A new method for ligand docking to flexible receptors by dual alanine scanning and refinement (SCARE).
ABSTRACT: Protein binding sites undergo ligand specific conformational changes upon ligand binding. However, most docking protocols rely on a fixed conformation of the receptor, or on the prior knowledge of multiple conformations representing the variation of the pocket, or on a known bounding box for the ligand. Here we described a general induced fit docking protocol that requires only one initial pocket conformation and identifies most of the correct ligand positions as the lowest score. We expanded a previously used diverse "cross-docking" benchmark to thirty ligand-protein pairs extracted from different crystal structures. The algorithm systematically scans pairs of neighbouring side chains, replaces them by alanines, and docks the ligand to each 'gapped' version of the pocket. All docked positions are scored, refined with original side chains and flexible backbone and re-scored. In the optimal version of the protocol pairs of residues were replaced by alanines and only one best scoring conformation was selected from each 'gapped' pocket for refinement. The optimal SCARE (SCan Alanines and REfine) protocol identifies a near native conformation (under 2 angstroms RMSD) as the lowest rank for 80% of pairs if the docking bounding box is defined by the predicted pocket envelope, and for as many as 90% of the pairs if the bounding box is derived from the known answer with approximately 5 angstroms margin as used in most previous publications. The presented fully automated algorithm takes about 2 h per pose of a single processor time, requires only one pocket structure and no prior knowledge about the binding site location. Furthermore, the results for conformationally conserved pockets do not deteriorate due to substantial increase of the pocket variability.
Project description:Modulating protein interaction pathways may lead to the cure of many diseases. Known protein-protein inhibitors bind to large pockets on the protein-protein interface. Such large pockets are detected also in the protein-protein complexes without known inhibitors, making such complexes potentially druggable. The inhibitor-binding site is primary defined by the side chains that form the largest pocket in the protein-bound conformation. Low-resolution ligand docking shows that the success rate for the protein-bound conformation is close to the one for the ligand-bound conformation, and significantly higher than for the apo conformation. The conformational change on the protein interface upon binding to the other protein results in a pocket employed by the ligand when it binds to that interface. This proof-of-concept study suggests that rather than using computational pocket-opening procedures, one can opt for an experimentally determined structure of the target co-crystallized protein-protein complex as a starting point for drug design.
Project description:Computational methods for docking ligands have been shown to be remarkably dependent on precise protein conformation, where acceptable results in pose prediction have been generally possible only in the artificial case of re-docking a ligand into a protein binding site whose conformation was determined in the presence of the same ligand (the "cognate" docking problem). In such cases, on well curated protein/ligand complexes, accurate dockings can be returned as top-scoring over 75% of the time using tools such as Surflex-Dock. A critical application of docking in modeling for lead optimization requires accurate pose prediction for novel ligands, ranging from simple synthetic analogs to very different molecular scaffolds. Typical results for widely used programs in the "cross-docking case" (making use of a single fixed protein conformation) have rates closer to 20% success. By making use of protein conformations from multiple complexes, Surflex-Dock yields an average success rate of 61% across eight pharmaceutically relevant targets. Following docking, protein pocket adaptation and rescoring identifies single pose families that are correct an average of 67% of the time. Consideration of the best of two pose families (from alternate scoring regimes) yields a 75% mean success rate.
Project description:The publication of the crystal structure of the beta2-adrenergic receptor (beta2-AR) proved that G protein-coupled receptors (GPCRs) share a structurally conserved rhodopsin-like 7TM core. Here, to probe to which extent realistic GPCR structures can be recreated through modeling, carazolol was docked at two rhodopsin-based homology models of the human beta 2-AR. The first featured a rhodopsin-like second extracellular loop, which interfered with ligand docking and with the orientation of several residues in the binding pocket. The second featured a second extracellular loop built completely de novo, which afforded a more accurate model of the binding pocket and a better docking of the ligand. Furthermore, incorporating available biochemical and computational data to the model by correcting the conformation of a single residue lining the binding pocket --Phe290(6.52)--, resulted in significantly improved docking poses. These results support the applicability of GPCR modeling to the design of site-directed mutagenesis experiments and to drug discovery.
Project description:Phytoestrogens are plant compounds with estrogenic effects found in many foods. We have previously reported phytoestrogen activity of blackcurrant anthocyanins (cyanidin-3-glucoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, and delphinidin-3-rutinoside) via the estrogen receptor (ER)α. In this study, we investigated the participation of ERβ in the phytoestrogen activity of these anthocyanins. Blackcurrant anthocyanin induced ERβ-mediated transcriptional activity, and the IC50 of ERβ was lower than that of ERα, indicating that blackcurrant anthocyanins have a higher binding affinity to ERβ. In silico docking analysis of cyanidin and delphinidin, the core portions of the compound that fits within the ligand-binding pocket of ERβ, showed that similarly to 17β-estradiol, hydrogen bonds formed with the ERβ residues Glu305, Arg346, and His475. No fitting placement of glucoside or rutinoside sugar chains within the ligand-binding pocket of ERβ-estradiol complex was detected. However, as the conformation of helices 3 and 12 in ERβ varies depending on the ligand, we suggest that the surrounding structure, including these helices, adopts a conformation capable of accommodating glucoside or rutinoside. Comparison of ERα and ERβ docking structures revealed that the selectivity for ERβ is higher than that for ERα, similar to genistein. These results show that blackcurrant anthocyanins exert phytoestrogen activity via ERβ.
Project description:Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8 angstroms resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.
Project description:Lipoxygenases (LOX) play pivotal roles in the biosynthesis of leukotrienes and other biologically active potent signalling compounds. Developing inhibitors for LOX is of high interest to researchers. Modelling the interactions between LOX and its substrate arachidonic acid is critical for developing LOX specific inhibitors. Currently, there are no LOX-substrate structures. Recently, the structure of a coral LOX, 8R-LOX, which is 41% sequence identical to the human 5-LOX was solved to 1.85Å resolution. This structure provides a foundation for modelling enzyme-substrate interactions.In this research, we applied a computational method, Internal Coordinate Mechanics (ICM), to model the interactions between 8R-LOX and its substrate arachidonic acid. Docking arachidonic acid to 8R-LOX was performed. The most favoured docked ligand conformations were retained. We compared the results of our simulation with a proposed model and concluded that the binding pocket identified in this study agrees with the proposed model partially.The results showed that the conformation of arachidonic acid docked into the ICM-identified docking site has less energy than that docked into the manually defined docking site for pseudo wild type 8R-LOX. The mutation at I805 resulted in no docking pocket found near Fe atom. The energy of the arachidonic acid conformation docked into the manually defined docking site is higher in mutant 8R-LOX than in wild type 8R-LOX. The arachidonic acid conformations are not productive conformations.We concluded that, for the wild type 8R-LOX, the conformation of arachidonic acid docked into the ICM-identified docking site is more stable than that docked into the manually defined docking site. Mutation affects the structure of the putative active site pocket of 8R-LOX, and leads no docking pockets around the catalytic Fe atom. The docking simulation in a mutant 8R-LOX demonstrated that the structural change due to the mutation impacts the enzyme activity. Further research and analysis is required to obtain the 8R-LOX-substrate model.
Project description:Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket properties for ligand binding.
Project description:Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA polymerase (pol) beta, a family X polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol beta insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol beta bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.
Project description:Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.
Project description:The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box. Mutations that weaken the TLX-Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression.