Modelling CreA protein–DNA recognition determinants. A molecular dynamics study of fully charged CreA–DNA model in water

CreA, the negative regulator mediating carbon catabolism repression in Aspergillus nidulans, is a protein that contains a DNA-binding domain comprising two zinc finger motifs. A 3D model for the CreA–G4 (5′-GCGGGGGCGT-3′) complex is constructed on the basis of the structure of the Zif268–DNA crystal complex [Science 252 (1991) 809] and using similarity analysis and computer assisted modelling techniques. The CreA–G4 model was then subjected to a set of molecular dynamics (MD) studies. Based on our previous nano second long Zif268–DNA MD simulation, a 170 ps long trajectory was deemed sufficient to test possible DNA–protein interactions. A screening of the static model and of the trajectory was performed for protein amino acids, nucleotide bases, phosphates backbone and water molecules mediating protein–DNA contacts. Energy, root mean square deviation (RMSD), principal inertial moment and distances were analysed. For this time span, the stability shown in fluctuation patterns reveals the presence of a complex behaving in a manner similar to Zif268–DNA.

An unambiguous characterisation of the amino acids involved in DNA-binding was obtained. These results could contribute towards the establishment of a code of protein–DNA recognition for this class of DNA-binding motifs.     

Water-mediated interactions in the CRP–cAMP–DNA complex: Does water mediate sequence-specific binding at the DNA primary-kink site?

The cyclic AMP receptor protein (CRP) of Escherichia coli binds preferentially to DNA sequences possessing a T:A base pair at position 6 (at which the DNA becomes kinked), but with which it does not form any direct interactions. It has been proposed that indirect readout is involved in CRP-DNA binding, in which specificity for this base pair is primarily related to sequence effects on the energetic susceptibility of the DNA to kink formation. In the current study, the possibility of contributions to indirect readout by water-mediated hydrogen bonding of CRP with the T:A base pair was investigated. A 1.0 ns molecular dynamics simulation of the CRP-cAMP-DNA complex in explicit solvent was performed, and assessed for water-mediated CRP-DNA hydrogen bonds; results were compared to several X-ray crystal structures of comparable complexes. While several water-mediated CRP-DNA hydrogen bonds were identified, none of these involved the T:A base pair at position 6. Therefore, the sequence specificity for this base pair is not likely enhanced by water-mediated hydrogen bonding with the CRP.     

The Mg2+ binding sites of the 5S rRNA loop E motif as investigated by molecular dynamics simulations

Molecular dynamics simulations have been used to investigate the binding of Mg(2+) ions to the deep groove of the eubacterial 5S rRNA loop E. The simulations suggest that long-lived and specific water-mediated interactions established between the hydrated ions and the RNA atoms lining up the binding sites contribute to the stabilization of this motif. The Mg(2+) binding specificity is modulated by two factors: (i) a required electrostatic complementarity and (ii) a structural correspondence between the hydrated ion and its binding pocket that can be estimated by its degree of dehydration and the resulting number and lifetime of the intervening water-mediated contacts. Two distinct binding modes for pentahydrated Mg(2+) ions that result in a significant freezing of the tumbling motions of the ions are described, and mechanistic details related to the stabilization of nucleic acids by divalent ions are provided.     

Energy-based prediction of amino acid-nucleotide base recognition

Despite decades of investigations, it is not yet clear whether there are rules dictating the specificity of the interaction between amino acids and nucleotide bases. This issue was addressed by determining, in a dataset consisting of 100 high-resolution protein-DNA structures, the frequency and energy of interaction between each amino acid and base, and the energetics of water-mediated interactions. The analysis was carried out using HINT, a non-Newtonian force field encoding both enthalpic and entropic contributions, and Rank, a geometry-based tool for evaluating hydrogen bond interactions. A frequency- and energy-based preferential interaction of Arg and Lys with G, Asp and Glu with C, and Asn and Gln with A was found. Not only favorable, but also unfavorable contacts were found to be conserved. Water-mediated interactions strongly increase the probability of Thr-A, Lys-A, and Lys-C contacts. The frequency, interaction energy, and water enhancement factors associated with each amino acid-base pair were used to predict the base triplet recognized by the helix motif in 45 zinc fingers, which represents an ideal case study for the analysis of one-to-one amino acid-base pair contacts. The model correctly predicted 70.4% of 135 amino acid-base pairs, and, by weighting the energetic relevance of each amino acid-base pair to the overall recognition energy, it yielded a prediction rate of 89.7%.     

