Explore the vast range of titles available.

Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections. We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group. The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection.

Transcription factors are central components of the intracellular regulatory networks that control gene expression. An increasingly recognized phenomenon among human transcription factors is the formation of structure upon target binding. Here, we study the folding and binding of the pKID domain of CREB to the KIX domain of the co-activator CBP. Our simulations of a topology-based Gō-type model predict a coupled folding and binding mechanism, and the existence of partially bound intermediates. From transition-path and Phi-value analyses, we find that the binding transition state resembles the unstructured state in solution, implying that CREB becomes structured only after committing to binding. A change of structure following binding is reminiscent of an induced-fit mechanism and contrasts with models in which binding occurs to pre-structured conformations that exist in the unbound state at equilibrium. Interestingly, increasing the amount of structure in the unbound pKID reduces the rate of binding, suggesting a \"fly-casting\"-like process. We find that the inclusion of attractive non-native interactions results in the formation of non-specific encounter complexes that enhance the on-rate of binding, but do not significantly change the binding mechanism. Our study helps explain how being unstructured can confer an advantage in protein target recognition. The simulations are in general agreement with the results of a recently reported nuclear magnetic resonance study, and aid in the interpretation of the experimental binding kinetics.

The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) gamma-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis. We report here a high-resolution crystal structure of the ClfA ligand binding segment in complex with a synthetic peptide mimicking the binding site in Fg. The residues in Fg required for binding to ClfA are identified from this structure and from complementing biochemical studies. Furthermore, the platelet integrin alpha(IIb)beta(3) and ClfA bind to the same segment in the Fg gamma-chain but the two cellular binding proteins recognize different residues in the common targeted Fg segment. Based on these differences, we have identified peptides that selectively antagonize the ClfA-Fg interaction. The ClfA-Fg binding mechanism is a variant of the \"Dock, Lock and Latch\" mechanism previously described for the Staphylococcus epidermidis SdrG-Fg interaction. The structural insights gained from analyzing the ClfANFg peptide complex and identifications of peptides that selectively recognize ClfA but not alpha(IIb)beta(3) may allow the design of novel anti-staphylococcal agents. Our results also suggest that different MSCRAMMs with similar structural organization may have originated from a common ancestor but have evolved to accommodate specific ligand structures.

Background: Human serum albumin (HSA) is the most abundant protein in blood plasma, having high affinity binding sites for several endogenous and exogenous compounds. Trimethoxy flavone (TMF) is a naturally occurring flavone isolated from Andrographis viscosula and used in the treatment of dyspepsia, influenza, malaria, respiratory functions and as an astringent and antidote for poisonous stings of some insects. Methodology/Principal Findings: The main aim of the experiment was to examine the interaction between TMF and HSA at physiological conditions. Upon addition of TMF to HSA, the fluorescence emission was quenched and the binding constant of TMF with HSA was found to be K(TMF) = 1.0±0.01×103 M−1, which corresponds to −5.4 kcal M−1 of free energy. Micro-TOF Q mass spectrometry results showed a mass increase of from 66,513 Da (free HSA) to 66,823 Da (HAS +Drug), indicating the strong binding of TMF with HSA resulting in decrease of fluorescence. The HSA conformation was altered upon binding of TMF to HSA with decrease in α-helix and an increase in β-sheets and random coils suggesting partial unfolding of protein secondary structure. Molecular docking experiments found that TMF binds strongly with HSA at IIIA domain of hydrophobic pocket with hydrogen bond and hydrophobic interactions. Among which two hydrogen bonds are formed between O (19) of TMF to Arg 410, Tyr 411 and another one from O (7) of TMF to Asn 391, with bond distance of 2.1 Å, 3.6 Å and 2.6 Å, respectively. Conclusions/Significance: In view of the evidence presented, it is imperative to assign a greater role of HSA's as a carrier molecule for many drugs to understand the interactions of HSA with TMF will be pivotal in the design of new TMF-inspired drugs.

Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be.

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb(3) in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb(3) clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb(3) lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb(3) (65:30:5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb(3) (40:35:20:5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb(3), STxB-Gb(3) complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.

The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules) would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock approximately 11,000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors.

Ligand virtual screening is a widely used tool to assist in new pharmaceutical discovery. In practice, virtual screening approaches have a number of limitations, and the development of new methodologies is required. Previously, we showed that remotely related proteins identified by threading often share a common binding site occupied by chemically similar ligands. Here, we demonstrate that across an evolutionarily related, but distant family of proteins, the ligands that bind to the common binding site contain a set of strongly conserved anchor functional groups as well as a variable region that accounts for their binding specificity. Furthermore, the sequence and structure conservation of residues contacting the anchor functional groups is significantly higher than those contacting ligand variable regions. Exploiting these insights, we developed FINDSITELHM that employs structural information extracted from weakly related proteins to perform rapid ligand docking by homology modeling. In large scale benchmarking, using the predicted anchor-binding mode and the crystal structure of the receptor, FINDSITELHM outperforms classical docking approaches with an average ligand RMSD from native of ∼2.5 Å. For weakly homologous receptor protein models, using FINDSITELHM, the fraction of recovered binding residues and specific contacts is 0.66 (0.55) and 0.49 (0.38) for highly confident (all) targets, respectively. Finally, in virtual screening for HIV-1 protease inhibitors, using similarity to the ligand anchor region yields significantly improved enrichment factors. Thus, the rather accurate, computationally inexpensive FINDSITELHM algorithm should be a useful approach to assist in the discovery of novel biopharmaceuticals. As an integral part of drug development, high-throughput virtual screening is a widely used tool that could in principle significantly reduce the cost and time to discovery of new pharmaceuticals. In practice, virtual screening algorithms suffer from a number of limitations. The high sensitivity of all-atom ligand docking approaches to the quality of the target receptor structure restricts the selection of drug targets to those for which high-quality X-ray structures are available. Furthermore, the predicted binding affinity is typically strongly correlated with the molecular weight of the ligand, independent of whether or not it really binds. To address these significant problems, we developed FINDSITELHM, a novel threading-based approach that employs structural information extracted from weakly related proteins to perform rapid ligand docking and ranking that is very much in the spirit of homology modeling of protein structures. Particularly for low-quality modeled receptor structures, FINDSITELHM outperforms classical all-atom ligand docking approaches in terms of the accuracy of ligand binding pose prediction and requires considerably less CPU time. As an attractive alternative to classical molecular docking, FINDSITELHM offers the possibility of rapid structure-based virtual screening at the proteome level to improve and speed up the discovery of new biopharmaceuticals.

The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GR-DNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior.