Structures closed into cycles in globular proteins

Different types of structures closed into cycles are widespread at all the levels of structural organization of proteins. β-Hairpins, triple-stranded β-sheets, and βαβ-units represent simple structural motifs closed into cycles by systems of hydrogen bonds. Secondary closing of these simple motifs into larger cycles by means of different superhelices, split β-hairpins, or SS-bridges results in formation of complex structural motifs such as abcd-units, φ-motifs, five- and seven-segment α/β-motifs, etc. At the level of tertiary structure many proteins and domains fold into structures closed into cylinders. Apparently, closing the motifs and domains into cycles and cylinders results in formation of more cooperative and stable structures as compared with open ones, and this may be the reason for high frequencies of occurrence of the motifs in proteins.     

The structural basis of substrate recognition in an exo-beta-D-glucosaminidase involved in chitosan hydrolysis

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with β-galactosidase activity (Escherichia coli LacZ), β-glucuronidase activity (Homo sapiens GusB), and β-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-β-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 Å resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural β-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-β-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.     

Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding

Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite − 1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)₈-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.     

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.     

Hydrophilic Linkers and Polar Contacts Affect Aggregation of FG Repeat Peptides

Transport of large proteins into the nucleus involves two events, binding of the cargo protein to a transport receptor in the cytoplasm and passage of the cargo-transporter complex through the selective permeability barrier of the nuclear pore complex. The permeability barrier is formed by largely disordered polypeptides, each containing a number of conserved hydrophobic phenylalanine-glycine (FG) sequence motifs, connected by hydrophilic linkers of varying sequence (FG nups). How the motifs interact to form the permeability barrier, however, is not yet known. We have, therefore, carried out molecular dynamics simulations on various model FG repeat peptides to study the aggregation propensity of FG nups and the specific roles of the hydrophobic FG motifs and the hydrophilic linkers. Our simulations show spontaneous aggregation of the model nups into hydrated aggregates, which exhibit structural features assumed to be part of the permeability barrier. Our simulations suggest that short β-sheets are an important structural feature of the aggregates and that Phe residues are sufficiently exposed to allow rapid binding of transport receptors. A surprisingly large influence of the amino acid composition of the hydrophilic linkers on aggregation is seen, as well as a major contribution of hydrogen-bonding patterns.     

Addition of α-O-GlcNAc to threonine residues define the post-translational modification of mucin-like molecules in Trypanosoma cruzi

Trypanosoma cruzi, an intracellular protozoan etiologic agent of Chagas disease is covered by a dense coat of mucin-type glycoproteins, which is important to promote the parasite entry and persistence in the mammalian host cells. The O-glycosylation of T. cruzi mucins (Tc-mucins) is initiated by enzymatic addition of α-O-N-acetylglucosamine (GlcNAc) to threonine (Thr) by the UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase (pp-α-GlcNAcT) in the Golgi. The Tc-mucin is characterized by the presence of a high structural diversity of O-linked oligosaccharides found among different parasite strains, comprising two O-glycan Cores. In the Core 1, from strains principally associated with the domestic transmission cycle of Chagas disease, the GlcNAc O-4 is substituted with a β-galactopyranose (βGalp) unit, and in the most complex oligosaccharides the GlcNAc O-6 is further processed by the addition of β1?→?2-linked Galp residues creating a short linear Galp-containing chain. In the Core 2 structures, expressed by strains isolated from T. cruzi sylvatic hosts, the GlcNAc O-4 carries a β-galactofuranose (βGalf) unit and the GlcNAc O-6 can carry a branched Galpβ1?→?3[Galpβ1?→?2]Galpβ1?→?6 motif. The O-glycans carrying nonreducing terminal βGalp are available for sialylation by a surface T. cruzi trans-sialidase activity. Based on structural results, this review summarizes available data on the highly conserved process, which adds the GlcNAc unit in α-linkage to Thr residues the basis of the post-translational modification system in T. cruzi mucins. In addition, a mechanism unique employed by the parasite to transfer exogenous sialic acid residues to Tc-mucins is presented.     

