu₂L₂(μ-1,5-dca)₂(dca)₂] dinuclear unit. Dimeric units are connected to each other by single μ-1,5-dicyanamido group to form infinite 1D chains which propagate parallel to the crystallographic c-axis. The variable temperature magnetic susceptibility measurements evidenced weakly antiferromagnetic interactions (J = −0.26 cm⁻¹) in {[Cu₂L₂(μ-1,5-dca)₂(dca)₂]}_n, 1.