Protein-induced DNA bending: the role of phosphate neutralisation

An important component of protein–nucleic acid interactions is the formation of salt bridges between cationic amino acid side chains and the anionic phosphate groups of the nucleic acid. We have used molecular mechanics to study the energetic and conformational impact of such interactions. Firstly, crystallographic protein–nucleic acid complexes from the Protein Data Bank were analysed in terms of DNA curvature and the presence of salt bridges. For complexes where the DNA is significantly bent, the contribution of salt bridges to this curvature was modelled by studying the effect of neutralising the appropriate phosphate groups. The number and the distribution of salt bridges vary widely for different DNA binding motifs and appear to have very different effects on DNA. In the case of homeodomain, bZIP and helix–loop–helix proteins, salt bridges induce DNA bending, whereas for prokaryotic helix–turn–helix proteins the number of salt bridges is much smaller and little bending is found. By analysing the components of the DNA deformation energy involved in protein binding we show that salt bridges consistently increase the flexibility of the DNA backbone. Received: 1 September 2000 / Accepted: 5 January 2001 / Published online: 3 May 2001     

A computational model of the 5-HT3 receptor extracellular domain: search for ligand binding sites

A three-dimensional model of the 5-HT₃ receptor extarcellular domain has been derived on the basis of the nicotinic acetylcholine receptor model recently published by Tsigelny et al. Maximum complementarity between the position and characteristics of mutated residues putatively involved in ligand interaction and the pharmacophoric elements derived by the indirect approach applied on several series of 5-HT₃ ligands have been exploited to gain insights into the ligand binding modalities and to speculate on the mechanistic role of the structural components. The analysis of the three-dimensional model allows one to distinguish among amino acids that exert key roles in ligand interactions, subunit architecture, receptor assembly and receptor dynamics. For some of these, alternative roles with respect to the ones hypothesized by experimentalists are assigned. Different binding modalities for agonists and antagonists are highlighted, and residues which probably play a role in the transduction of binding into a change in conformational state of the receptor are suggested. Received: 27 July 2000 / Accepted: 15 September 2000 / Published online: 21 December 2000     

Mapping a Noncovalent Protein–Peptide Interface by Top-Down FTICR Mass Spectrometry Using Electron Capture Dissociation

Noncovalent protein–ligand and protein–protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein–ligand interactions. In this way the site of protein–ligand interfaces can be identified. To date, protein–ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein–peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein–peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide–protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein–protein interfaces.     

Stabilization centers and protein stability

The well-balanced stability of protein structures allows large-scale fluctuations, which are indispensable in many biochemical functions, ensures the long-term persistence of the equilibrium structure and it regulates the degradation of proteins to provide amino acids for biosynthesis. This balance is studied in the present work with two sets of proteins by analyzing stabilization centers, defined as certain clusters of residues involved in cooperative long-range interactions. One data set contains 56 proteins, which belong to 16 families of homologous proteins, derived from organisms of various physiological temperatures. The other set is composed of 31 major histocompatibility complex (MHC)–peptide complexes, which represent peptide transporters complexed with peptide ligands that apparently contribute to the stabilization of the MHC proteins themselves. We show here that stabilization centers, which had been identified as special clusters of residues that protect the protein structure, evolved to serve also as regulators of function – related degradation of useless protein as part of protein housekeeping. Received: 25 August 2000 / Accepted: 6 September 2000 / Published online: 21 December 2000     

How accurate can molecular dynamics/linear response and Poisson–Boltzmann/solvent accessible surface calculations be for predicting relative binding affinities? Acetylcholinesterase huprine inhibitors as a test case

