Mass spectrometry and non-covalent protein-ligand complexes: confirmation of binding sites and changes in tertiary structure

An experimental approach is described for determining protein-small molecule non-covalent ligand binding sites and protein conformational changes induced by ligand binding. The methodology utilizes time resolved limited proteolysis and the high throughput analysis capability of MALDI TOF MS to determine the binding site in a tetanus toxin C-fragment (51 kDa)-doxorubicin (543 Da) non-covalent complex. Comparing relative ion abundances of peptides released from the time resolved limited proteolysis of tetanus toxin C-fragment (TetC) and the TetC-doxorubicin complex every 10 min from 10 to 120 min of digestion revealed that the binding of doxorubicin induced a significant change in surface topology of TetC. Four of the twenty-nine peptides observed by MALDI MS, including amino acids 351-360, 299-304, 305-311 and 312-316, had a lower abundance in the TetC-doxorubicin complex relative to TetC from 10 to 100 min of digestion. A decrease in ion abundance suggests doxorubicin obstructs the access of the protease to one or both termini of these peptides, identifying doxorubicin binding site(s). Conversely, five peptide ions, including amino acids 335-350, 364-375, 364-376, 281-298, and 316-328, all had a greater abundance in the digest of the complex, indicating an increase in accessibility to these sites. These five peptides flank regions of decreased ion abundance, suggesting that doxorubicin not only binds to the surface, but also induces a conformational change in TetC.     

Mass spectrometry based tools to investigate protein-ligand interactions for drug discovery

The initial stages of drug discovery are increasingly reliant on development and improvement of analytical methods to investigate protein-protein and protein-ligand interactions. For over 20 years, mass spectrometry (MS) has been recognized as providing a fast, sensitive and high-throughput methodology for analysis of weak non-covalent complexes. Careful control of electrospray ionization conditions has enabled investigation of the structure, stability and interactions of proteins and peptides in a solvent free environment. This critical review covers the use of mass spectrometry for kinetic, dynamic and structural studies of proteins and protein complexes. We discuss how conjunction of mass spectrometry with related techniques and methodologies such as ion mobility, hydrogen-deuterium exchange (HDX), protein footprinting or chemical cross-linking can provide us with structural information useful for drug development. Along with other biophysical techniques, such as NMR or X-ray crystallography, mass spectrometry provides a powerful toolbox for investigation of biological problems of medical relevance (204 references).     

Evaluation of automated nano-electrospray mass spectrometry in the determination of non-covalent protein-ligand complexes

The use of electrospray ionization mass spectrometry (ESI-MS) for studying non-covalent interactions between macromolecules and ligands is well established. ESI-MS can be a useful tool for the determination of dissociation constants between molecules in the gas phase. We validate this method by studying the binding of the catalytic domain of cellobiohydrolase I (CBH I) from Trichoderma reesei to the disaccharide inhibitor cellobiose. The method was further applied to study two newly synthesized cellobiose derivatives (m-iodobenzyl 2-deoxy-2-azido-beta-cellobioside and p-benzyloxybenzyl beta-cellobioside). In a titration experiment, peak areas of different charge states of the free enzyme and the complex were summed in order to determine the dissociation constant. For cellobiose and m-iodobenzyl 2-deoxy-2-azido-beta-cellobioside, the calculated values are in good agreement with those reported from either displacement titration or equilibrium binding experiments in solution. Due to non-specific binding, the dissociation constant of p-benzyloxybenzyl beta-cellobioside does not correspond with the solution-based value. Our results indicate the need for careful interpretation of data sets when using nanoESI to study non-covalent interactions.     