On the structural repertoire of pools of short, random RNA sequences

A detailed knowledge of the mapping between sequence and structure spaces in populations of RNA molecules is essential to better understand their present-day functional properties, to envisage a plausible early evolution of RNA in a prebiotic chemical environment and to improve the design of in vitro evolution experiments, among others. Analysis of natural RNAs, as well as in vitro and computational studies, show that certain RNA structural motifs are much more abundant than others, pointing out a complex relation between sequence and structure. Within this framework, we have investigated computationally the structural properties of a large pool (10⁸ molecules) of single-stranded, 35 nt-long, random RNA sequences. The secondary structures obtained are ranked and classified into structure families. The number of structures in main families is analytically calculated and compared with the numerical results. This permits a quantification of the fraction of structure space covered by a large pool of sequences. We further show that the number of structural motifs and their frequency is highly unbalanced with respect to the nucleotide composition: simple structures such as stem-loops and hairpins arise from sequences depleted in G, while more complex structures require an enrichment of G. In general, we observe a strong correlation between subfamilies—characterized by a fixed number of paired nucleotides—and nucleotide composition. Our results are compared to the structural repertoire obtained in a second pool where isolated base pairs are prohibited.     

Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins

β-Barrel proteins are frequently found in the outer membrane of mitochondria, chloroplasts and Gram-negative bacteria. In Escherichia coli, these proteins are inserted in the outer membrane by the Bam (β-barrel assembly machinery) complex, a multiprotein machinery formed by the β-barrel protein BamA and the four peripheral membrane proteins BamB, BamC, BamD and BamE. The periplasmic part of BamA binds prefolded β-barrel proteins by a β-augmentation mechanism, thereby stabilizing the precursors prior to their membrane insertion. However, the role of the associated proteins within the Bam complex remains unknown. Here, we describe the crystal structure of BamB, a nonessential component of the Bam complex. The structure shows a typical eight-bladed β-propeller fold. Two sequence stretches of BamB were previously identified to be important for interaction with BamA. In our structure, both motifs are located in close proximity to each other and contribute to a conserved region forming a narrow groove on the top of the propeller. Moreover, crystal contacts reveal two interaction modes of how BamB might bind unfolded β-barrel proteins. In the crystal lattice, BamB binds to exposed β-strands by β-augmentation, whereas peptide stretches rich in aromatic residues can be accommodated in hydrophobic pockets located at the bottom of the propeller. Thus, BamB could simultaneously bind to BamA and prefolded β-barrel proteins, thereby enhancing the folding and membrane insertion capability of the Bam complex.     

Evolution of β-amylase: Patterns of variation and conservation in subfamily sequences in relation to parsimony mechanisms

Soybean and sweet potato β-amylases are structured as α/β barrels and the same kind of folding may account for all known β-amylases. We provide a comprehensive analysis of both protein and DNA (coding region) sequences of β-amylases. The aim of the study is to contribute to the knowledge of the evolutionary molecular relationships among all known β-amylases. Our approach combines the identification of the putative eightfold structural core formed by β-strands with a complete multi-alignment analysis of all known sequences. Comparing putative β-amylase (α/β)₈ cores from plants and microorganisms, two differentiated versions of residues at the packing sites, and a unique set of eight identical residues at the C-terminal catalytical site are observed, indicating early evolutionary divergence and absence of localized three-dimensional evolution, respectively. A new analytical approach has been developed in order to work out conserved motifs for β-amylases, mostly related with the enzyme activity. This approach appears useful as a new routine to find sets of motifs (each set being known as a fingerprint) in protein families. We demonstrate that the evolutionary mechanism for β-amylases is a combination of parsimonious divergence at three distinguishable rates in relation to the functional signatures, the barrel scaffold, and α-helix-containing loops. © 1996 Wiley-Liss, Inc.     

Characterization of fucosyl glycopeptides from cell surface and cellular material of rat fibroblasts

Approximately 70% of fucose-labeled glycopeptides from the cell surface and cellular material of rat fibroblasts (3Y1B cells) were hydrolyzed by endo-β-N-acetylglucosaminidase D in the presence of neuraminidase, β-glactosidase and β-N-acetylglucosaminidase. Structure of the suspceptible glycopeptides were found to be very similar to non-membrane glycopeptides of the complex heteropolysaccharide unit, such as the sialylated glycopeptides of thyroglobulin. On the other hand, the resistant glycopeptides were also refractory toward endo-β-N-acethylglucosaminidase H and α-mannosidase, and appeared to be a mixture of glycopeptides with unique structures.     

