Crystal structure of mouse CD1d bound to the self ligand phosphatidylcholine: a molecular basis for NKT cell activation

NKT cells are immunoregulatory lymphocytes whose activation is triggered by the recognition of lipid Ags in the context of the CD1d molecules by the TCR. In this study we present the crystal structure to 2.8 A of mouse CD1d bound to phosphatidylcholine. The interactions between the ligand acyl chains and the CD1d molecule define the structural and chemical requirements for the binding of lipid Ags to CD1d. The orientation of the polar headgroup toward the C terminus of the alpha1 helix provides a rationale for the structural basis for the observed Valpha chain bias in invariant NKT cells. The contribution of the ligand to the protein surface suggests a likely mode of recognition of lipid Ags by the NKT cell TCR.     

Structural basis for ligand recognition and discrimination of a quorum-quenching antibody

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using “quorum sensing,” which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key Glu^H95 provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11·AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.     

Structural basis for ligand recognition and activation of RAGE

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor involved in?inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling events upon binding of a variety of ligands, such as glycated proteins, amyloid-β, HMGB1, and S100 proteins. The X-ray crystal structure of the VC1 ligand-binding region of the human RAGE ectodomain was determined at 1.85?? resolution. The VC1 ligand-binding surface was mapped onto the structure from titrations with S100B monitored by heteronuclear NMR spectroscopy. These NMR chemical shift perturbations were used as input for restrained docking calculations to generate a model for the VC1-S100B complex. Together, the arrangement of VC1 molecules in the crystal and complementary biochemical studies suggest a role for self-association in RAGE function. Our results enhance understanding of the functional outcomes of S100 protein binding to RAGE and provide insight into mechanistic models for how the receptor is activated.     

Targeting effectors: the molecular recognition of Type III secreted proteins

The Type III secretion system (TTSS) facilitates the export of effector proteins from pathogenic and symbiotic Gram-negative bacteria into the cytosol of eukaryotic host cells. The current functional and evolutionary knowledge on the molecular recognition of TTSS substrates and computational models of the secretion signal are discussed in this review.     

Structural basis for ligand recognition in a mushroom lectin: solvent structure as specificity predictor

Lectins are able to recognize specific carbohydrate structures through their carbohydrate recognition domain (CRD). The lectin from the mushroom Agaricus bisporus (ABL) has the remarkable ability of selectively recognizing the TF-antigen, composed of Galβ1-3GalNAc, Ser/Thr linked to proteins, specifically exposed in neoplastic tissues. Strikingly, the recently solved crystal structure of tetrameric ABL in the presence of TF-antigen and other carbohydrates showed that each monomer has two CRDs, each being able to bind specifically to different monosaccharides that differ only in the configuration of a single hydroxyl, like N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc). Understanding how lectin CRDs bind and discriminate mono and/or (poly)-saccharides is an important issue in glycobiology, with potential impact in the design of better and selective lectin inhibitors with potential therapeutic properties. In this work, and based on the unusual monosaccharide epimeric specificity of the ABL CRDs, we have performed molecular dynamics simulations of the natural (crystallographic) and inverted (changing GalNAc for GlcNAc and vice-versa) ABL–monosaccharide complexes in order to understand the selective ligand recognition properties of each CRD. We also performed a detailed analysis of the CRD local solvent structure, using previously developed methodology, and related it with the recognition mechanism. Our results provide a detailed picture of each ABL CRD specificity, allowing a better understanding of the carbohydrate selective recognition process in this particular lectin.     

The binding of synthetic triiodo l-thyronine analogs to human transthyretin: molecular basis of cooperative and non-cooperative ligand recognition

Transthyretin (TTR) is a tetrameric β-sheet-rich transporter protein directly involved in human amyloid diseases. Several classes of small molecules can bind to TTR delaying its amyloid fibril formation, thus being promising drug candidates to treat TTR amyloidoses. In the present study, we characterized the interactions of the synthetic triiodo L-thyronine analogs and thyroid hormone nuclear receptor TRβ-selective agonists GC-1 and GC-24 with the wild type and V30M variant of human transthyretin (TTR). To achieve this aim, we conducted in vitro TTR acid-mediated aggregation and isothermal titration calorimetry experiments and determined the TTR:GC-1 and TTR:GC-24 crystal structures. Our data indicate that both GC-1 and GC-24 bind to TTR in a non-cooperative manner and are good inhibitors of TTR aggregation, with dissociation constants for both hormone binding sites (HBS) in the low micromolar range. Analysis of the crystal structures of TTRwt:GC-1(24) complexes and their comparison with the TTRwt X-ray structure bound to its natural ligand thyroxine (T4) suggests, at the molecular level, the basis for the cooperative process displayed by T4 and the non-cooperative process provoked by both GC-1 and GC-24 during binding to TTR.     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.     

