G protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets; however, progress in identifying new small molecule drugs has been disappointing. The past four years have seen remarkable progress in the structural biology of GPCRs, raising the possibility of applying structure-based approaches to GPCR drug discovery efforts. Of the various structure-based approaches that have been applied to soluble protein targets, such as proteases and kinases, in silico docking is among the most ready applicable to GPCRs. Early studies suggest that GPCR binding pockets are well suited to docking, and docking screens have identified potent and novel compounds for these targets. This review will focus on the current state of in silico docking for GPCRs.
Molecular docking remains an important tool for structure-based screening to find new ligands and chemical probes. As docking ambitions grow to include new scoring function terms, and to address ever more targets, the reliability and extendability of the orientation sampling, and the throughput of the method, become pressing. Here we explore sampling techniques that eliminate stochastic behavior in DOCK3.6, allowing us to optimize the method for regularly variable sampling of orientations. This also enabled a focused effort to optimize the code for efficiency, with a three-fold increase in the speed of the program. This, in turn, facilitated extensive testing of the method on the 102 targets, 22,805 ligands and 1,411,214 decoys of the Directory of Useful Decoys - Enhanced (DUD-E) benchmarking set, at multiple levels of sampling. Encouragingly, we observe that as sampling increases from 50 to 500 to 2000 to 5000 to 20000 molecular orientations in the binding site (and so from about 1×1010 to 4×1010 to 1×1011 to 2×1011 to 5×1011 mean atoms scored per target, since multiple conformations are sampled per orientation), the enrichment of ligands over decoys monotonically increases for most DUD-E targets. Meanwhile, including internal electrostatics in the evaluation ligand conformational energies, and restricting aromatic hydroxyls to low energy rotamers, further improved enrichment values. Several of the strategies used here to improve the efficiency of the code are broadly applicable in the field.
A docking screen identified reversible, non-covalent inhibitors (e.g. 1) of the parasite cysteine protease cruzain. Chemical optimization of 1 led to a series of oxadiazoles possessing interpretable SAR and potencies as much as 500-fold greater than 1. Detailed investigation of the SAR series subsequently revealed that many members of the oxadiazole class (and surprisingly also 1) act via divergent modes of inhibition – competitive or via colloidal aggregation – depending on the assay conditions employed.
Protein classification typically uses structural, sequence, or functional similarity. Here we introduce an orthogonal method that organizes proteins by ligand similarity, focusing here on the class A G protein-coupled receptor (GPCR) protein family. Comparing a ligand-based dendogram to a sequence-based one, we sought examples of GPCRs that were distantly linked by sequence but neighbors by ligand similarity. Experimental testing of compounds predicted to link three of these new pairs confirmed the predicted association, with potencies ranging from the low-nanomolar to low-micromolar. We then identified hundreds of non-GPCRs closely related to GPCRs by ligand similarity, including the CXCR2 chemokine receptor to Casein kinase I, the cannabinoid receptors to epoxide hydrolase 2, and the α2 adrenergic receptor to phospholipase D. These, too, were confirmed experimentally. Ligand similarities among these targets may reflect a chemical integration in the time domain of molecular signaling.
Penicillin-binding protein 6 (PBP6) is one of the two main dd-carboxypeptidases in Escherichia coli, which are implicated in maturation of bacterial cell wall and formation of cell shape. Here, we report the first X-ray crystal structures of PBP6, capturing its apo state (2.1 Å), an acyl-enzyme intermediate with the antibiotic ampicillin (1.8 Å), and for the first time for a PBP, a preacylation complex (a “Michaelis complex”, determined at 1.8 Å) with a peptidoglycan substrate fragment containing the full pentapeptide, NAM-(l-Ala-d-isoGlu-l-Lys-d-Ala-d-Ala). These structures illuminate the molecular interactions essential for ligand recognition and catalysis by dd-carboxypeptidases, and suggest a coupling of conformational flexibility of active site loops to the reaction coordinate. The substrate fragment complex structure, in particular, provides templates for models of cell wall recognition by PBPs, as well as substantiating evidence for the molecular mimicry by β-lactam antibiotics of the peptidoglycan acyl-d-Ala-d-Ala moiety.
Normal cilia length and motility are critical for proper cellular function. Prior studies of the regulation of ciliary structure and length have primarily focused on the intraflagellar transport machinery and motor proteins required for ciliary assembly and disassembly. However, several mutants with abnormal length flagella highlight the importance of signaling proteins as well. In this study, an unbiased chemical screen was performed to uncover signaling pathways that are critical for ciliogenesis and length regulation using flagella of the green alga Chlamydomonas reinhardtii as a model. The annotated Sigma LOPAC1280 chemical library was screened for effects on flagellar length, motility and severing as well as cell viability. Assay data were clustered to identify pathways regulating flagella. The most frequently target found to be involved in flagellar length regulation was the family of dopamine binding G-protein coupled receptors (GPCRs). In mammalian cells, cilium length could indeed be altered with expression of the dopamine D1 receptor. Our screen thus reveals signaling pathways that are potentially critical for ciliary formation, resorption, and length maintenance, which represent candidate targets for therapeutic intervention of disorders involving ciliary malformation and malfunction.
PDE4 is one of eleven known cyclic nucleotide phosphodiesterase families and plays a pivotal role in mediating hydrolytic degradation of the important cyclic nucleotide second messenger, cyclic 3′5′ adenosine monophosphate (cAMP). PDE4 inhibitors are known to have anti-inflammatory properties, but their use in the clinic has been hampered by mechanism-associated side effects that limit maximally tolerated doses. In an attempt to initiate the development of better-tolerated PDE4 inhibitors we have surveyed existing approved drugs for PDE4-inhibitory activity. With this objective, we utilised a high-throughput computational approach that identified moexipril, a well tolerated and safe angiotensin-converting enzyme (ACE) inhibitor, as a PDE4 inhibitor. Experimentally we showed that moexipril and two structurally related analogues acted in the micro molar range to inhibit PDE4 activity. Employing a FRET-based biosensor constructed from the nucleotide binding domain of the type 1 exchange protein activated by cAMP, EPAC1, we demonstrated that moexipril markedly potentiated the ability of forskolin to increase intracellular cAMP levels. Finally, we demonstrated that the PDE4 inhibitory effect of moexipril is functionally able to induce phosphorylation of the small heat shock protein, Hsp20, by cAMP dependent protein kinase A. Our data suggest that moexipril is a bona fide PDE4 inhibitor that may provide the starting point for development of novel PDE4 inhibitors with an improved therapeutic window.
Phosphodiesterase inhibitor; Protein kinase A (PKA), PDE4; Catechol ether; Cyclic 3′5′ adenosine monophosphate (cAMP)
Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from Listeria monocytogenes str. 4b F2365 and Bh0225 from Bacillus halodurans C-125, were cloned, expressed and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 Å with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 Å away from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275 and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high energy intermediate (HEI) form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, d-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The kcat/Km values for the cobalt-substituted enzymes with these substrates are ~105 M−1 s−1.
Applications in structural biology and medicinal chemistry require protein-ligand scoring functions for two distinct tasks: (i) ranking different poses of a small molecule in a protein binding site; and (ii) ranking different small molecules by their complementarity to a protein site. Using probability theory, we developed two atomic distance-dependent statistical scoring functions: PoseScore was optimized for recognizing native binding geometries of ligands from other poses and RankScore was optimized for distinguishing ligands from nonbinding molecules. Both scores are based on a set of 8,885 crystallographic structures of protein-ligand complexes, but differ in the values of three key parameters. Factors influencing the accuracy of scoring were investigated, including the maximal atomic distance and non-native ligand geometries used for scoring, as well as the use of protein models instead of crystallographic structures for training and testing the scoring function. For the test set of 19 targets, RankScore improved the ligand enrichment (logAUC) and early enrichment (EF1) scores computed by DOCK 3.6 for 13 and 14 targets, respectively. In addition, RankScore performed better at rescoring than each of seven other scoring functions tested. Accepting both the crystal structure and decoy geometries with all-atom root-mean-square errors of up to 2 Å from the crystal structure as correct binding poses, PoseScore gave the best score to a correct binding pose among 100 decoys for 88% of all cases in a benchmark set containing 100 protein-ligand complexes. PoseScore accuracy is comparable to that of DrugScoreCSD and ITScore/SE, and superior to 12 other tested scoring functions. Therefore, RankScore can facilitate ligand discovery, by ranking complexes of the target with different small molecules; PoseScore can be used for protein-ligand complex structure prediction, by ranking different conformations of a given protein-ligand pair. The statistical potentials are available through the Integrative Modeling Platform (IMP) software package (http://salilab.org/imp/) and the LigScore web server (http://salilab.org/ligscore/).
statistical potential; reference state; binding pose; ligand enrichment
Discovering the unintended “off-targets” that predict adverse drug reactions (ADRs) is daunting by empirical methods alone. Drugs can act on multiple protein targets, some of which can be unrelated by traditional molecular metrics, and hundreds of proteins have been implicated in side effects. We therefore explored a computational strategy to predict the activity of 656 marketed drugs on 73 unintended “side effect” targets. Approximately half of the predictions were confirmed, either from proprietary databases unknown to the method or by new experimental assays. Affinities for these new off-targets ranged from 1 nM to 30 μM. To explore relevance, we developed an association metric to prioritize those new off-targets that explained side effects better than any known target of a given drug, creating a Drug-Target-ADR network. Among these new associations was the prediction that the abdominal pain side effect of the synthetic estrogen chlorotrianisene was mediated through its newly discovered inhibition of the enzyme COX-1. The clinical relevance of this inhibition was borne-out in whole human blood platelet aggregation assays. This approach may have wide application to de-risking toxicological liabilities in drug discovery.
The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts.
To compare virtual and high-throughput screening in an unbiased way, 50,000 compounds were docked into the 3-dimensional structure of dihydrofolate reductase prospectively, and the results were compared to a subsequent experimental screening of the same library. Undertaking these calculations demanded careful database curation and control calculations with annotated inhibitors. These ultimately led to a ranked list of more likely and less likely inhibitors and to the prediction that relatively few inhibitors would be found in the empirical screen. The latter prediction turned out to be correct, with arguably no validated inhibitors found experimentally. Subsequent retesting of high-scoring docked molecules may have found 2 true inhibitors, although this remains uncertain due to experimental ambiguities. The implications of this study for screening campaigns are considered. (Journal of Biomolecular Screening 2005:667-674)
high-throughput screening; HTS; virtual screening; molecular docking; database preparation
Target identification is a core challenge in chemical genetics. Here we use chemical similarity to predict computationally the targets of 586 compounds active in a zebrafish behavioral assay. Of 20 predictions tested, 11 had activities ranging from 1 to 10,000nM on the predicted targets. The role of two of these targets was tested in the original zebrafish phenotype. Prediction of targets from chemotype is rapid and may be generally applicable.
Ligand enrichment among top-ranking hits is a key metric of molecular docking. To avoid bias, decoys should resemble ligands physically, so that enrichment is not simply a separation of gross features, yet be chemically distinct from them, so that they are unlikely to be binders. We have assembled a directory of useful decoys (DUD), with 2950 ligands for 40 different targets. Every ligand has 36 decoy molecules that are physically similar but topologically distinct, leading to a database of 98,266 compounds. For most targets, enrichment was at least half a log better with uncorrected databases such as the MDDR than with DUD, evidence of bias in the former. These calculations also allowed forty-by-forty cross docking, where the enrichments of each ligand set could be compared for all 40 targets, enabling a specificity metric for the docking screens. DUD is freely available online as a benchmarking set for docking at http://blaster.docking.org/dud/.
virtual screening; molecular docking; docking decoy; automation; enrichment; binding pose
G-Protein coupled receptors (GPCRs) are intensely studied as drug targets and for their role in signaling. With the determination of the first crystal structures, interest in structure-based ligand discovery has increased. Unfortunately, most GPCRs lack experimental structures. The determination of the D3 receptor structure, and a community challenge to predict it, enabled a fully prospective comparison of ligand discovery from a modeled structure versus that of the subsequently released crystal structure. Over 3.3 million molecules were docked against a homology model, and 26 of the highest ranking were tested for binding. Six had affinities from 0.2 to 3.1μM. Subsequently, the crystal structure was released and the docking screen repeated. Of the 25 compounds selected, five had affinities from 0.3 to 3.0μM. One of the novel ligands from the homology model screen was optimized for affinity to 81nM. The feasibility of docking screens against modeled GPCRs more generally is considered.
Two enzymes of unknown function from the amidohydrolase superfamily were discovered to catalyze the deamination of N-6-methyladenine to hypoxanthine and methyl amine. The methylation of adenine in bacterial DNA is a common modification for the protection of host DNA against restriction endonucleases. The enzyme from Bacillus halodurans, Bh0637, catalyzes the deamination of N-6-methyladenine with a kcat of 185 s−1 and a kcat/Km of 2.5 × 106 M−1 s−1. Bh0637 catalyzes the deamination of N-6-methyladenine two orders of magnitude faster than adenine. A comparative model of Bh0637 was computed using the three-dimensional structure of Atu4426 (PDB code: 3NQB) as a structural template and computational docking was used to rationalize the preferential utilization of N-6-methyladenine over adenine. This is the first identification of an N-6-methyladenine deaminase (6-MAD).