Chromosomal arrangement of the chicken β-type globin genes

We have isolated the chicken β-type globin genes from a library of chicken DNA-λ Charon 4A recombinant bacteriophage. There are four β-type genes within this segment of the genome; we believe this represents all of the β-type genes of the chicken. The recombinant λCβG1 contains the embryonic ?- and adult β-globin genes. The hatching β^H and embryonic p-globin genes are found in the recombinant λCβG2. Although λCβG1 and λCβG2 do not physically overlap, we present evidence that all four genes are closely linked and transcribed from the same DNA strand. These experiments demonstrate that the chromosomal regions represented by λCβG1 and λCβG2 lie approximately 1.6 kb apart in the chicken genome. A third recombinant λCβG3 extends the genomic locus studied in the vicinity of the β-type globin genes to approximately 39 kb. The physical order of the chicken β-type globin genes within this segment of the chromosome is 5′ … ?-β^H-β-? … 3′. This arrangement is unique among the vertebrate β-type globin gene clusters thus far examined, in that embryonic genes are located at the 5′ and 3′ ends of the cluster while the hatching and adult genes occupy central positions.     

Uptake of beta-ecdysone by the fat body and membrane vesicles of fat body cells of Sarcophaga peregrina larvae.

The fat body of Sarcophaga peregrina larvae was shown to incorporate ³H-β-ecdysone when it was incubated with the hormone in vitro. Most of the incorporated radioactivity was found in the cytoplasmic fraction as free β-ecdysone, not as a protein-β-ecdysone complex.Rapid uptake and accumulation of β-ecdysone was observed in the membrane vesicles of fat body cells in vitro. The apparent K_m value for uptake was estimated to be 1·25 × 10^?7 M. The β-ecdysone in the membrane vesicles was rapidly released when 2,4-dinitrophenol was added. These results suggest that β-ecdysone was incorporated into the membrane vesicles by active transport and not by free diffusion. The hormone is probably incorporated into larval tissues by the same mechanism as it is incorporated into the membrane vesicles of fat body cells.     

Colored motifs reveal computational building blocks in the C. elegans brain

Background

Complex networks can often be decomposed into less complex sub-networks whose structures can give hints about the functional organization of the network as a whole. However, these structural motifs can only tell one part of the functional story because in this analysis each node and edge is treated on an equal footing. In real networks, two motifs that are topologically identical but whose nodes perform very different functions will play very different roles in the network.

Methodology/Principal Findings

Here, we combine structural information derived from the topology of the neuronal network of the nematode C. elegans with information about the biological function of these nodes, thus coloring nodes by function. We discover that particular colorations of motifs are significantly more abundant in the worm brain than expected by chance, and have particular computational functions that emphasize the feed-forward structure of information processing in the network, while evading feedback loops. Interneurons are strongly over-represented among the common motifs, supporting the notion that these motifs process and transduce the information from the sensor neurons towards the muscles. Some of the most common motifs identified in the search for significant colored motifs play a crucial role in the system of neurons controlling the worm''s locomotion.

Conclusions/Significance

The analysis of complex networks in terms of colored motifs combines two independent data sets to generate insight about these networks that cannot be obtained with either data set alone. The method is general and should allow a decomposition of any complex networks into its functional (rather than topological) motifs as long as both wiring and functional information is available.     

Solution structure and dynamics of glia maturation factor from Caenorhabditis elegans

BackgroundThe GMF class of the ADF-H domain family proteins regulate actin dynamics by binding to the Arp2/3 complex and F-actin through their Site-1 and Site-2, respectively. CeGMF of C. elegans is analogous to GMFγ of human and mouse and is 138 amino acids in length.MethodsWe have characterized the solution structure and dynamics of CeGMF by solution NMR spectroscopy and its thermal stability by DSC.ResultsThe solution structure of CeGMF shows canonical ADF-H fold with two additional β-strands in the β4-β5 loop region. The Site-1 of CeGMF is well formed and residues of all three regions of Site-1 show dynamic flexibility. However, the β4-β5 loop of Site-2 is less inclined towards the C-terminal, as the latter is truncated by four residues in comparison to GMF isoforms of human and mouse. Regions of Site-2 show motions on ns-ps timescale, but dynamic flexibility of β4-β5 loop is low in comparison to corresponding F-loop region of ADF/cofilin UNC-60B. A general difference in packing of α3 and α1 between GMF and ADF/cofilins was noticed. Additionally, thermal stability of CeGMF was significantly higher than its ADF/cofilin homologs.ConclusionWe have presented the first solution structure of GMF from C. elegans, which highlights the structural differences between the Site-2 of CeGMF and mammalian GMF isoforms. Further, we have seen the differences in structure, dynamics, and thermal stability of GMF and ADF/cofilin.General significanceThis study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.     

Genetic analysis of provitamin A carotenoid β-cryptoxanthin concentration and relationship with other carotenoids in maize grain (<Emphasis Type="Italic">Zea mays</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Provitamin A (proVA) carotenoids are converted into retinol (vitamin A) in the human body, are the subject of human nutrition studies, and are targets for biofortification of staple crops. β-Carotene has been the principal target for enhancing levels of proVA. There is recent interest in enhancing the proVA carotenoid β-cryptoxanthin since it has excellent bioavailability, and in maize may be nearly as effective as β-carotene in providing retinol to humans. This study was designed to enhance our understanding of the genetic control of: levels of β-cryptoxanthin, conversion of β-carotene into β-cryptoxanthin and zeaxanthin, conversion of β-cryptoxanthin into zeaxanthin, and flux into and within the β-branch of carotenoid pathway. A biparental population derived from two inbreds with relatively high levels of β-cryptoxanthin and different ratios of β-carotene to β-cryptoxanthin and β-cryptoxanthin to zeaxanthin was studied. Three field replications of this F_2:3 population were grown, grain analyzed by liquid chromatography (LC), and composite interval mapping (CIM) performed to identify 90 quantitative trait loci (QTL) for carotenoids. We detected QTL for β-carotene/(β-cryptoxanthin + zeaxanthin) and (β-carotene + β-cryptoxanthin)/zeaxanthin ratios that contain candidate gene hydroxylase 4 (hyd4), which has not been previously associated with QTL for carotenoids in maize grain. Two color assessment methods, visual score and chromameter reading, were used to phenotype one replicate of the population for initial assessment as simple alternative measuring procedures. A common finding for LC and chromameter analysis included QTL on chromosome 5 that contain candidate gene lycopene β cyclase (lcyβ).     

Ultra-high resolution crystal structure of recombinant caprine β-lactoglobulin

β-Lactoglobulin (βlg) is the most abundant whey protein in the milks of ruminant animals. While bovine βlg has been subjected to a vast array of studies, little is known about the caprine ortholog. We present an ultra-high resolution crystal structure of caprine βlg complemented by analytical ultracentrifugation and small-angle X-ray scattering data. In both solution and crystalline states caprine βlg is dimeric (K_D < 5 μM); however, our data suggest a flexible quaternary arrangement of subunits within the dimer. These structural findings will provide insight into relationships among structural, processing, nutritional and immunological characteristics that distinguish cow’s and goat’s milk.     

STD-NMR studies of two acceptor substrates of GlfT2, a galactofuranosyltransferase from Mycobacterium tuberculosis: Epitope mapping studies

The major structural component of the mycobacterial cell wall, the mycolyl–arabinogalactan–peptidoglycan complex, possesses a galactan core composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating β-(1→6) and β-(1→5) linkages. Recent studies have shown that the entire galactan is synthesized by two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation transfer difference (STD) NMR studies GlfT2 using two trisaccharide acceptor substrates, β-d-Galf-(1→6)-β-d-Galf-(1→5)-β-d-Galf-O(CH₂)₇CH₃ (2) and β-d-Galf-(1→5)-β-d-Galf-(1→6)-β-d-Galf-O(CH₂)₇CH₃ (3), as well as the donor substrate for the enzyme, UDP-Galf. Epitope mapping demonstrated a greater enhancement toward the ‘reducing’ ends of both trisaccharides, and that UDP-galactofuranose (UDP-Galf) made more intimate contacts through its nucleotide moiety. This observation is consistent with the greater flexibility required within the active site of the reaction between the growing polymer acceptor and the UDP-Galf donor. The addition of UDP-Galf to either 2 or 3 in the presence of GlfT2 generated a tetrasaccharide product, indicating that the enzyme was catalytically active.