Selective detection of specific protein-ligand complexes by electrosonic spray-precursor ion scan tandem mass spectrometry

A novel mass spectrometric method for the selective detection of specific protein-ligand complexes is presented. The new method is based on electrosonic spray ionization of samples containing protein and ligand molecules, and mass spectrometric detection using the precursor ion scanning function on a triple quadrupole instrument. Mass-selected intact protein-ligand complex ions are subjected to fragmentation by means of collision-induced dissociation in the collision cell of the instrument, while the second mass analyzer is set to the m/z of protonated ligand ions or their alkali metal adducts. The method allows for the detection of only those ions which yield ions characteristic of the ligand molecules upon fragmentation. Since the scan range of first analyzer is set well above the m/z of the ligand ion, and the CID conditions are established to permit fragmentation of only loosely bound, noncovalent complexes, the method is specific to the detection of protein-ligand complexes under described conditions. Behavior of biologically specific and nonspecific complexes was compared under various instrumental settings. Parameters were optimized to obtain maximal selectivity for specific complexes. Specific and nonspecific complexes were found to show markedly different fragmentation characteristics, which can be a basis for selective detection of complexes with biological relevance. Preparation of specific and nonspecific complexes containing identical building blocks was attempted. Complex ions with identical stoichiometry but different origin showed the expected difference in fragmentation characteristics, which gives direct evidence for the different mechanism of specific versus nonspecific complex ion formation.     

2D depiction of protein-ligand complexes

A method is presented for the automated preparation of schematic diagrams for protein-ligand complexes, in which the ligand is displayed in conventional 2D form, and the interactions to and between the residues in its vicinity are summarized in a concise and information-rich manner. The structural entities are arranged to maximize aesthetic ideals and to properly convey important distance relationships. The diagram is annotated with calculated hydrogen bonds, a substitution contour, solvent exposure, chelated metals, covalently bound linkages, pi-pi and pi-cation interactions, and, for series of complexes, conserved residues and interactions. Residues, cofactors, ions, and solvent components are drawn in cartoon form as adjuncts to the ligand. The method can be applied to aligned sets which contain multiple ligands, or multiple members of a protein family, in which case the ligand orientations and protein residue placement will show consistent trends throughout the series.     

Binding constant determination of high-affinity protein-ligand complexes by electrospray ionization mass spectrometry and ligand competition

We describe an approach for the determination of binding constants for protein-ligand complexes with electrospray ionization mass spectrometry, based on the observation of unbound ligands competing for binding to a protein target. For the first time, dissociation constants lower than picomolar could be determined with good accuracy by electrospray ionization mass spectrometry. The presented methodology relies only on the determination of signal intensity ratios for free ligands in the low mass region. Therefore, all the advantages of measuring low masses with mass spectrometry, such as high resolution are preserved. By using a reference ligand with known binding affinity, the affinity of a second ligand can be determined. Since no noncovalently bound species are observed, assumptions about response factors are not necessary. The method is validated with ligands binding to avidin and applied to ligands binding to p38 mitogen-activated protein kinase.     

Isomer separation and gas-phase configurations of organoruthenium anticancer complexes: Ion mobility mass spectrometry and modeling

We have used ion mobility-mass spectrometry combined with molecular modeling for the separation and configurational analysis of three low-molecular-weight isomeric organoruthenium anticancer complexes containing ortho-, meta-, or para-terphenyl arene ligands. The isomers were separated using ion mobility based on traveling-wave technology and the experimentally determined collision cross sections were compared to theoretical calculations. Excellent agreement was observed between the experimentally and theoretically derived measurements.     

Dication induced stabilization of gas-phase ternary beta-cyclodextrin inclusion complexes observed by electrospray mass spectrometry

Electrospray mass spectrometry (ES-MS) is an important tool for characterization of non-covalent binding in the gas phase. In this study, iron (II) has been introduced as a dication to enhance the detection of cyclodextrin (CD) plus aromatic compound complexes in ES-MS. Evidence that a novel ternary complex comprised of one beta-CD, one iron (II) and one toluene exists as an inclusion complex has been compiled via ES-MS and ES-MS/MS experiments as well as by a computational approach. This evidence strongly suggests that iron (II) serves to modify the conformation of the beta-CD ring, and that toluene inclusion is stabilized by dication interaction with the toluene pi-system and by crimping of the beta-CD ring leading to stronger van der Waals interactions with toluene. Mg(II), another dication of similar radius, showed similar behavior, while added group one cations (H(+) and Na(+)) were ineffective at producing observable ions representative of the complex. The ternary beta-CD complex with iron (II) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) has also been examined. ES-MS and ES-MS/MS experiments suggest that it is the polar portion of 2,4,5-T (i.e., the carboxylic acid moiety) that is favored for inclusion in the beta-CD cavity, rather than the non-polar aromatic part.     

CSAR benchmark exercise of 2010: selection of the protein-ligand complexes

A major goal in drug design is the improvement of computational methods for docking and scoring. The Community Structure Activity Resource (CSAR) aims to collect available data from industry and academia which may be used for this purpose ( www.csardock.org ). Also, CSAR is charged with organizing community-wide exercises based on the collected data. The first of these exercises was aimed to gauge the overall state of docking and scoring, using a large and diverse data set of protein-ligand complexes. Participants were asked to calculate the affinity of the complexes as provided and then recalculate with changes which may improve their specific method. This first data set was selected from existing PDB entries which had binding data (K(d) or K(i)) in Binding MOAD, augmented with entries from PDB bind. The final data set contains 343 diverse protein-ligand complexes and spans 14 pK(d). Sixteen proteins have three or more complexes in the data set, from which a user could start an inspection of congeneric series. Inherent experimental error limits the possible correlation between scores and measured affinity; Pearson R is limited to ~ 0.91 (Pearson R2 0.83) when fitting to the data set without over parameterizing. Pearson R is limited to ~ 0.83(Pearson R2 ~ 0.70) when scoring the data set with a method trained on outside data [corrected]. The details of how the data set was initially selected, and the process by which it matured to better fit the needs of the community are presented. Many groups generously participated in improving the data set, and this underscores the value of a supportive, collaborative effort in moving our field forward.     

Mass spectrometric study of gas-phase thermal stability of yttrium(III) tris(dipivaloylmethanate)

The processes of thermal decomposition of yttrium(III) tris(dipivaloylmethanate) (Y(dpm)₃) vapour and its mixtures with oxygen were investigated by using the two-temperature variant of the double-chamber Knudsen cell, with mass spectrometric recording of the gasphase composition within the temperature range 150°–650°C. The threshold temperatures of the stability of the complex vapour were determined. The temperature dependence of the rate constant was obtained for the thermolysis of Y(dpm)₃ vapour, and the effective activation energy of the process was determined. Qualitative data were obtained on the influence of Cu(dpm)₂ vapour on the thermolysis of Y(dpm)₃ vapour in oxygen atmosphere.

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Zusammenfassung Unter Verwendung einer Zweitemperaturenvariante der Knudsenschen Doppelkammerzelle wurde mittels MS-Untersuchung der Gasphasenzusammensetzung im Temperaturbereich 150°–650°C der thermische Abbau von Yttrium(III)-tris(dipivaloylmethanat) (Y(dpm)₃) untersucht. Dabei wurden die Schwellentemperaturen der Stabilität des Komplex-Dampfes ermittelt. Für die Thermolyse des Y(dpm)₃-Dampfes wurde die Temperaturabhängigkeit der Geschwindigkeitskonstante sowie die effektive Aktivierungsenergie des Prozesses bestimmt. Die qualitativen Angaben wurden anhand des Einflusses von Cu(dpm)₂-Dampf auf die Thermolyse von Y(dpm)₃ in Sauerstoffatmosphäre erhalten.

     

Zwitterion formation in gas-phase cyclodextrin complexes

Protonated complexes of amino acids and underivatized beta-cyclodextrin, produced by electrospray ionization and trapped in the Fourier transform mass spectrometer, undergo formation of ternary complexes when reacted with alkyl amine. Based on the reactivities of the protonated amino acid complexes with alkylamines, the reactivities of the corresponding amino acid esters, and partially derivatized beta-cyclodextrin hosts, we conclude that the ternary complexes are salt-bridge zwitterionic species composed of amino acid zwitterions and protonated alkylamine all interacting with the hydroxyl groups on the narrow rim of the cyclodextrin. Molecular modeling calculations and experimental results suggest that the interactions of the amino acids with the rims contribute greatly to the formation of the zwitterionic species.     

Structure determination of protein-ligand complexes by transferred paramagnetic shifts

Rational drug design depends on the knowledge of the three-dimensional (3D) structure of complexes between proteins and lead compounds of low molecular weight. A novel nuclear magnetic resonance (NMR) spectroscopy strategy based on the paramagnetic effects from lanthanide ions allows the rapid determination of the 3D structure of a small ligand molecule bound to its protein target in solution and, simultaneously, its location and orientation with respect to the protein. The method relies on the presence of a lanthanide ion in the protein target and on fast exchange between bound and free ligand. The binding affinity of the ligand and the paramagnetic effects experienced in the bound state are derived from concentration-dependent (1)H and (13)C spectra of the ligand at natural isotopic abundance. Combined with prior knowledge of the crystal or solution structure of the protein and of the magnetic susceptibility tensor of the lanthanide ion, the paramagnetic data define the location and orientation of the bound ligand molecule with respect to the protein from simple 1D NMR spectra. The method was verified with the ternary 30 kDa complex between the lanthanide-labeled N-terminal domain of the epsilon exonuclease subunit from the Escherichia coli DNA polymerase III, the subunit theta, and thymidine. The binding mode of thymidine was found to be very similar to that of thymidine monophosphate present in the crystal structure.     

Studies of gas-phase reactions of cationic iron complexes of 2-pyrimidinyloxy-N-arylbenzylamines by electrospray ionization tandem mass spectrometry

Electrospray ionization triple quadrupole mass spectrometry (ESI‐TSQ‐MS) and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICR‐MS) were used to investigate the interesting gas‐phase reactions of the cationic iron (Fe) complexes of 2‐pyrimidinyloxy‐N‐arylbenzylamines (1–6), which are generated by ESI when mixing their methanolic solutions. Further studies of these Fe complexes by collision‐induced dissociation (CID) show that Fe(III) complexes undergo an interesting gas‐phase single electron transfer (SET) reaction to give 1^?+–6^?+,with loss of neutral FeCl₂, whereas Fe(II) can catalyze gas‐phase Smiles rearrangement reactions of compounds 1–6. By using different Fe(II)X₂ salts (X = Cl or Br) with a set of reactants, the role of the counterion (X^?) and the structure effect of the reactants on Fe(II)‐catalyzed gas‐phase Smiles rearrangement reactions are studied. Evidence obtained from by TSQ‐MS and FTICR‐MS experiments, hydrogen/deuterium (H/D) exchange experiments and theoretical computations supported some unique gas‐phase chemistries initiated by introduction of Fe(II) into 1. Importantly, by comparing the distinct gas‐phase reaction results of the cationic Fe(III) complexes with those of Fe(II) complexes, the charge state effects of iron on the gas‐phase chemistries of Fe complexes are revealed. Copyright © 2010 John Wiley & Sons, Ltd.     

DSX: a knowledge-based scoring function for the assessment of protein-ligand complexes

We introduce the new knowledge-based scoring function DSX that consists of distance-dependent pair potentials, novel torsion angle potentials, and newly defined solvent accessible surface-dependent potentials. DSX pair potentials are based on the statistical formalism of DrugScore, extended by a much more specialized set of atom types. The original DrugScore-like reference state is rather unstable with respect to modifications in the used atom types. Therefore, an important method to overcome this problem and to allow for robust results when deriving pair potentials for arbitrary sets of atom types is presented. A validation based on a carefully prepared test set is shown, enabling direct comparison to the majority of other popular scoring functions. Here, DSX features superior performance with respect to docking- and ranking power and runtime requirements. Furthermore, the beneficial combination with torsion angle-dependent and desolvation-dependent potentials is demonstrated. DSX is robust, flexible, and capable of working together with special features of popular docking engines, e.g., flexible protein residues in AutoDock or GOLD. The program is freely available to the scientific community and can be downloaded from our Web site www.agklebe.de .     

Postprocessing of docked protein-ligand complexes using implicit solvation models

Molecular docking plays an important role in drug discovery as a tool for the structure-based design of small organic ligands for macromolecules. Possible applications of docking are identification of the bioactive conformation of a protein-ligand complex and the ranking of different ligands with respect to their strength of binding to a particular target. We have investigated the effect of implicit water on the postprocessing of binding poses generated by molecular docking using MM-PB/GB-SA (molecular mechanics Poisson-Boltzmann and generalized Born surface area) methodology. The investigation was divided into three parts: geometry optimization, pose selection, and estimation of the relative binding energies of docked protein-ligand complexes. Appropriate geometry optimization afforded more accurate binding poses for 20% of the complexes investigated. The time required for this step was greatly reduced by minimizing the energy of the binding site using GB solvation models rather than minimizing the entire complex using the PB model. By optimizing the geometries of docking poses using the GB(HCT+SA) model then calculating their free energies of binding using the PB implicit solvent model, binding poses similar to those observed in crystal structures were obtained. Rescoring of these poses according to their calculated binding energies resulted in improved correlations with experimental binding data. These correlations could be further improved by applying the postprocessing to several of the most highly ranked poses rather than focusing exclusively on the top-scored pose. The postprocessing protocol was successfully applied to the analysis of a set of Factor Xa inhibitors and a set of glycopeptide ligands for the class II major histocompatibility complex (MHC) A(q) protein. These results indicate that the protocol for the postprocessing of docked protein-ligand complexes developed in this paper may be generally useful for structure-based design in drug discovery.     

Conserved core substructures in the overlay of protein-ligand complexes

The method of conserved core substructure matching (CSM) for the overlay of protein-ligand complexes is described. The method relies upon distance geometry to align structurally similar substructures without regard to sequence similarity onto substructures from a reference protein empirically selected to include key determinants of binding site location and geometry. The error in ligand position is reduced in reoriented ensembles generated with CSM when compared to other overlay methods. Since CSM can only succeed when the selected core substructure is geometrically conserved, misalignments only rarely occur. The method may be applied to reliably overlay large numbers of protein-ligand complexes in a way that optimizes ligand position at a specific binding site or subsite or to align structures from large and diverse protein families where the conserved binding site is localized to only a small portion of either protein. Core substructures may be complex and must be chosen with care. We have created a database of empirically selected core substructures to demonstrate the utility of CSM alignment of ligand binding sites in important drug targets. A Web-based interface can be used to apply CSM to align large collections of protein-ligand complexes for use in drug design using these substructures or to evaluate the use of alternative core substructures that may then be shared with the larger user community. Examples show the benefit of CSM in the practice of structure-based drug design.     

A deconvolution method for the separation of specific versus nonspecific interactions in noncovalent protein-ligand complexes analyzed by ESI-FT-ICR mass spectrometry

A method to separate specific and nonspecific noncovalent interactions observed in ESI mass spectra between a protein and its ligands is presented. Assuming noncooperative binding, the specific ligand binding is modeled as a statistical distribution on identical binding sites. For the nonspecific fraction we assume a statistical distribution on a large number of "nonspecific" interacting sites. The model was successfully applied to the noncovalent interaction between the protein creatine kinase (CK) and its ligands adenosine diphosphate (ADP) and adenosine triphosphate (ATP) that both exhibit nonspecific binding in the mass spectrum. The two sequential dissociation constants obtained by applying our method are K(1,diss) = 11.8 +/- 1.5 microM and K(2,diss) = 48 +/- 6 microM for ADP. For ATP, the constants are K(1,diss) = 27 +/- 7 microM and K(2,diss) = 114 +/- 27 microM. All constants are in good correlation with reported literature values. The model should be valuable for systems with a large dissociation constant that require high ligand concentrations and thus have increased potential of forming nonspecific adducts.