This study examines the accuracy of molecular dynamics-linear response (MD/LR) and Poisson–Boltzmann/solvent accessible surface (PB/SAS) calculations to predict relative binding affinities. A series of acetylcholinesterase (AChE) huprine inhibitors has been chosen as a test system owing to the availability of free-energy (thermodynamic integration) calculations. The results obtained with the MD/LR approach point out a clear relationship between the experimental affinity and the electrostatic interaction energy alone for a subset of huprines, but the suitability of the MD/LR approach to predict the binding affinity of the whole series of compounds is limited. On the other hand, PB/SAS calculations show a marked dependence on both the computational protocol and the nature of the inhibitor–enzyme complex. Received: 2 August 2000 / Accepted: 8 September 2000 / Published online: 21 December 2000     

Ab initio calculations with effective core potentials on trivalent lanthanide–terpyridine complexes

 The electronic structures of complexes of terpyridine (tpy) with trivalent lanthanides (Ln) were calculated using ab initio methods with effective core potentials at Hartree–Fock and post-Hartree–Fock levels of theory. The quasirelativistic large-core (with 4f electrons included in the core) pseudopotentials of the Stuttgart group were chosen for the Ln atoms. The variation of several properties of the Ln(tpy)³⁺ complexes was studied for the whole Ln series. It was shown that there was a monotonous variation for all properties (geometrical and energetic) along the Ln series, except for Mulliken charges on the metal atom. Calculations were performed on three complexes of known solid-state structure. The difference between experimental and calculated geometries is discussed; for all structures, it is found to be lower than 0.2 ?. In all cases, the relative order from one complex to another is conserved. Received: 13 September 1999 / Accepted: 3 February 2000 / Published online: 19 April 2000     

Assessment of conformation and energetics of the N-terminal part of elafin via computer simulations

Elafin, a specific inhibitor of elastase, is thought to play a regulatory role in inflammation. An NMR-derived solution structure of recombinant elafin has been reported [Francart et al. (1997) J Mol Biol 268:666 ], although the conformation of its flexible N-terminal part is not established. There is experimental evidence that the N terminus (residues 1–15) of elafin interacts with the cell membrane. To explore the conformational preferences of residues in this region, we have performed Monte Carlo simulations of the peptide in water, in cyclohexane, and in a model membrane. Additionally, 3.7-ns molecular dynamics with explicit water was carried out. The main results were that the hydrophobic environment stabilizes an α helix in the region 6–11, the peptide is unordered in water, and it is attached to the membrane via the amphiphilic α-helix 6–11, which inserts with its N terminus forming an angle of about 60° to the membrane plane. We therefore assume that in nonpolar media the N-terminal part of elafin forms a short α helix which might act as a membrane anchor. Received: 5 July 2000 / Accepted: 4 October 2000 / Published online: 28 February 2001     

Reparameterization of hybrid functionals based on energy differences of states of different multiplicity

 Low-spin/high-spin energy splittings for Fe(II) transition-metal complexes – particularly in weak ligand fields – cannot be well described by density functional methods. Different density functionals yield results which differ by up to 1 eV in transition-metal complexes with sulfur-rich first coordination spheres. We attribute this failure to the fact that the high-spin state is systematically favoured in Hartree–Fock-type theories, because Fermi correlation is included in the exact exchange, while Coulomb correlation is not. We thus expect that the admixture of exact exchange to a given density functional will heavily influence the energy splitting between states of different multiplicity. We demonstrate that the energy splitting depends linearly on the coefficient of exact exchange admixture. This remarkable result is found for all the Fe(II)–S complexes studied. From this observation we conclude in connection with experimental results that Becke's 20% admixture should be reduced to about 15% if meaningful energetics are sought for transition-metal compounds. We rationalize that this reduction by 5% will not affect the quality of the hybrid functional since we arrive at a slightly modified functional, which lies between the pure density functional and the hybrid density functional, which both give good results for “standard” systems. Received: 13 July 2001 / Accepted: 31 August 2001 /  Published online: 16 November 2001     

Thermodynamics of Mixed Ligand Complex Formation of Copper(II) and Nickel Ions with Glycine and Histidine in Solution

Mixed ligand complexes of copper(II) and nickel(II) with glycine (Gly) and histidine (His) have been studied in aqueous solution using calorimetric, potentiometric and spectrophotometric data at 298.15 K and the ionic strength of I = 0.5 mol·L–1 (KNO3). The thermodynamic parameters of formation of various mixed complexes have been determined. The probable coordination modes of amino acid residues in mixed ligand complexes is discussed.     

