Impact of protein and ligand impurities on ITC-derived protein–ligand thermodynamics

Background

The thermodynamic characterization of protein–ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations.

Methods

Ligands synthesized to deviating purity and protein of different pureness were titrated by ITC. Data curation was attempted also considering information from analytical techniques to correct stoichiometry.

Results and conclusions

We used trypsin and tRNA-guanine transglycosylase (TGT), together with high affinity ligands to investigate the effect of errors in protein concentration as well as the impact of ligand impurities on the apparent thermodynamics. We found that errors in protein concentration did not change the thermodynamic properties obtained significantly. However, most ligand impurities led to pronounced changes in binding enthalpy. If protein binding of the respective impurity is not expected, the actual ligand concentration was corrected for and the thus revised data compared to thermodynamic properties obtained with the respective pure ligand. Even in these cases, we observed differences in binding enthalpy of about 4 kJ ⋅ mol^− 1, which is considered significant.

General significance

Our results indicate that ligand purity is the critical parameter to monitor if accurate thermodynamic data of a protein–ligand complex are to be recorded. Furthermore, artificially changing fitting parameters to obtain a sound interaction stoichiometry in the presence of uncharacterized ligand impurities may lead to thermodynamic parameters significantly deviating from the accurate thermodynamic signature.     

Evaluation of small ligand–protein interaction by ligation reaction with DNA-modified ligand

A method for the evaluation of interactions between protein and ligand using DNA-modified ligands, including signal enhancement of the DNA ligation reactions, is described. For proof of principle, a DNA probe modified by biotin was used. Two DNA probes were prepared with complementary sticky-ends. While one DNA probe was modified at the 5′-end of the sticky-end, the other was not modified. The probes could be ligated together by T4 DNA ligase along the strand without biotin modification. However, in the presence of streptavidin or anti-biotin Fab, the ligation reaction joining the two probes could not occur on either strand.     

Receptor–ligand molecular docking

Docking methodology aims to predict the experimental binding modes and affinities of small molecules within the binding site of particular receptor targets and is currently used as a standard computational tool in drug design for lead compound optimisation and in virtual screening studies to find novel biologically active molecules. The basic tools of a docking methodology include a search algorithm and an energy scoring function for generating and evaluating ligand poses. In this review, we present the search algorithms and scoring functions most commonly used in current molecular docking methods that focus on protein–ligand applications. We summarise the main topics and recent computational and methodological advances in protein–ligand docking. Protein flexibility, multiple ligand binding modes and the free-energy landscape profile for binding affinity prediction are important and interconnected challenges to be overcome by further methodological developments in the docking field.     

Variation of protein binding cavity volume and ligand volume in protein–ligand complexes

We have systematically analyzed the variation of protein binding cavity volume of 200 protein–ligand complexes belonging to eight protein families. Wide variation in protein binding cavity volume for the same protein is observed on binding different ligands. Analysis of individual protein families shows high correlation between atom–atom interactions in binding site and ligand volume. This study implies the significance of protein flexibility in docking small molecule inhibitors on the basis of protein binding cavity volume with respect to ligand volume.     

Do mammalian cytochrome P450s show multiple ligand access pathways and ligand channelling?

Understanding substrate binding and product release in cytochrome P450 (CYP) enzymes is important for explaining their key role in drug metabolism, toxicity, xenobiotic degradation and biosynthesis. Here, molecular simulations of substrate and product exit from the buried active site of a mammalian P450, the microsomal CYP2C5, identified a dominant exit channel, termed pathway (pw) 2c. Previous simulations with soluble bacterial P450s showed a different dominant egress channel, pw2a. Combining these, we propose two mechanisms in CYP2C5: (i) a one-way route by which lipophilic substrates access the enzyme from the membrane by pw2a and hydroxylated products egress along pw2c; and (ii) a two-way route for access and egress, along pw2c, for soluble compounds. The proposed differences in substrate access and product egress routes between membrane-bound mammalian P450s and soluble bacterial P450s highlight the adaptability of the P450 fold to the requirements of differing cellular locations and substrate specificity profiles.     

LICOS, a primordial costimulatory ligand?

In mammals, the classical B7 molecules expressed on antigen-presenting cells, B7-1 (CD80) and B7-2 (CD86), bind the structurally related glycoproteins CD28 and CTLA-4 (CD152), generating costimulatory signals that regulate the activation state of T cells. A recently identified human CD28-like protein, ICOS, also induces costimulatory signals in T cells when crosslinked with antibodies, but it is unclear whether ICOS is part of a B7-mediated regulatory pathway of previously unsuspected complexity, or whether it functions independently and in parallel. Here, we report that, rather than binding B7-1 or B7-2, ICOS binds a new B7-related molecule of previously unknown function that we call LICOS (for ligand of ICOS). At 37 degrees C, LICOS binds only to ICOS but, at lower, non-physiological temperatures, it also binds weakly to CD28 and CTLA-4. Sequence comparisons suggest that LICOS is the homologue of a molecule expressed by avian macrophages and of a murine protein whose expression is induced in non-lymphoid organs by tumour necrosis factor alpha (TNFalpha). Our results define the components of a distinct and novel costimulatory pathway and raise the possibility that LICOS, rather than B7-1 or B7-2, is the contemporary homologue of a primordial vertebrate costimulatory ligand.     