Role of water mediated interactions in protein-protein recognition landscapes

The energy landscape picture of protein folding and binding is employed to optimize a number of pair potentials for direct and water-mediated interactions in protein complex interfaces. We find that water-mediated interactions greatly complement direct interactions in discriminating against various types of trap interactions that model those present in the cell. We highlight the context dependent nature of knowledge-based binding potentials, as contrasted with the situation for autonomous folding. By performing a Principal Component Analysis (PCA) of the corresponding interaction matrixes, we rationalize the strength of the recognition signal for each combination of the contact type and reference trap states using the differential in the idealized "canonical" amino acid compositions of native and trap layers. The comparison of direct and water-mediated contact potential matrixes emphasizes the importance of partial solvation in stabilizing charged groups in the protein interfaces. Specific water-mediated interresidue interactions are expected to influence significantly the kinetics as well as thermodynamics of protein association.     

Molecular dynamics simulation of thymine glycol-lesioned DNA reveals specific hydration at the lesion

One nanosecond molecular dynamics (MD) simulation of a thymine glycol (TG)-lesioned part of human lymphoblast AG9387 was performed to determine structural changes in DNA molecule caused by the presence of a lesion. These changes can be significant for proper recognition of lesions by a repair enzyme. Thymine glycol is the DNA oxidative lesion formed by addition of OH radicals to C5 and C6 atoms of the thymine base. This lesion is known as causing Cockayne Syndrome-inherited genetic disorder. Distribution of water molecules in a hydration shell around the DNA molecule was analyzed for its contribution to the recognition of the TG lesion by the repair enzyme. The results of MD simulation show there is a specific DNA structural configuration formed at the lesion. After 500 ps the DNA is bent in a kink at the TG site. This change dislocates the glycosyl bond at C5' to a position closer to the DNA surface, and thus its atoms are more exposed to the surrounding water shell. The increased number of water molecules that are close to the TG site indicates that the glycosyl bond may be easily contacted by the repair enzyme. In addition, the higher number of water molecules at the TG site substantiates the importance of water-mediated hydrogen bonds created between the repair enzyme and the DNA upon formation of the complex. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1723-1731, 2001     

Ab initio fragment molecular orbital (FMO) method applied to analysis of the ligand-protein interaction in a pheromone-binding protein

Full quantum computation of the electronic state of proteins has recently become possible by the advent of the ab initio fragment molecular orbital (FMO) method. We applied this method to the analysis of the interaction between the Bombyx mori pheromone-binding protein and its ligand, bombykol. The protein–ligand interaction of this molecular complex was minutely analyzed by the FMO method, and the analysis revealed several important interactions between the ligand and amino acid residues.     

The role of hydrophobic surfaces in altering water-mediated peptide-peptide interactions in an aqueous environment

Using Born-Oppenheimer molecular dynamics within the density functional framework, we calculated the effective force acting on water-mediated peptide-peptide interaction between antiparallel β-sheets in an aqueous environment and also in the vicinity of a hydrophobic surface. From the magnitude of the effective force (corresponding to the slope of the free energy as a function of the interpeptide distance) and its sign (a negative value indicates an effective attraction, whereas a positive value indicates an effective repulsion) we can elucidate the fundamental differences of the water-mediated peptide-peptide interactions in those two environments. The computed effective forces indicate that the water-mediated interaction between peptides in an aqueous environment is attractive in the range of interpeptide distance d = 7-8 ? when hydrophobic surfaces are not nearby. Due to the stabilization of the water molecules bridging between the two β-sheets, a free energy barrier exists between the direct and indirect (water-mediated) interpeptide interactions. However, when the peptides are in the proximity of hydrophobic surfaces, this free energy barrier decreases because the hydrophobic surfaces enhance the interpeptide attraction by the destabilization and ease-to-libration of the bridging water molecules between them.     

Tryptophan–water interaction in Monellin: Hydration patterns from molecular dynamics simulation

Femtosecond spectroscopy carried out earlier on Monellin and some other systems has given insights into the hydration dynamics of the proteins. In the present work, molecular dynamics simulations have been performed on Monellin to study the hydration dynamics. A method has been described to follow up the molecular events of the protein–water interactions in detail. The time constants of the survival correlation function match well with the reported experimental values. This validates the procedure, adapted here for Monellin, to investigate the hydration dynamics in general.     

Conformational and energetic effects of truncating nonbonded interactions in an aqueous protein dynamics simulation

In a previous aqueous protein dynamics study, we compared the rms deviation relative to the crystal structure for distance-dependent and constant dielectric models with and without a nonbonded cutoff. The structures obtained from a constant dielectric simulation with a cutoff were substantially different from the structures obtained from a distance-dependent dielectric simulation, with and without cutoff, and a constant dielectric model without a cutoff. In fact, structures from the distance-dependent dielectric simulations were insensitive to the nonbonded cutoff and in good agreement with the structures generated from the constant dielectric simulation without a cutoff. In addition, the solute-solvent temperature differential and solvent evaporation artifacts, characteristic of the constant dielectric simulation with a cutoff, were not present for the distance-dependent dielectric simulations. In this current work, we explore whether this dielectric-dependent cutoff-sensitive behavior for a constant dielectric model arises from the discontinuities in the forces at the nonbonded cutoff or from neglecting the structure-stabilizing interactions beyond the nonbonded cutoff. We also examine the origin of the dielectric-dependent artifacts, and its potential influence on the structural disparity. Several protocols for protein dynamics simulations are compared using both constant and distance-dependent dielectric models, including implementation of a switching function and a nonbonded cutoff and two different temperature coupling algorithms. We show that the distance-dependent dielectric model conserves energy in the SPASMS molecular mechanics and dynamics software for the time steps and nonbonded cutoffs commonly used in macromolecule simulations. Although the switching function simulation also conserved energy over a range of commonly used cutoffs, the constant dielectric model with a switching function yielded conformational results more similar to a constant dielectric simulation without a switching function than to a constant dielectric model without a nonbonded cutoff. Therefore, the conformational disparity between the dielectric models arises from neglecting important structure-stabilizing interactions beyond the cutoff, rather than differences in energy conservation. © 1993 John Wiley & Sons, Inc.     

Strong binding in the DNA minor groove by an aromatic diamidine with a shape that does not match the curvature of the groove

A combination of biophysical techniques has been used to characterize the interaction of an antitrypanosomal agent, CGP 40215A, with DNA. The results from a broad array of methods (DNase I footprinting, surface plasmon resonance, X-ray crystallography, and molecular dynamics) indicate that this compound binds to the minor groove of AT DNA sequences. Despite its unusual linear shape that is not complementary to that of the DNA groove, a high binding affinity was observed in comparison with other similar but more curved diamidine compounds. The amidine groups at both ends of the ligand and the -NH groups on the linker are involved in extensive and dynamic H-bonds to the DNA bases. Complementary and consistent results were obtained from both the X-ray and molecular dynamics studies; both of these methods reveal direct and water-mediated H-bonds between the ligand and the DNA.     

Molecular dynamics study of DNA binding by INT‐DBD under a polarized force field

The DNA binding domain of transposon Tn916 integrase (INT‐DBD) binds to DNA target site by positioning the face of a three‐stranded antiparallel β‐sheet within the major groove. As the negatively charged DNA directly interacts with the positively charged residues (such as Arg and Lys) of INT‐DBD, the electrostatic interaction is expected to play an important role in the dynamical stability of the protein–DNA binding complex. In the current work, the combined use of quantum‐based polarized protein‐specific charge (PPC) for protein and polarized nucleic acid‐specific charge (PNC) for DNA were employed in molecular dynamics simulation to study the interaction dynamics between INT‐DBD and DNA. Our study shows that the protein–DNA structure is stabilized by polarization and the calculated protein–DNA binding free energy is in good agreement with the experimental data. Furthermore, our study revealed a positive correlation between the measured binding energy difference in alanine mutation and the occupancy of the corresponding residue's hydrogen bond. This correlation relation directly relates the contribution of a specific residue to protein–DNA binding energy to the strength of the hydrogen bond formed between the specific residue and DNA. © 2013 Wiley Periodicals, Inc.     

Behaviour of milk protein/polysaccharide systems in high sucrose

The stability of dairy emulsions containing both proteins and polysaccharides is mainly controlled by the nature of the protein–polysaccharide interaction. This work describes the behaviour of a micellar casein/locust bean gum (LBG) model system, and examines the effect of adding sucrose. For such systems a very high incompatibility has been demonstrated leading to thermodynamic separation into two distinct phases, one rich in protein and the other in polysaccharide. The second virial coefficient approach described by Edmond and Ogston was adopted to clarify the effect of sucrose on the micellar casein/LBG phase diagrams.     

Insights into the acylation mechanism of class A beta-lactamases from molecular dynamics simulations of the TEM-1 enzyme complexed with benzylpenicillin

Herein, we present results from molecular dynamics MD simulations ( approximately 1 ns) of the TEM-1 beta-lactamase in aqueous solution. Both the free form of the enzyme and its complex with benzylpenicillin were studied. During the simulation of the free enzyme, the conformation of the Omega loop and the interresidue contacts defining the complex H-bond network in the active site were quite stable. Most interestingly, the water molecule connecting Glu166 and Ser70 does not exchange with bulk solvent, emphasizing its structural and catalytic relevance. In the presence of the substrate, Ser130, Ser235, and Arg244 directly interact with the beta-lactam carboxylate via H-bonds, whereas the Lys234 ammonium group has only an electrostatic influence. These interactions together with other specific contacts result in a very short distance ( approximately 3 A) between the attacking hydroxyl group of Ser70 and the beta-lactam ring carbonyl group, which is a favorable orientation for nucleophilic attack. Our simulations also gave insight into the possible pathways for proton abstraction from the Ser70 hydroxyl group. We propose that either the Glu166 carboxylate-Wat1 or the substrate carboxylate-Ser130 moieties could abstract a proton from the nucleophilic Ser70.     

Docking glycosaminoglycans to proteins: analysis of solvent inclusion

Glycosaminoglycans (GAGs) are anionic polysaccharides, which participate in key processes in the extracellular matrix by interactions with protein targets. Due to their charged nature, accurate consideration of electrostatic and water-mediated interactions is indispensable for understanding GAGs binding properties. However, solvent is often overlooked in molecular recognition studies. Here we analyze the abundance of solvent in GAG-protein interfaces and investigate the challenges of adding explicit solvent in GAG-protein docking experiments. We observe PDB GAG-protein interfaces being significantly more hydrated than protein–protein interfaces. Furthermore, by applying molecular dynamics approaches we estimate that about half of GAG-protein interactions are water-mediated. With a dataset of eleven GAG-protein complexes we analyze how solvent inclusion affects Autodock 3, eHiTs, MOE and FlexX docking. We develop an approach to de novo place explicit solvent into the binding site prior to docking, which uses the GRID program to predict positions of waters and to locate possible areas of solvent displacement upon ligand binding. To investigate how solvent placement affects docking performance, we compare these results with those obtained by taking into account information about the solvent position in the crystal structure. In general, we observe that inclusion of solvent improves the results obtained with these methods. Our data show that Autodock 3 performs best, though it experiences difficulties to quantitatively reproduce experimental data on specificity of heparin/heparan sulfate disaccharides binding to IL-8. Our work highlights the current challenges of introducing solvent in protein-GAGs recognition studies, which is crucial for exploiting the full potential of these molecules for rational engineering.     