Structural basis for molecular recognition in an affibody:affibody complex

Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K(d) approximately 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large ( approximately 1670 A(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine Halpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium N(eta)H(2) groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.     

Computational inference of the molecular logic for synaptic connectivity in C. elegans

MOTIVATION: The nematode C. elegans is an ideal model organism in which to investigate the biomolecular mechanisms underlying the connectivity of neurons, because synaptic connections are described in a comprehensive wiring diagram and methods for defining gene expression profiles of individual neurons are now available. RESULTS: Here we present computational techniques linking these two types of information. A systems-based approach (EMBP: Entropy Minimization and Boolean Parsimony) identifies sets of synergistically interacting genes whose joint expression predicts neural connectivity. We introduce an information theoretic measure of the multivariate synergy, a fundamental concept in systems biology, connecting the members of these gene sets. We present and validate our preliminary results based on publicly available information, and demonstrate that their synergy is exceptionally high indicating joint involvement in pathways. Our strategy provides a robust methodology that will yield increasingly more accurate results as more neuron-specific gene expression data emerge. Ultimately, we expect our approach to provide important clues for universal mechanisms of neural interconnectivity.     

The sense of smell: molecular basis of odorant recognition

Most animal species rely on odorant compounds to locate food, predators, or toxins. The sense of smell is also involved in animal communication, and revealing the underlying mechanisms will therefore facilitate a deeper understanding of animal behaviour. Since the 1940s different theories have speculated on the fundamental basis of olfaction. It was assumed that odorant molecules were recognized by selective protein receptors in the nose, triggering a nervous signal processed by the brain. The discovery of these receptors in the early 1990s allowed great progress in understanding the physiological and biochemical principles of olfaction. An overview of the different mechanisms involved in the coding of odour character as well as odour intensity is presented here, focusing on the biochemical basis of odorant recognition. Despite the enormous progress achieved in recent years, details of odorant-receptor interaction at the molecular level and the mechanisms of olfactory receptor activation are poorly understood. The likely role of metal ions in odorant recognition is discussed, and also the perireceptor events involved in odorant transport and biotransformation, with a view to providing a comprehensive overview of mammalian olfaction to guide future computational structural models and the design of functional experiments. Recent studies have analysed the olfactory genome of several species, providing information about the evolution of olfaction. The role of the olfactory system in animal communication is also described.     

The molecular basis for ligand specificity in a mouse olfactory receptor: a network of functionally important residues

Sequence differences between members of the mouse olfac-tory receptor MOR42 subfamily (MOR42-3 and MOR42-1) are likely to be the basis for variation in ligand binding preference among these receptors. We investigated the specificity of MOR42-3 for a variety of dicarboxylic acids. We used site-directed mutagenesis, guided by homology modeling and ligand docking studies, to locate functionally important residues. Receptors were expressed in Xenopus oocytes and assayed using high throughput electrophysiology. The importance of the Val-113 residue, located deep within the receptor, was analyzed in the context of interhelical interactions. We also screened additional residues predicted to be involved in ligand binding site, based on comparison of ortholog/paralog pairs from the mouse and human olfactory receptor genomes (Man, O., Gilad, Y., and Lancet, D. (2004) Protein Sci. 13, 240-254). A network of 8 residues in transmembrane domains III, V, and VI was identified. These residues form part of the ligand binding pocket of MOR42-3. C12 dicarboxylic acid did not activate the receptor in our functional assay, yet our docking simulations predicted its binding site in MOR42-3. Binding without activation implied that C12 dicarboxylic acid might act as an antagonist. In our functional assay, C12 dicarboxylic acid did indeed act as an antagonist of MOR42-3, in agreement with molecular docking studies. Our results demonstrate a powerful approach based on the synergy between computational predictions and physiological assays.     

The molecular basis of CaMKII function in synaptic and behavioural memory

Long-term potentiation (LTP) in the CA1 region of the hippocampus has been the primary model by which to study the cellular and molecular basis of memory. Calcium/calmodulin-dependent protein kinase II (CaMKII) is necessary for LTP induction, is persistently activated by stimuli that elicit LTP, and can, by itself, enhance the efficacy of synaptic transmission. The analysis of CaMKII autophosphorylation and dephosphorylation indicates that this kinase could serve as a molecular switch that is capable of long-term memory storage. Consistent with such a role, mutations that prevent persistent activation of CaMKII block LTP, experience-dependent plasticity and behavioural memory. These results make CaMKII a leading candidate in the search for the molecular basis of memory.     

A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.

The mammalian olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants presumably results from the association of odorous ligands with specific receptors on olfactory sensory neurons. To address the problem of olfactory perception at a molecular level, we have cloned and characterized 18 different members of an extremely large multigene family that encodes seven transmembrane domain proteins whose expression is restricted to the olfactory epithelium. The members of this novel gene family are likely to encode a diverse family of odorant receptors.     

Molecular basis of ligand recognition by OASS from E. histolytica: Insights from structural and molecular dynamics simulation studies

Background

O-acetyl serine sulfhydrylase (OASS) is a pyridoxal phosphate (PLP) dependent enzyme catalyzing the last step of the cysteine biosynthetic pathway. Here we analyze and investigate the factors responsible for recognition and different conformational changes accompanying the binding of various ligands to OASS.

Methods

X ray crystallography was used to determine the structures of OASS from Entamoeba histolytica in complex with methionine (substrate analog), isoleucine (inhibitor) and an inhibitory tetra-peptide to 2.00 Å, 2.03 Å and 1.87 Å resolutions, respectively. Molecular dynamics simulations were used to investigate the reasons responsible for the extent of domain movement and cleft closure of the enzyme in presence of different ligands.

Results

Here we report for the first time an OASS-methionine structure with an unmutated catalytic lysine at the active site. This is also the first OASS structure with a closed active site lacking external aldimine formation. The OASS-isoleucine structure shows the active site cleft in open state. Molecular dynamics studies indicate that cofactor PLP, N88 and G192 form a triad of energy contributors to close the active site upon ligand binding and orientation of the Schiff base forming nitrogen of the ligand is critical for this interaction.

Conclusions

Methionine proves to be a better binder to OASS than isoleucine. The β branching of isoleucine does not allow it to reorient itself in suitable conformation near PLP to cause active site closure.

General significance

Our findings have important implications in designing better inhibitors against OASS across all pathogenic microbial species.     

The 1.3 A crystal structure of a biotin-binding pseudoknot and the basis for RNA molecular recognition

A pseudoknot-containing aptamer isolated from a pool of random sequence molecules has been shown previously to represent an optimal RNA solution to the problem of binding biotin. The affinity of this RNA molecule is nonetheless orders of magnitude weaker than that of its highly evolved protein analogs, avidin and streptavidin. To understand the structural basis for biotin binding and to compare directly strategies for ligand recognition available to proteins and RNA molecules, we have determined the 1.3 A crystal structure of the aptamer complexed with its ligand. Biotin is bound at the interface between the pseudoknot's stacked helices in a pocket defined almost entirely by base-paired nucleotides. In comparison to the protein avidin, the aptamer packs more tightly around the biotin headgroup and makes fewer contacts with its fatty acid tail. Whereas biotin is deeply buried within the hydrophobic core in the avidin complex, the aptamer relies on a combination of hydrated magnesium ions and immobilized water molecules to surround its ligand. In addition to demonstrating fundamentally different approaches to molecular recognition by proteins and RNA, the structure provides general insight into the mechanisms by which RNA function is mediated by divalent metals.     

A radioassay for synaptic core complex assembly: screening of herbal extracts for effectors

Synaptic core complex formation between syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) on the plasma membrane and synaptobrevin on the vesicle membrane is responsible for membrane fusion and neurotransmitter release. A radiolabeled protein binding assay for synaptic core complex formation was developed. The components of this assay included recombinant glutathione S-transferase (GST)-syntaxin immobilized on glutathione agarose beads, SNAP25 and (125)I-labeled synaptobrevin. Reactions were performed in tubes containing filter inserts to facilitate removal of unbound protein. The radiolabeled protein bound was then quantified by gamma counter. A K(d) of 1.6 microM was determined for the GST-syntaxin/SNAP25/synaptobrevin complex, and a K(d) of 12 microM was determined for the GST-syntaxin/synaptobrevin complex. The assay was used to screen 14 herbal extracts for effectors of core complex formation. Herbs traditionally used to treat neurological conditions such as depression, anxiety, and stress were chosen. A Hypericum perforatum extract was found to have a nonspecific effect via protein complexation, whereas an Albizzia julibrissin extract was found to reduce the level of core complex formation. The assay was used to further investigate the effect of the A. julibrissin extract. The discovery of an inhibitor of core complex formation demonstrates the efficacy of the assay in screening natural products for substances that affect core complex formation.