Discovering protein−ligand chalcogen bonding in the protein data bank using endocyclic sulfur-containing heterocycles as ligand search subsets

The chalcogen bond, the noncovalent, electrostatic attraction between covalently bonded atoms in group 16 and Lewis bases, is present in protein?ligand interactions based on X-ray structures deposited in the Protein Data Bank (PDB). Discovering protein?ligand chalcogen bonding in the PDB employed a strategy that focused on searching the database for protein complexes of five-membered, heterocyclic ligands containing endocyclic sulfur with endo electron-withdrawing groups (isothiazoles; thiazoles; 1,2,3-, 1,2.4-, 1,2,5-, 1,3,4-thiadiazoles) and thiophenes with exo electron-withdrawing groups, e.g., 2-chloro, 2-bromo, 2-amino, 2-alkylthio. Out of 930 ligands investigated, 33 or 3.5% have protein?ligand S---O interactions of which 31 are chalcogen bonds and two appear to be S---HO hydrogen bonds. The bond angles for some of the chalcogen bonds found in the PDB are less than 90°, and an electrostatic model is proposed to explain this phenomenon.     

Mass spectrometry-based approaches to protein–ligand interactions

One of the greatest current challenges in proteomics is to develop an understanding of cellular communication and regulation processes, most of which involve noncovalent interactions of proteins with various binding partners. Mass spectrometry plays an important role in all aspects of these research efforts. This article provides a survey of mass spectrometry-based approaches for exploring protein–ligand interactions. A wide array of techniques is available, and the choice of method depends on the specific problem at hand. For example, the high-throughput screening of compound libraries for binding to a specific receptor requires different approaches than structural studies on multiprotein complexes. This review is directed to readers wishing to obtain a concise yet comprehensive overview of existing experimental techniques. Specific emphasis is placed on emerging methods that have been developed within the last few years.     

Fas ligand: Can it ever be tamed?

Fas ligand (FasL) was first described functionally as an inducible cell surface molecule used by cytotoxic T cells to induce apoptotic cell death in tumor cells and activated lymphocytes. With the identification of Fas as the Ipr gene product, FasL became recognized as a molecule involved in down-regulation of the immune system. While FasL can be used to efficiently kill Fas-expressing tumor cells as well as activated T and B lymphocytes in vitro, attempts to use FasL therapeutically to treat cancer or to prevent transplant     

Molecular basis of ligand dissociation in β-adrenergic receptors

The important and diverse biological functions of β-adrenergic receptors (βARs) have promoted the search for compounds to stimulate or inhibit their activity. In this regard, unraveling the molecular basis of ligand binding/unbinding events is essential to understand the pharmacological properties of these G protein-coupled receptors. In this study, we use the steered molecular dynamics simulation method to describe, in atomic detail, the unbinding process of two inverse agonists, which have been recently co-crystallized with β(1) and β(2)ARs subtypes, along four different channels. Our results indicate that this type of compounds likely accesses the orthosteric binding site of βARs from the extracellular water environment. Importantly, reconstruction of forces and energies from the simulations of the dissociation process suggests, for the first time, the presence of secondary binding sites located in the extracellular loops 2 and 3 and transmembrane helix 7, where ligands are transiently retained by electrostatic and Van der Waals interactions. Comparison of the residues that form these new transient allosteric binding sites in both βARs subtypes reveals the importance of non-conserved electrostatic interactions as well as conserved aromatic contacts in the early steps of the binding process.     

LigandRNA: computational predictor of RNA–ligand interactions

RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA–small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA–ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a “meta-predictor” leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.     

A platinum–quinacridine hybrid as a G-quadruplex ligand

A novel platinum–quinacridine hybrid, comprising a monofunctional Pt moiety and a G-quadruplex ligand (mono-para-quinacridine or MPQ), has been synthesized and shown to interact with quadruplex DNA via a dual noncovalent/covalent binding mode. Denaturing gel electrophoresis was used to separate the various platination products of 22AG (an oligonucleotide that mimics the human telomeric repeat) by Pt-MPQ, and it was shown that two platinated adducts are highly stable quadruplex structures. Dimethylsulfate/piperidine treatment and 3′-exonuclease digestion of the isolated adducts allowed us to precisely determine the platination pattern of 22AG by Pt-MPQ, which displays three main sites G2, G10 and G22. Data presented herein support the hypothesis that Pt-MPQ traps preferentially the antiparallel structure of the 22AG quadruplex. Finally, the kinetics of Pt-MPQ platination using a construct containing both quadruplex DNA and a duplex DNA parts provide the first insights into the Pt-MPQ preference for quadruplex DNA over duplex DNA.     

What is the natural ligand of GPR55?

GPR55 is a seven transmembrane G protein-coupled receptor and was originally identified as a putative third cannabinoid receptor. Recently, lysophosphatidylinositol (LPI) was reported to be a GPR55 ligand. Stimulation of GPR55 by LPI activates G(12/13) and G(q/11) proteins, induces phosphorylation of the extracellular signal-regulated kinase and increases intracellular calcium concentration. Lysophospholipids are molecularly quite diverse across species and tissues. A recent report showed that the predominant fatty acyl moiety of LPI in rat brain is stearic acid followed by arachidonic acid. The biological activity of arachidonic acid-containing LPI species towards GPR55 was shown to be markedly higher than that of LPI species containing other fatty acyl groups, suggesting that 2-arachidonolyl LPI is the most likely natural ligand of GPR55.