Mapping the energetics of water-protein and water-ligand interactions with the "natural" HINT forcefield: predictive tools for characterizing the roles of water in biomolecules

The energetics and hydrogen bonding pattern of water molecules bound to proteins were mapped by analyzing structural data (resolution better than 2.3A) for sets of uncomplexed and ligand-complexed proteins. Water-protein and water-ligand interactions were evaluated using hydropatic interactions (HINT), a non-Newtonian forcefield based on experimentally determined logP(octanol/water) values. Potential water hydrogen bonding ability was assessed by a new Rank algorithm. The HINT-derived binding energies and Ranks for second shell water molecules were -0.04 kcal mol(-1) and 0.0, respectively, for first shell water molecules -0.38 kcal mol(-1) and 1.6, for active site water molecules -0.45 kcal mol(-1) and 2.3, for cavity water molecules -0.55 kcal mol(-1) and 3.3, and for buried water molecules -0.56 kcal mol(-1) and 4.4. For the last four classes, similar energies indicate that internal and external water molecules interact with protein almost equally, despite different degrees of hydrogen bonding. The binding energies and Ranks for water molecules bridging ligand-protein were -1.13 kcal mol(-1) and 4.5, respectively. This energetic contribution is shared equally between protein and ligand, whereas Rank favors the protein. Lastly, by comparing the uncomplexed and complexed forms of proteins, guidelines were developed for prediction of the roles played by active site water molecules in ligand binding. A water molecule with high Rank and HINT score is unlikely to make further interactions with the ligand and is largely irrelevant to the binding process, while a water molecule with moderate Rank and high HINT score is available for ligand interaction. Water molecule displaced for steric reasons were characterized by lower Rank and HINT score. These guidelines, tested by calculating HINT score and Rank for 50 water molecules bound in the active site of four uncomplexed proteins (for which the structures of the liganded forms were also available), correctly predicted the ultimate roles (in the complex) for 76% of water molecules. Some failures were likely due to ambiguities in the structural data.     

Studies on ligand binding to histidine triad nucleotide binding protein 1

Histidine triad nucleotide binding protein (HINT1) is an intracellular protein that binds purine mononucleotides. Strong sequence conservation suggests that these proteins play a fundamental role in cell biology, however its exact cellular function continues to remain elusive. nuclear magnetic resonance (NMR) studies using STD and HSQC were conducted to observe ligand binding to HINT1. These studies were confirmed using fluorescence spectroscopy titrations. We found that AICAR, the first non-phosphate containing ligand, binds to mouse histidine triad nucleotide binding protein 1 (HINT1). Chemical shift perturbations are mapped onto the X-ray structure showing AICAR binds at the same site as GMP. The NMR results demonstrated that this method will be valuable for the future screening of small molecules that can be used to modulate the function of HINT1.     

Explaining cyclodextrin-mycotoxin interactions using a 'natural' force field

Docking techniques and the HINT (Hydropathic Interaction) program were used to explain interactions of aflatoxin B(1) and ochratoxin A with beta- and gamma-cyclodextrins. The work was aimed at designing a chemosensor to identify very low concentrations of these mycotoxins by exploiting the affinity of the cyclodextrin cavity for many small organic molecules. Actually, the inclusion of the fluorescent portion of these toxins into the cavity may lower the quenching effect of the solvent, thus enhancing the luminescence. HINT is a 'natural' force field, based on experimentally determined LogP(octanol/water) values, that is able to consider both enthalpic and entropic contributions to the binding free energy with an unified approach. HINT is normally applied to predict the DeltaG degrees of binding for protein-ligand, protein-protein, and protein-DNA interactions. The leading forces in biomolecular processes are the same as those involved in organic host-guest inclusion phenomena, therefore we applied this methodology for the first time to cyclodextrin complexes. The results allowed us to explain spectroscopic data in absence of available crystallographic or NMR structural data.     

HINT predictive analysis of binding between retinol binding protein and hydrophobic ligands

The interaction between the retinol binding protein and four ligands was evaluated using HINT, a software based on experimental LogP values of individual atoms. A satisfactory correlation was found between the HINT scores and the experimental dissociation constants of three of the ligands, fenretinide, N-ethylretinamide and all-trans retinol, despite their hydrophobic nature. A prediction is made for the binding affinity of the fourth ligand, axerophtene, not yet determined in solution.     

A novel and efficient tool for locating and characterizing protein cavities and binding sites

Systematic investigation of a protein and its binding site characteristics are crucial for designing small molecules that modulate protein functions. However, fundamental uncertainties in binding site interactions and insufficient knowledge of the properties of even well‐defined binding pockets can make it difficult to design optimal drugs. Herein, we report the development and implementation of a cavity detection algorithm built with HINT toolkit functions that we are naming Vectorial Identification of Cavity Extents (VICE). This very efficient algorithm is based on geometric criteria applied to simple integer grid maps. In testing, we carried out a systematic investigation on a very diverse data set of proteins and protein–protein/protein–polynucleotide complexes for locating and characterizing the indentations, cavities, pockets, grooves, channels, and surface regions. Additionally, we evaluated a curated data set of unbound proteins for which a ligand‐bound protein structures are also known; here the VICE algorithm located the actual ligand in the largest cavity in 83% of the cases and in one of the three largest in 90% of the cases. An interactive front‐end provides a quick and simple procedure for locating, displaying and manipulating cavities in these structures. Information describing the cavity, including its volume and surface area metrics, and lists of atoms, residues, and/or chains lining the binding pocket, can be easily obtained and analyzed. For example, the relative cross‐sectional surface area (to total surface area) of cavity openings in well‐enclosed cavities is 0.06 ± 0.04 and in surface clefts or crevices is 0.25 ± 0.09. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Identification and hydropathic characterization of structural features affecting sequence specificity for doxorubicin intercalation into DNA double-stranded polynucleotides.

The computer molecular modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field function that includes hydrogen bonding, coulombic and hydrophobic terms, was used to study sequence-selective doxorubicin binding/intercalation in the 64 unique CAxy, CGxy, TAxy, TGxy base pair quartet combinations. The CAAT quartet sequence is shown to have the highest binding score of the 64 combinations. Of the two regularly alternating polynucleotides, d(CGCGCG)2and d(TATATA)2, the HINT calculated binding scores reveal doxorubicin binds preferentially to d(TATATA)2. Although interactions of the chromophore with the DNA base pairs defining the intercalation site [I-1] [I+1] and the neighboring [I+2] base pair are predominant, the results obtained with HINT indicate that the base pair [I+3] contributes significantly to the sequence selectivity of doxorubicin by providing an additional hydrogen bonding opportunity for the N3' ammonium of the daunosamine sugar moiety in approximately 25% of the sequences. This observation, that interactions involving a base pair [I+3] distal to the intercalation site play a significant role in stabilizing/destabilizing the intercalation of doxorubicin into the various DNA sequences, has not been previously reported. In general terms, this work shows that molecular modeling and careful analysis of molecular interactions can have a significant role in designing and evaluating nucleotides and antineoplastic agents.     

Interchain hydrophobic clustering promotes rigidity in HIV-1 protease flap dynamics: new insights from Molecular Dynamics

The dynamics of HIV-1 protease (HIV-pr), a drug target for HIV infection, has been studied extensively by both computational and experimental methods. The flap dynamics of HIV-pr is considered to be more important for better ligand binding and enzymatic actions. Moreover, it has been demonstrated that the drug-induced mutations can change the flap dynamics of HIV-pr affecting the binding affinity of the ligands. Therefore, detailed understanding of flap dynamics is essential for designing better inhibitors. Previous computational investigations observed significant variation in the flap opening in nanosecond time scale indicating that the dynamics is highly sensitive to the simulation protocols. To understand the sensitivity of the flap dynamics on the force field and simulation protocol, molecular dynamics simulations of HIV-pr have been performed with two different AMBER force fields, ff99 and ff02. Two different trajectories (20?ns each) were obtained using the ff99 and ff02 force field. The results showed polarizable force field (ff02) make the flap tighter than the nonpolarizable force field (ff99). Some polar interactions and hydrogen bonds involving flap residues were found to be stronger with ff02 force field. The formation of interchain hydrophobic cluster (between flap tip of one chain and active site wall of another chain) was found to be dominant in the semi-open structures obtained from the simulations irrespective of the force field. It is proposed that an inhibitor, which will promote this interchain hydrophobic clustering, may make the flaps more rigid, and presumably the effect of mutation would be small on ligand binding.     

Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system

A study of the binding of the antibacterial agent trimethoprim to Escherichia coli dihydrofolate reductase was carried out using energy minimization techniques with both a full, all-atom valence force field and a united atom force field. Convergence criteria ensured that no significant structural or energetic changes would occur with further minimization. Root-mean-square (RMS) deviations of both minimized structures with the experimental structure were calculated for selected regions of the protein. In the active site, the all-atom minimized structure fit the experimental structure much better than did the united atom structure. To ascertain what constitutes a good fit, the RMS deviations between crystal structures of the same enzyme either from different species or in different crystal environments were compared. The differences between the active site of the all-atom minimized structure and the experimental structure are similar to differences observed between crystal structures of the same protein. Finally, the energetics of ligand binding were analyzed for the all-atom minimized coordinates. Strain energy induced in the ligand, the corresponding entropy loss due to shifts in harmonic frequencies, and the role of specific residues in ligand binding were examined. Water molecules, even those not in direct contact with the ligand, were found to have significant interaction energies with the ligand. Thus, the inclusion of at least one shell of waters may be vital for accurate simulations of enzyme complexes.     

Differences in hydropathic properties of ligand binding at four independent sites in wheat germ agglutinin-oligosaccharide crystal complexes.

The binding interactions of N-acetyl-D-neuraminic acid and N,N' diacetyl-chitobiose (GlcNAc-beta-1,4-GlcNAc), observed in crystal complexes of wheat germ agglutinin (WGA) at four independent sites/monomer, were analyzed and compared with the modeling program HINT (Hydropathic INTeractions). This empirical method allows assessment of relative ligand binding strength and is particularly applicable to cases of weak binding where experimental data is absent. Although the four WGA binding sites are interrelated by a fourfold sequence repeat (eight sites/dimer), similarity extends only to the presence of an aromatic amino acid-rich pocket and a conserved serine. Strong binding requires additional interactions from a contacting domain in the second subunit. Ligand positions were either derived from crystal structures and further optimized by modeling and molecular mechanics, or from comparative modeling. Analysis of the overall HINT binding scores for the two types of ligands are consistent with the presence of two high-affinity and two low-affinity sites per monomer. Identity of these sites correlates well with crystal structure occupancies. The high-affinity sites are roughly equivalent, as predicted from solution binding studies. Binding scores for the low-affinity sites are weaker by at least a factor of two. Quantitative estimates for polar, nonpolar, and ionic interactions revealed that H-bonding makes the largest contribution to complex stabilization in the seven bound configurations, consistent with published thermodynamic data. Although the observed nonpolar interactions are small, they may play a critical role in orienting the ligand optimally.     

Gating of HCN channels by cyclic nucleotides: residue contacts that underlie ligand binding, selectivity, and efficacy

Cyclic nucleotides (cNMPs) regulate the activity of various proteins by interacting with a conserved cyclic nucleotide-binding domain (CNBD). Although X-ray crystallographic studies have revealed the structures of several CNBDs, the residues responsible for generating the high efficacy with which ligand binding leads to protein activation remain unknown. Here, we combine molecular dynamics simulations with mutagenesis to identify ligand contacts important for the regulation of the hyperpolarization-activated HCN2 channel by cNMPs. Surprisingly, out of 7 residues that make strong contacts with ligand, only R632 in the C helix of the CNBD is essential for high ligand efficacy, due to its selective stabilization of cNMP binding to the open state of the channel. Principal component analysis suggests that a local movement of the C helix upon ligand binding propagates through the CNBD of one subunit to the C linker of a neighboring subunit to apply force to the gate of the channel.     

Protein conformation change of myoglobin upon ligand binding probed by ultraviolet resonance Raman spectroscopy

Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding, while Trp14 did not. Tyr151 in the C-terminus also exhibited a definite change upon CO binding, but Tyr103 and Tyr146 did not. The spectral change of Tyr residues was characterized through solvent effects of a model compound. The corresponding spectral differences between CO- and n-butyl isocyanide-bound forms were much smaller than those between the deoxy and CO-bound forms, suggesting that the conformation change in the C- and N-terminal regions is induced by the proximal side of the heme through the movement of iron. Although the swinging up of His64 upon binding of a bulky ligand is noted by X-ray crystallographic analysis, UVRR spectra of His for the n-butyl isocyanide-bound form did not detect the exposure of His64 to solvent.     

Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin

The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography.     

Critical role for polar residues in coupling leukotriene B4 binding to signal transduction in BLT1

Leukotriene B(4) (LTB(4)) mediates a variety of inflammatory diseases such as asthma, arthritis, atherosclerosis, and cancer through activation of the G-protein-coupled receptor, BLT1. Using in silico molecular dynamics simulations combined with site-directed mutagenesis we characterized the ligand binding site and activation mechanism for BLT1. Mutation of residues predicted as potential ligand contact points in transmembrane domains (TMs) III (H94A and Y102A), V (E185A), and VI (N241A) resulted in reduced binding affinity. Analysis of arginines in extracellular loop 2 revealed that mutating arginine 156 but not arginine 171 or 178 to alanine resulted in complete loss of LTB(4) binding to BLT1. Structural models for the ligand-free and ligand-bound states of BLT1 revealed an activation core formed around Asp-64, displaying multiple dynamic interactions with Asn-36, Ser-100, and Asn-281 and a triad of serines, Ser-276, Ser-277, and Ser-278. Mutagenesis of many of these residues in BLT1 resulted in loss of signaling capacity while retaining normal LTB(4) binding function. Thus, polar residues within TMs III, V, and VI and extracellular loop 2 are critical for ligand binding, whereas polar residues in TMs II, III, and VII play a central role in transducing the ligand-induced conformational change to activation. The delineation of a validated binding site and activation mechanism should facilitate structure-based design of inhibitors targeting BLT1.     

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.     

Multiple binding modes for palmitate to barley lipid transfer protein facilitated by the presence of proline 12

Molecular dynamics simulations have been used to characterise the binding of the fatty acid ligand palmitate in the barley lipid transfer protein 1 (LTP) internal cavity. Two different palmitate binding modes (1 and 2), with similar protein–ligand interaction energies, have been identified using a variety of simulation strategies. These strategies include applying experimental protein–ligand atom–atom distance restraints during the simulation, or protonating the palmitate ligand, or using the vacuum GROMOS 54B7 force‐field parameter set for the ligand during the initial stages of the simulations. In both the binding modes identified the palmitate carboxylate head group hydrogen bonds with main chain amide groups in helix A, residues 4 to 19, of the protein. In binding mode 1 the hydrogen bonds are to Lys 11, Cys 13, and Leu 14 and in binding mode 2 to Thr 15, Tyr 16, Val 17, Ser 24 and also to the OH of Thr 15. In both cases palmitate binding exploits irregularity of the intrahelical hydrogen‐bonding pattern in helix A of barley LTP due to the presence of Pro 12. Simulations of two variants of barley LTP, namely the single mutant Pro12Val and the double mutant Pro12Val Pro70Val, show that Pro 12 is required for persistent palmitate binding in the LTP cavity. Overall, the work identifies key MD simulation approaches for characterizing the details of protein–ligand interactions in complexes where NMR data provide insufficient restraints.     

Hydropathic interaction analyses of small organic activators binding to antithrombin

Recently we designed the first small organic ligands, sulfated flavanoids and flavonoids, that act as activators of antithrombin for accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade [Gunnarsson and Desai, Bioorg. Med. Chem. Lett. (2003) 13:579]. To better understand the binding properties of these activators at a molecular level, we have utilized computerized hydropathic interaction (HINT) analyses of the sulfated molecules interacting in two plausible electropositive regions, the pentasaccharide- and extended heparin-binding sites, of antithrombin in its native and activated forms. HINT analyses indicate favorable multi-point interactions of the activators in both binding sites of the two forms of antithrombin. Yet, HINT predicts better interaction of most activators, except for (-)-catechin sulfate, with the activated form of antithrombin than with the native form supporting the observation in solution that these molecules function as activators of the inhibitor. Further, whereas (+)-catechin sulfate recognized the activated form of antithrombin better in both the pentasaccharide- and extended heparin- binding sites, the native form was better recognized by (-)-catechin sulfate, thus explaining its weaker binding and activation potential in solution. A reasonable linear correlation between the overall HINT score and the solution free energy of binding of the sulfated activators was evident. This investigation indicates that HINT is a useful tool in understanding interactions of antithrombin with small sulfated organic ligands at a molecular level, has some good predictive properties, and is likely to be useful for rational design purposes.     

A hierarchical method for generating low-energy conformers of a protein-ligand complex

A novel dynamical protocol for finding the low-energy conformations of a protein-ligand complex is described. The energy functions examined consist of an empirical force field with four different dielectric screening models; the generalized Born/surface area model also is examined. Application of the method to three complexes of known crystal structure provides insights into the energy functions used for selecting low-energy docked conformations and into the structure of the binding-energy surface. Evidence is presented that the local energy minima of a ligand in a binding site are arranged in a hierarchical fashion. This observation motivates the construction of a hierarchical docking algorithm that substantially enriches the population of ligand conformations close to the crystal conformation. The algorithm is also adapted to permit docking into a flexible binding site and preliminary tests of this method are presented. Proteins 33:475–495, 1998. © 1998 Wiley-Liss, Inc.