Identification of common structural features of binding sites in galactose-specific proteins

Galactose-binding proteins characterize an important subgroup of sugar-binding proteins that are involved in a variety of biological processes. Structural studies have shown that the Gal-specific proteins encompass a diverse range of primary and tertiary structures. The binding sites for galactose also seem to vary in different protein-galactose complexes. No common binding site features that are shared by the Gal-specific proteins to achieve ligand specificity are so far known. With the assumption that common recognition principles will exist for common substrate recognition, the present study was undertaken to identify and characterize any unique galactose-binding site signature by analyzing the three-dimensional (3D) structures of 18 protein-galactose complexes. These proteins belong to 7 nonhomologous families; thus, there is no sequence or structural similarity across the families. Within each family, the binding site residues and their relative distances were well conserved, but there were no similarities across families. A novel, yet simple, approach was adopted to characterize the binding site residues by representing their relative spatial dispositions in polar coordinates. A combination of the deduced geometrical features with the structural characteristics, such as solvent accessibility and secondary structure type, furnished a potential galactose-binding site signature. The signature was evaluated by incorporation into the program COTRAN to search for potential galactose-binding sites in proteins that share the same fold as the known galactose-binding proteins. COTRAN is able to detect galactose-binding sites with a very high specificity and sensitivity. The deduced galactose-binding site signature is strongly validated and can be used to search for galactose-binding sites in proteins. PROSITE-type signature sequences have also been inferred for galectin and C-type animal lectin-like fold families of Gal-binding proteins.     

Potency of arachidonic acid in polyunsaturated fatty acid-induced death of human monocyte-macrophages: implications for atherosclerosis

Evidence suggests that oxidation of LDL is involved in the progression of atherosclerosis by inducing apoptosis in macrophages. Polyunsaturated fatty acids (PUFAs) are prominent components of LDL and are highly peroxidisable. We therefore tested PUFAs for induction of apoptosis in human monocyte-macrophages in vitro. Arachidonic acid (AA) induced the highest levels of apoptosis followed by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), despite DHA and EPA being more peroxidisable than AA. alpha-Linolenic acid induced lower levels of apoptosis. Linoleic and oleic acids were innocuous. Results of experiments with AA products and enzyme inhibitors suggest roles for peroxidation, cyclooxygenase and lipoxygenase in AA-induced apoptosis. Our results further suggest activation of PPARgamma by AA and DHA associated with apoptosis induction. These findings may be relevant to potential mechanisms of fatty acid influences on plaques and may suggest strategies for combating atherosclerosis progression.     

Infectivity analysis of two variable DNA B components of<Emphasis Type="Italic">Mungbean yellow mosaic virus-Vigna</Emphasis> in<Emphasis Type="Italic">Vigna mungo</Emphasis> and<Emphasis Type="Italic">Vigna radiata</Emphasis>

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), aBegomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85·6%) for KA27 DNA B. The Rep-binding domain had three complete 11 -nt (5’-TGTATCGGTGT-3′) iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5’-ATCGGTGT-3’) had a 3-nt deletion. KA27 DNA B, which exhibited 93·9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94·1%) and BC1 (97·6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMV-Vig is an important determinant of host-range betweenV. mungo andV. radiata.     

Divergence in gene expression related to variation in host specificity of an ectomycorrhizal fungus

Ectomycorrhizae are formed by mutualistic interactions between fungi and the roots of woody plants. During symbiosis the two organisms exchange carbon and nutrients in a specific tissue that is formed at the contact between a compatible fungus and plant. There is considerable variation in the degree of host specificity among species and strains of ectomycorrhizal fungi. In this study, we have for the first time shown that this variation is associated with quantitative differences in gene expression, and with divergence in nucleotide sequences of symbiosis-regulated genes. Gene expression and sequence evolution were compared in different strains of the ectomycorrhizal fungus Paxillus involutus; the strains included Nau, which is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. On a genomic level, Nau and Maj were very similar. The sequence identity was 98.9% in the 16 loci analysed, and only three out of 1075 genes analysed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes in different strains showed that two of them have evolved at an enhanced rate in Nau. The sequence divergence can be explained by a decreased selection pressure, which in turn is determined by lower functional constraints on these proteins in Nau as compared to the compatible strains.     

Conformation, orientation and dynamics of dodecylphosphocholine in micellar aggregate: a 3.2 ns molecular dynamics simulation study

A dodecylphosphocholine micelle of 86 monomers with 5776 water molecules has been simulated under NPT conditions for 3.2 ns using GROMACS2.0. The micelle was found to be very dynamic. Some of the C-C bonds, independent of their position in the DPC monomer, adopt gauche conformation and the trans <--> gauche transitions are quite frequent. An average of about 11% of the C-C bonds in the micelle are observed to be in the gauche conformation (i.e., |dihedral angle|< 120 degrees). The terminal methyl groups are randomly distributed all over the micelle whereas the nitrogen atom of phosphocholine headgroup atoms is restricted to the interface region. Some of the monomers were found to lie on the surface. The shape of micelle, influenced by the packing considerations, shows deviations from spherical shape. The phosphocholine headgroup is well solvated and there is no water penetration into the micelle core. The overall features of the micelle of 86 DPC monomers conforms to the lattice model of micelle proposed by Dill and Flory [Dill K A, Flory P J (1981) Proc Natl Acad Sci USA 78, 676-680] and is similar to DPC micelles of smaller aggregate sizes except for the positional preference of the C-C bonds for the gauche conformation and the trans<-->gauche transition times [Tieleman D P, van der Spoel D, Berendsen H J C (2000) J Phys Chem B 104, 6380-6388; Wymore T, Gao X F, Wong T C (1999) J Mol Struct (Theochem) 485-486, 195-210]. It appears that packing considerations play a predominant role in determining the shape and dynamics of the micelle.     

Structure and regulation of the cAMP-binding domains of Epac2

Cyclic adenosine monophosphate (cAMP) is a universal second messenger that, in eukaryotes, was believed to act only on cAMP-dependent protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Recently, guanine nucleotide exchange factors specific for the small GTP-binding proteins Rap1 and Rap2 (Epacs) were described, which are also activated directly by cAMP. Here, we have determined the three-dimensional structure of the regulatory domain of Epac2, which consists of two cyclic nucleotide monophosphate (cNMP)-binding domains and one DEP (Dishevelled, Egl, Pleckstrin) domain. This is the first structure of a cNMP-binding domain in the absence of ligand, and comparison with previous structures, sequence alignment and biochemical experiments allow us to delineate a mechanism for cyclic nucleotide-mediated conformational change and activation that is most likely conserved for all cNMP-regulated proteins. We identify a hinge region that couples cAMP binding to a conformational change of the C-terminal regions. Mutations in the hinge of Epac can uncouple cAMP binding from its exchange activity.