Clozapine, by virtue of its absence of extrapyramidal side effects and greater efficacy, revolutionized the treatment of schizophrenia, although the mechanisms underlying this exceptional activity remain controversial. Combining an unbiased cheminformatics and physical screening approach, we evaluated clozapine's activity at >2350 distinct molecular targets. Clozapine, and the closely related atypical antipsychotic drug olanzapine, interacted potently with a unique spectrum of molecular targets. This distinct pattern, which was not shared with the typical antipsychotic drug haloperidol, suggested that the serotonergic neuronal system was a key determinant of clozapine's actions. To test this hypothesis, we used pet1−/− mice, which are deficient in serotonergic presynaptic markers. We discovered that the antipsychotic-like properties of the atypical antipsychotic drugs clozapine and olanzapine were abolished in a pharmacological model that mimics NMDA-receptor hypofunction in pet1−/− mice, whereas haloperidol's efficacy was unaffected. These results show that clozapine's ability to normalize NMDA-receptor hypofunction, which is characteristic of schizophrenia, depends on an intact presynaptic serotonergic neuronal system.
serotonin; schizophrenia; clozapine; pet1; antipsychotic drugs; receptor pharmacology; schizophrenia; antipsychotics; drug discovery; development; molecular & cellular neurobiology; chemical biology
We investigated a series of sulfonamide boronic acids that resulted from the merging of two unrelated AmpC β-lactamase inhibitor series. The new boronic acids differed in the replacement of the canonical carboxamide, found in all penicillin and cephalosporin antibiotics, with a sulfonamide. Surprisingly, these sulfonamides had a highly distinct structure-activity relationship from the previously explored carboxamides, high ligand efficiencies (up to 0.91), Ki values down to 25 nM and up to 23 times better for smaller analogs. Conversely, Ki values were 10 to 20 times worse for larger molecules than in the carboxamide congener series. X-ray crystal structures (1.6–1.8 Å) of AmpC with three of the new sulfonamides suggest that this altered structure-activity relationship results from the different geometry and polarity of the sulfonamide versus the carboxamide. The most potent inhibitor reversed β-lactamase-mediated resistance to third generation cephalosporins, lowering their minimum inhibitory concentrations up to 32-fold in cell culture.
AmpC; structure-based inhibitor design; structure-based drug discovery; β-lactamase inhibition; X-ray crystallography; antimicrobial; antibiotic resistance
A small set of boronic acids acting as low nanomolar inhibitors of AmpC β-lactamase were designed and synthesized in the effort to improve affinity, pharmacokinetic properties, and to provide a valid lead compound. X-ray crystallography revealed the binary complex of the best inhibitor bound to the enzyme, highlighting possibilities for its further rational derivatization and chemical optimization.
Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed and purified to homogeneity. The enzyme crystallized in the space group P3121 and the structure was determined to a resolution of 2.1 Å. The protein folds as a (β/α)7β-barrel and a binuclear metal center is found at the C-terminal end of the β-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the β-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of δ- and γ-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was δ-nonanoic lactone with a kcat/Km of 1.6 × 106 M−1 s−1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of kcat and kcat/Km of 0.07 min−1 and 0.8 M−1 s−1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is ~30%. The eight substrate specificity loops were transplanted from PTE to Dr0930 but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a kcat of 1.14 min−1, an increase of 16-fold over the wild type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.
The Similarity Ensemble Approach (SEAa) relates proteins based on the set-wise chemical similarity among their ligands. It can be used to rapidly search large compound databases and to build cross-target similarity maps. The emerging maps relate targets in ways that reveal relationships one might not recognize based on sequence or structural similarities alone. SEA has previously revealed cross talk between drugs acting primarily on G-protein coupled receptors (GPCRs). Here we used SEA to look for potential off-target inhibition of the enzyme protein farnesyltransferase (PFTase) by commercially available drugs. The inhibition of PFTase has profound consequences for oncogenesis, as well as a number of other diseases. In the present study, two commercial drugs, Loratadine and Miconazole, were identified as potential ligands for PFTase and subsequently confirmed as such experimentally. These results point towards the applicability of SEA for the prediction of not only GPCR-GPCR drug cross talk, but also GPCR-enzyme and enzyme-enzyme drug cross talk.
An enzyme from Pseudomonas aeruginosa, Pa0142 (gi|9945972) has been identified for the first time that is able to catalyze the deamination of 8-oxoguanine (8-oxoG) to uric acid. 8-Oxoguanine is formed by the oxidation of guanine residues within DNA by reactive oxygen species and this lesion results in the G:C to T:A transversions. The value of kcat/Km for the deamination of 8-oxoG by Pa0142 at pH 8.0 and 30 °C is 2.0 × 104 M−1 s−1. This enzyme can also catalyze the deamination of isocystosine and guanine at rates that are approximately an order of magnitude slower. The three-dimensional structure of a homologous enzyme (gi|44264246) from the Sargasso Sea has been determined by x-ray diffraction methods to a resolution of 2.2Å (PDB code: 3h4u). The enzyme folds as a (β/α)8− barrel and it is a member of the amidohydrolase superfamily with a single zinc in the active site. This enzyme catalyzes the deamination of 8-oxoG with a value of kcat/Km of 2.7 × 105 M−1 s−1. Computational docking of potential high energy intermediates for the deamination reaction to the x-ray crystal structure suggests that the active site binding of 8-oxoG is facilitated by hydrogen bond interactions from a conserved glutamine that follows β-strand 1 with O6, a conserved tyrosine that follows β-strand 2 with N7, and a conserved cysteine residue that follows β-strand 4 with O8. A bioinformatic analysis of available protein sequences suggest that approximately 200 other bacteria possess an enzyme capable of catalyzing the deamination of 8-oxoG.
Pre-organization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein, and consequently enzymes can be dramatically stabilized by substitutions that attenuate the size and pre-organization “strain” of the active site. How this stability-activity trade-off constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities, or how these new activities manifest structurally. These questions are both germane and easy to study in β-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of β-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C β-lactamases which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100- to 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability – from 1.7 to 4.1 kcal/mol – consistent with the function-stability hypothesis. Intriguingly, several of the substitutions were 10 – 20 Å from the catalytic serine; the question arose how they conferred extended-spectrum activity. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by x-ray crystallography. Distinct mechanisms of action are revealed for each mutant, including changes in the flexibility and ground state structures of the enzyme. These results explain the structural bases for the antibiotic resistance conferred by these substitutions, and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.
protein stability; AmpC beta-lactamase; antibiotic resistance; evolution; action-at-a-distance
Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of β-lactams, is similar in sequence to a cluster of ~400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of L-Xaa-L-Xaa, L-Xaa-D-Xaa and D-Xaa-L-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an L-amino acid at the N-terminus and a D-amino acid at the C-terminus. The best substrate identified was L-Arg-D-Asp (kcat/Km = 7.6 × 105 M−1 s−1). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of L-Ala-D-Asp. The enzyme folds as a (β/α)8-barrel and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223 and Asp-320). Solvent viscosity and kinetic effects by D2O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for kcat and kcat/Km indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of L/D-Ala-L/D-Ala to the three dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.
A model binding site was used to investigate charge–charge interactions in molecular docking. This simple site, a small (180 Å3) engineered cavity in cyctochrome c peroxidase (CCP), is negatively charged and completely buried from solvent, allowing us to explore the balance between electrostatic energy and ligand desolvation energy in a system where many of the common approximations in docking do not apply. A database with about 5300 molecules was docked into this cavity. Retrospective testing with known ligands and decoys showed that overall the balance between electrostatic interaction and desolvation energy was captured. More interesting were prospective docking scre”ens that looked for novel ligands, especially those that might reveal problems with the docking and energy methods. Based on screens of the 5300 compound database, both high-scoring and low-scoring molecules were acquired and tested for binding. Out of 16 new, high-scoring compounds tested, 15 were observed to bind. All of these were small heterocyclic cations. Binding constants were measured for a few of these, they ranged between 20 μM and 60 μM. Crystal structures were determined for ten of these ligands in complex with the protein. The observed ligand geometry corresponded closely to that predicted by docking. Several low-scoring alkyl amino cations were also tested and found to bind. The low docking score of these molecules owed to the relatively high charge density of the charged amino group and the corresponding high desolvation penalty. When the complex structures of those ligands were determined, a bound water molecule was observed interacting with the amino group and a backbone carbonyl group of the cavity. This water molecule mitigates the desolvation penalty and improves the interaction energy relative to that of the “naked” site used in the docking screen. Finally, six low-scoring neutral molecules were also tested, with a view to looking for false negative predictions. Whereas most of these did not bind, two did (phenol and 3-fluorocatechol). Crystal structures for these two ligands in complex with the cavity site suggest reasons for their binding. That these neutral molecules do, in fact bind, contradicts previous results in this site and, along with the alkyl amines, provides instructive false negatives that help identify weaknesses in our scoring functions. Several improvements of these are considered.
molecular docking; electrostatic; solvation; cyctochrome c peroxidase; X-ray crystallography