Effects of hydrophobic and dipole-dipole interactions on the conformational transitions of a model polypeptide

We studied the effects of hydrophobicity and dipole-dipole interactions between the nearest-neighbor amide planes on the secondary structures of a model polypeptide by calculating the free energy differences between different peptide structures. The free energy calculations were performed with low computational costs using the accelerated Monte Carlo simulation (umbrella sampling) method, with a bias-potential method used earlier in our accelerated molecular dynamics simulations. It was found that the hydrophobic interaction enhances the stability of alpha helices at both low and high temperatures but stabilizes beta structures only at high temperatures at which alpha helices are not stable. The nearest-neighbor dipole-dipole interaction stabilizes beta structures under all conditions, especially in the low temperature region where alpha helices are the stable structures. Our results indicate clearly that the dipole-dipole interaction between the nearest neighboring amide planes plays an important role in determining the peptide structures. Current research provides a more unified and quantitative picture for understanding the effects of different forms of interactions on polypeptide structures. In addition, the present model can be extended to describe DNA/RNA, polymer, copolymer, and other chain systems.     

The natural DNA bending angle in the lac repressor headpiece-O1 operator complex is determined by protein-DNA contacts and water release

We performed molecular dynamics simulations of the lac repressor headpiece-O1 operator complex for natural, over and underbent DNA to assess the factors that determine the natural DNA bending angle. At the natural angle, the specific and nonspecific contacts between the protein and DNA are optimized. Protein-DNA contacts show different angle dependences in the right and left sites, with the left site generally getting weaker and the right site getting stronger as the bending angle increases. Two entropic factors were identified as well: at the natural bending angle, water release and the quasiharmonic protein configurational entropy are maximized. The gain in protein configurational entropy might stem from an entropy-entropy compensation mechanism, in which a reduction in protein fluctuations is offset by a loss in correlations between the right and left sites.     

DNA recognition by the anthracycline antibiotic respinomycin D: NMR structure of the intercalation complex with d(AGACGTCT)2

Respinomycin D is a member of the anthracycline family of antitumour antibiotics that interact with double stranded DNA through intercalation. The clinical agents daunomycin and doxorubicin are the most well-studied of this class but have a relatively simple molecular architecture in which the pendant daunosamine sugar resides in the DNA minor groove. Respinomycin D, which belongs to the nogalamycin group of anthracyclines, possesses additional sugar residues at either end of the aglycone chromophore that modulate the biological activity but whose role in molecular recognition is unknown. We report the NMR structure of the respinomycin D-d(AGACGTCT)2 complex in solution derived from NOE restraints and molecular dynamics simulations. We show that the drug threads through the DNA double helix forming stabilising interactions in both the major and minor groove, the latter through a different binding geometry to that previously reported. The bicycloaminoglucose sugar resides in the major groove and makes specific contacts with guanine at the 5'-CpG intercalation site, however, the disaccharide attached at the C4 position plays little part in drug binding and DNA recognition and is largely solvent exposed.     

Insights into the recognition and association of transmembrane alpha-helices. The free energy of alpha-helix dimerization in glycophorin A

The free energy of alpha-helix dimerization of the transmembrane (TM) region of glycophorin A was estimated from a 125-ns molecular dynamics (MD) simulation in a membrane mimetic. The free energy profile was obtained by allowing the TM helical segments to diffuse reversibly along the reaction pathway. Partition of the potential of mean force into free energy components illuminates the critical steps of alpha-helix recognition and association. At large separations, the TM segments are pushed together by the solvent, allowing initial, but not necessarily native, interhelical interactions to occur. This early recognition stage precedes the formation of native contacts, which is accompanied by a tilt of the helices, characteristic of the dimeric structure. This step is primarily driven by the van der Waals helix-helix interactions. Free energy perturbation calculations of the L75A and I76A point mutations reveal a disruption in helix-helix association due to a loss of favorable dispersion interactions. Additional MD simulations of the native TM dimer and of a single alpha-helix confirm that, prior to association, individual alpha-helices are independently stable, in agreement with the "two-stage" model of integral membrane protein folding.