Inexpensive and accurate predictions of optical excitations in transition-metal complexes: the TDDFT/PBE0 route

Time-dependent density functional theory (TDDFT) is applied to calculate vertical excitation energies of three representative transition metal complexes. The computational model (PBE0) is obtained by combining the Perdew-Burke-Erzenrhof (PBE) generalized gradient functional with a predetermined amount of exact exchange. Our results show that the TDDFT/PBE0 model represents a cheap and reliable tool for the computation of optical excitations for transition metal complexes. Received: 8 August 2000 / Accepted: 7 September 2000 / Published online: 23 November 2000     

A study of DNA tethered to a surface by an all-atom molecular dynamics simulation

 In order to understand the structure of DNAs and their interactions when on microarray surfaces, we performed the first all-atom molecular dynamics simulation of DNA tethered to a surface. On the surface, the binding of the DNA was enhanced, and its average equilibrium conformation was the B form. The DNA duplex spontaneously tilted towards its nearest neighbor and settled in a leaning position with a interaxial distance of 2.2 nm. This close packing of the DNAs, which affects both in situ synthesis and deposition of probes on microarray surfaces, can thus be explained by salted-induced colloidlike DNA–DNA attractions. Received: 30 November 2000 / Accepted: 7 February 2001 / Published online: 22 May 2001     

Hydration of platinum(II) complexes: a molecular mechanics study using atom-based force-field parameters

 This work is related to the interaction of water with two platinum(II) complexes, [Pt(NH₃)₄]²⁺ (denoted 1) and trans-[Pt(OH)₂(NH₃)₂] (denoted 2). We have considered two approaches of a water molecule to complexes 1 and 2 along the z-axis normal to the platinum(II) coordination plane: approach I, with the water oxygen oriented towards Pt, and approach II, with one water hydrogen directed towards Pt. Calculations have been performed within a molecular mechanics method based upon the interaction potentials proposed earlier by Claverie et al. and subsequently adjusted to results obtained with symmetry – adapted perturbational theory as well as with supermolecule (up to second-order M?ller–Plesset, MP2) methods. We discuss some possible simplifications of the potentials mentioned. The results relative to the hydration of Pt complexes 1 and 2 following approach I or II are discussed and compared to recent (MP2) ab initio energy–distance curves that we have recently determined. The MP2 calculations have shown that besides exchange–repulsion contributions, which are very similar in all hydrated complexes, approach I is mainly governed by electrostatics, whereas for approach II both electrostatic and dispersion contributions are important. Received: 16 September 1999 / Accepted: 3 February 2000 / Published online: 5 June 2000     

Relativistic calculations on thallium hydride

The spectroscopic parameters of the ground state of thallium hydride are obtained using the four-component relativistic Dirac–Coulomb–Gaunt–coupled-cluster wave function with single and double excitations and an estimated triples correction method. Core correlation effects make the bond weaker but have little effect on the bond length. Inclusion of the distance dependence of the Gaunt part of the two-electron Breit interaction has an opposite but smaller effect on these properties. Received: 8 September 2000 / Revised version: 5 October 2000 / Published online: 21 December 2000     

Theoretical study on the complexation of the hexafluorophosphate,hexafluoroarsenate, and hexafluoroantimonate anions with dodecabenzylbambus[6] uril

Applying quantum chemical calculations, the most likely conformations of the anionic complex species dodecabenzylbambus[6]uril–PF6−, dodecabenzylbambus[6]uril–AsF6−, and dodecabenzylbambus[6]uril–SbF6− were found. In all these complexes, the considered anions, incorporated in the ligand cavity, are bound by hydrogen bonds between methine hydrogen atoms on the convex face of glycoluril units of the dodecabenzylbambus[6]uril ligand and fluorine atoms of the respective “central” anion. Moreover, the interaction energies of the investigated three anionic complexes were calculated. The absolute values of these calculated energies increase in the series of SbF6− < AsF6− < PF6−.     

A first principles study of Pd deposition on the TiO2(1 1 0) surface

 The adsorption of isolated Pd atoms on the (1 1 0) surface of rutile TiO₂ was investigated through ab initio embedded-cluster calculations performed at the Hartree–Fock, second-order M?ller–Plesset and Becke's three parameter hybrid method with the Lee–Yang–Parr correlation functional levels. The role played by the magnitude of the surrounding charges used in the embedding procedure was carefully analyzed. The most stable site for adsorption consisted of a fourfold hollow site in which the Pd atom was coordinated to a fivefold Ti atom, two basal oxygens, and a protruding oxygen atom. However, the adsorption energies computed after basis set superposition error corrections seemed to favor a bridge site in which the Pd atom binds two protruding oxygen atoms. A periodic slab calculation using gradient-corrected functionals and plane-wave basis sets confirmed that for full coverage, the hollow site was more stable, although Pd displacement along the fivefold Ti channels was almost free. These results agree with the experimental data obtained from scanning tunneling microscopy. Finally, the adsorption energy computed from the periodic calculations was found to be 1.88 eV. Received: 14 September 1999 / Accepted: 3 February 2000 / Published online: 19 April 2000     

Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography–mass spectrometry for solution-based ligand screening against multiple proteins

We present herein a novel bioseparation/chemical analysis strategy for protein–ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography–mass spectrometry (LC–MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC–MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5 s, and on-line prepare the “clean” sample to be directly compatible with the LC–MS analysis. The improvement in performance of this 2D-TFC/LC–MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC–MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.     

The role of human in the loop: lessons from D3R challenge 4

The rapid development of new machine learning techniques led to significant progress in the area of computer-aided drug design. However, despite the enormous predictive power of new methods, they lack explainability and are often used as black boxes. The most important decisions in drug discovery are still made by human experts who rely on intuitions and simplified representation of the field. We used D3R Grand Challenge 4 to model contributions of human experts during the prediction of the structure of protein–ligand complexes, and prediction of binding affinities for series of ligands in the context of absence or abundance of experimental data. We demonstrated that human decisions have a series of biases: a tendency to focus on easily identifiable protein–ligand interactions such as hydrogen bonds, and neglect for a more distributed and complex electrostatic interactions and solvation effects. While these biases still allow human experts to compete with blind algorithms in some areas, the underutilization of the information leads to significantly worse performance in data-rich tasks such as binding affinity prediction.     

Sulfur-containing alkenes—A new class of chelating ligands: Synthesis,coordination to palladium,and structure of the resulting complexes

Stable 1,2-disulfanylalkene palladium complexes [(RS-CH=CR′-SR)PdCl₂] were synthesized in 85–94% yield by reaction of palladium(II) chloride with sulfur-containing ligands RS-CH=C(R′)-SR (analogs of dithiolate ligands). The structure of the complexes was studied by NMR spectroscopy and quantum-chemical methods. The binding energy in palladium complexes with bis(arylsulfanyl)- and bis(alkylsulfanyl)alkenes was estimated (DFT) at 50 and 56 kcal/mol, respectively. Variation of substituents on the sulfur atoms is a convenient tool for fine tuning of the ligand properties and controlling the strength of the complex. The bite angle of the ligands does not depend on the substituent nature and is 88–89°, which is typical of square-planar complexes. According to the bite angle, the examined ligands are analogs of well known bidentate phosphine ligands, but the former are more labile since the corresponding binding energy is lower by 36 kcal/mol.     

Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein–protein and protein–immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein–protein and protein–immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS.