Crystal structure of enteric adenovirus serotype 41 short fiber head

Human enteric adenoviruses of species F contain two fibers in the same virion, a long fiber which binds to coxsackievirus and adenovirus receptor (CAR) and a short fiber of unknown function. We have determined the high-resolution crystal structure of the short fiber head of human adenovirus serotype 41 (Ad41). The short fiber head has the characteristic fold of other known fiber heads but has three unusual features. First, it has much shorter loops between the beta-strands. Second, one of the usually well-ordered beta-strands on the distal face of the fiber head is highly disordered and this same region is sensitive to digestion with pepsin, an enzyme occurring naturally in the intestinal tract, the physiological environment of Ad41. Third, the AB loop has a deletion giving it a distinct conformation incompatible with CAR binding.     

Crystal structure of the YchF protein reveals binding sites for GTP and nucleic acid

The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 A resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced alpha-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an alpha-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.     

Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob

Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a K_D (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.     

Crystal structure of the bacterial YhcH protein indicates a role in sialic acid catabolism

The yhcH gene is part of the nan operon in bacteria that encodes proteins involved in sialic acid catabolism. Determination of the crystal structure of YhcH from Haemophilus influenzae was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. The structure was determined at 2.2-A resolution by multiple-wavelength anomalous diffraction. The protein fold is a variation of the double-stranded beta-helix. Two antiparallel beta-sheets form a funnel opened at one side, where a putative active site contains a copper ion coordinated to the side chains of two histidine and two carboxylic acid residues. A comparison to other proteins with a similar fold and analysis of the genomic context suggested that YhcH may be a sugar isomerase involved in processing of exogenous sialic acid.     

Crystal structure of reovirus attachment protein sigma1 reveals evolutionary relationship to adenovirus fiber.

Reovirus attaches to cellular receptors with the sigma1 protein, a fiber-like molecule protruding from the 12 vertices of the icosahedral virion. The crystal structure of a receptor-binding fragment of sigma1 reveals an elongated trimer with two domains: a compact head with a new beta-barrel fold and a fibrous tail containing a triple beta-spiral. Numerous structural and functional similarities between reovirus sigma1 and the adenovirus fiber suggest an evolutionary link in the receptor-binding strategies of these two viruses. A prominent loop in the sigma1 head contains a cluster of residues that are conserved among reovirus serotypes and are likely to form a binding site for junction adhesion molecule, an integral tight junction protein that serves as a reovirus receptor. The fibrous tail is mainly responsible for sigma1 trimer formation, and it contains a highly flexible region that allows for significant movement between the base of the tail and the head. The architecture of the trimer interface and the observed flexibility indicate that sigma1 is a metastable structure poised to undergo conformational changes upon viral attachment and cell entry.     

Crystal structure of a lead-dependent ribozyme revealing metal binding sites relevant to catalysis

The leadzyme is a small RNA motif that catalyzes a site-specific, Pb2+-dependent cleavage reaction. As such, it is an example of a metal-dependent RNA enzyme. Here we describe the X-ray crystallographic structure of the leadzyme, which reveals two independent molecules per asymmetric unit. Both molecules feature an internal loop in which a bulged purine base stack twists away from the helical stem. This kinks the backbone, rendering the phosphodiester bond susceptible to cleavage. The independent molecules have different conformations: one leadzyme copy coordinates Mg2+, whereas the other binds only Ba2+ or Pb2+. In the active site of the latter molecule, a single Ba2+ ion coordinates the 2'-OH nucleophile, and appears to mimic the binding of catalytic lead. These observations allow a bond cleavage reaction to be modeled, which reveals the minimal structural features necessary for catalysis by this small ribozyme.     

Crystal structure of the human adenovirus proteinase with its 11 amino acid cofactor.

The three-dimensional structure of the human adenovirus-2 proteinase complexed with its 11 amino acid cofactor, pVIc, was determined at 2.6 A resolution by X-ray crystallographic analysis. The fold of this protein has not been seen before. However, it represents an example of either subtly divergent or powerfully convergent evolution, because the active site contains a Cys-His-Glu triplet and oxyanion hole in an arrangement similar to that in papain. Thus, the adenovirus proteinase represents a new, fifth group of enzymes that contain catalytic triads. pVIc, which extends a beta-sheet in the main chain, is distant from the active site, yet its binding increases the catalytic rate constant 300-fold for substrate hydrolysis. The structure reveals several potential targets for antiviral therapy.     

Role of sialic acid in bovine sperm-zona pellucida binding

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.     

Insight into the interaction sites between fatty acid binding proteins and their ligands

Fatty acid binding proteins (FABPs), are evolutionarily conserved small cytoplasmic proteins that occur in many tissue-specific types. One of their primary functions is to facilitate the clearance of the cytoplasmic matrix from free fatty acids and of other detergent-like compounds. Crystallographic studies of FABP proteins have revealed a well defined binding site located deep inside their β-clam structure that is hardly exposed to the bulk solution. However, NMR measurements revealed that, when the protein is equilibrated with its ligands, residues that are clearly located on the outer surface of the protein do interact with the ligand. To clarify this apparent contradiction we applied molecular dynamics simulations to follow the initial steps associated with the FABP–fatty acid interaction using, as a model, the interaction of toad liver basic FABP, or chicken liver bile acid binding protein, with a physiological concentration of palmitate ions. The simulations (~200 ns of accumulated time) show that fatty acid molecules interact, unevenly, with various loci on the protein surface, with the favored regions being the portal and the anti-portal domains. Random encounters with palmitate at these regions led to lasting adsorption to the surface, while encounters at the outer surface of the β-clam were transient. Therefore, we suggest that the protein surface is capable of sequestering free fatty acids from solution, where brief encounters evolve into adsorbed states, which later mature by migration of the ligand into a more specific binding site.     

Crystal structure of a secondary vitamin D3 binding site of milk beta-lactoglobulin

Beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 A resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 A). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an alpha-helix and a beta-strand I with 17.91 A in length, while the span of vitamin D(3) is about 12.51 A. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).     

Sperm binding to oviductal epithelial cells in the rat: role of sialic acid residues on the epithelial surface and sialic acid-binding sites on the sperm surface

The aim of this study was to assess the participation of carbohydrate residues in the adhesion of spermatozoa to the oviductal epithelium in the rat. We first examined, by lectin labeling, the distribution of glycoconjugates in rat oviducts obtained under various hormonal environments. Several classes of glycoconjugates were abundant in the epithelium, and the expression of some of these molecules varied differentially in ampulla and isthmus, along the estrous cycle and with estradiol and progesterone treatment. Proestrous rats were intraoviductally injected with lectins Dolichos biflorus, Erythrina cristagalli, Helix pomatia, Arachis hypogea, Ulex europaeus I, Triticum vulgaris, or Tritrichomonas mobilensis and were inseminated with 10-20 million epididymal spermatozoa in each uterine horn. Three hours later, the total number of spermatozoa present in the oviduct and the proportion adhering to the epithelium were determined. Intraoviductal administration of lectins did not affect the total number of spermatozoa recovered from the oviduct and only the sialic acid-binding lectin TML decreased the percentage of sperm cells adhering to the epithelium. The involvement of sialic acid in sperm-oviduct adhesion was further explored, inseminating spermatozoa preincubated with mannose, galactose, sialic acid, fucose, fetuin, or asialofetuin. Sialic acid and fetuin inhibited sperm-oviduct binding while other carbohydrates had no effect. Using TML lectin immunohistochemistry, we found that sialic acid-rich glycoconjugates are equally localized in the epithelium of ampulla and isthmus of proestrous rats. The electrophoretic pattern of sialic acid-rich glycoproteins of the epithelium showed 15 major protein bands, for which molecular mass ranged from 200 to 50 kDa with no difference between ampulla and isthmus or between estrous cycle stages. Binding sites for sialic acid-fluorescein isothiocyanate were demonstrated on the surface of rat spermatozoa, and biotinylated sialic acid recognized 11 plasma membrane proteins of sperm cells. These groups of sialic acid-rich glycoproteins in the oviductal epithelium and of sialic acid-binding proteins in the plasma membrane of sperm cells are good candidates for further studies to characterize the molecules responsible for sperm binding. We conclude that there are segment-specific changes of sugar residues present in the oviductal epithelium associated with different endocrine environments. Sperm-oviduct adhesion in the rat occurs by interaction of sialoglycoconjugates present in the epithelial cells with sialic acid-binding proteins on the sperm surface. This replicates the situation previously found in hamsters, disclosing for the first time that species-specificity in the sugar involved in sperm binding is not absolute.     

A single amino acid in the adenovirus type 37 fiber confers binding to human conjunctival cells

A 46-kDa receptor, coxsackievirus-adenovirus (Ad) receptor (CAR), mediates cell attachment of a number of different Ad serotypes; however, not all Ad serotypes utilize this receptor for infection. Moreover, the precise amino acid sequences in the Ad fiber protein that mediate cell attachment have yet to be identified. We investigated the interaction of subgroup D Ads with human ocular cells. Ad serotype 37 (Ad37), a virus associated with epidemic keratoconjunctivitis, but not a closely related virus serotype, Ad19p, exhibited preferential binding to and infection of human conjunctival cells. A single amino acid substitution in the Ad19p fiber distal domain (knob), Glu240 to Lys, conferred binding to conjunctival cells, while the reverse substitution in the Ad37 fiber abrogated cell binding. These findings provide new information on the fiber sequences that regulate Ad host cell tropism.     

tRNA structure and binding sites for cations

Equilibrium dialysis and electronic and nuclear resonance spectroscopy show that tRNA cooperatively binds divalent metal ions at very low concentrations (free metal concentration 3 × 10 ^?6 M). The first two methods show that different purified tRNAs have a very similar behavior, including initiator tRNA_F^met. tRNAs with an extra arm in the clover-leaf model, however, appear to have a slightly different behavior. The binding can be described in terms of two classes of sites. The cooperative association of divalent ions binding first does not parallel a cooperative change in the hyperchromism of the tRNA, while the non-cooperative association of the second class of divalent ions corresponds to the concentrations needed to obtain a cooperative melting of the tRNA. The temperature dependence of the number of binding sites and of their binding constants is also presented. The nature of the divalent ion gives the following efficiency: for the cooperativity Co⁺⁺>Mg⁺⁺>Mn⁺⁺ for the weak binding sites Mn⁺⁺>Co⁺⁺>Mg⁺⁺     

Amino acid sequence homology between ligands and their receptors: potential identification of binding sites

Mice were immunized with alpha-bungarotoxin (BGT), a nearly irreversible antagonist of the acetylcholine receptor (AChR), to produce monoclonal antibodies (Mabs). One of the Mabs (JMC2.7) bound not only to BGT, but to the AChR as well. To understand the molecular basis for this novel cross-reaction, the amino acid sequences of these proteins were searched for areas of similarity which might constitute the shared epitope. A number of short segments of sequence homology were found, one of them representing the BGT-binding site of the AChR. Because a portion of BGT resembles that part of the AChR that binds toxin, the self-binding of BGT was evaluated. As shown here, BGT binds specifically to itself to form dimers. In order to extend these observations, other ligand-receptor pairs were examined for sequence homology. The sodium channel and alpha-scorpion toxins were found to have distinct areas of similarity, as do interleukin 2 (IL-2) and the IL-2 receptor. As a general principle, we propose that peptide ligands and their receptors may often share amino acid sequence homology. In fact, the sites of interaction between two proteins may largely be determined by these regions of similarity.     

Influence of sialic acid on the binding activity of estrogen receptors

The influence of sialidase and sialyltransferase on the binding of 3H-estradiol to estrogen receptors in baboon uterus was investigated to ascertain if sialylation was involved. Specific binding capacity increased approximately 37% in the presence of sialidase, although Kd values essentially remained unchanged. 3H-Estradiol binding was correlated with free sialic acid in the presence of either sialidase or sialyltransferase. As sialidase concentrations were increased, 3H-estradiol binding and free sialic acid concentration increased linearly (r = 0.937, p less than 0.001). Incubation of 22 x 10(-5) U sialidase with its inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, decreased binding capacity and sialic acid concentration (r = 0.929, p less than 0.001). Although a decrease in binding capacity and free sialic acid concentration was observed in the presence of increasing amounts of sialyltransferase, a positive correlation was found between these two parameters (r = 0.839, p less than 0.035). A negative trend that was statistically insignificant was observed between binding capacity and sialic acid concentration when 2 x 10(-4) U sialyltransferase was incubated with the inhibitor, acetylsalicylic acid (r = -0.571, p = 0.195). The sialic acid concentration increased, while the 3H-estradiol binding capacity decreased. Collectively, these results show that both sialidase and sialyltransferase affect the binding of estradiol to its receptor in opposite directions. We suggest that biological activities of estrogen receptors in target cells may be regulated by the extent of sialylation of the receptor molecule itself. This posttranslational alteration may represent a new type of control mechanism for estrogen action.     

Understanding of the roles of phospholipase D and phosphatidic acid through their binding partners

Phospholipase D (PLD) is a phosphatidyl choline (PC)-hydrolyzing enzyme that generates phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling. Through interactions with signaling molecules, both PLD and PA can mediate a variety of cellular functions, such as, growth/proliferation, vesicle trafficking, cytoskeleton modulation, development, and morphogenesis. Therefore, systemic approaches for investigating PLD networks including interrelationship between PLD and PA and theirs binding partners, such as proteins and lipids, can enhance fundamental knowledge of roles of PLD and PA in diverse biological processes. In this review, we summarize previously reported protein-protein and protein-lipid interactions of PLD and PA and their binding partners. In addition, we describe the functional roles played by PLD and PA in these interactions, and provide PLD network that summarizes these interactions. The PLD network suggests that PLD and PA could act as a decision maker and/or as a coordinator of signal dynamics. This viewpoint provides a turning point for understanding the roles of PLD-PA as a dynamic signaling hub.     

Fucose and sialic acid contents of intestinal microvillus membranes from different animal species

Fucose and sialic acid contents of intestinal microvillus membranes isolated from different animal species have been analysed. Expressed on protein basis, brush borders from fish contained considerably high amounts of sialic acid (298 +/- 16 nmole/mg protein), while rat, goat, sheep and guinea pig membranes showed 41-61 nmole/mg protein. Pig, frog, monkey rabbit and chicken membranes exhibited low levels of sialic acid (10-13 nmole/mg protein). Fucose content of the brush borders was quite high (203-212 nmole/mg protein) in frog and fish intestines. It was least in rabbit (54 +/- 3) and of intermediate levels (80-122 nmole/mg protein) in various other animal species analysed. Fucose to sialic acid molar ratio was less than 1 in fish microvillus membranes. In all other animal species, the ratio was however, greater than one and ranged between 1.65 and 15.20.