Conformational flexibility of a protein-carbohydrate complex and the structure and ordering of surrounding water

Protein-carbohydrate non-covalent interactions are important to understand various biological processes in living organisms. One of the important issues in protein-carbohydrate binding is how the protein identifies the target carbohydrate and recognizes its conformational features. Surrounding water molecules are expected to play a critical role not only in mediating the recognition process but also in maintaining the structure of the complex. We carried out atomistic molecular dynamics (MD) simulations of an aqueous solution of the protein-carbohydrate complex formed between the hyaluronan binding domain (HABD) of the murine Cd44 protein and the octasaccharide hyaluronan (HA(8)). The conformational flexibilities of the protein and the carbohydrate, and the microscopic structure and ordering of water molecules around them in the complexed form have been explored. It is revealed that the formation of the complex is associated with significant immobilization of the monosaccharide units of the carbohydrate moiety that are involved in binding. Further, reduction in water densities around the binding residues of the two molecules in the complex with respect to their free forms clearly demonstrated that the recognition between the protein and the carbohydrate is facilitated by removal of a fraction of water molecules from regions around the binding domains.     

Dynamic properties of water around a protein-DNA complex from molecular dynamics simulations

Formation of protein-DNA complex is an important step in regulation of genes in living organisms. One important issue in this problem is the role played by water in mediating the protein-DNA interactions. In this work, we have carried out atomistic molecular dynamics simulations to explore the heterogeneous dynamics of water molecules present in different regions around a complex formed between the DNA binding domain of human TRF1 protein and a telomeric DNA. It is demonstrated that such heterogeneous water motions around the complex are correlated with the relaxation time scales of hydrogen bonds formed by those water molecules with the protein and DNA. The calculations reveal the existence of a fraction of extraordinarily restricted water molecules forming a highly rigid thin layer in between the binding motifs of the protein and DNA. It is further proved that higher rigidity of water layers around the complex originates from more frequent reformations of broken water-water hydrogen bonds. Importantly, it is found that the formation of the complex affects the transverse and longitudinal degrees of freedom of surrounding water molecules in a nonuniform manner.     

Differential flexibility of the secondary structures of lysozyme and the structure and ordering of surrounding water molecules

We have performed an atomistic molecular dynamics simulation of an aqueous solution of hen egg-white lysozyme at room temperature with explicit water molecules. Several analyses have been carried out to explore the differential flexibility of the secondary structural segments of the protein and the structure and ordering of water around them. It is found that the overall flexibility of the protein molecule is primarily controlled by few large-amplitude bistable motions exhibited by two coils; one connecting two α-helical segments in domain-1 and the other connecting a 3(10) helix and a β-sheet in domain-2 of the protein. The heterogeneous structuring of water around the segments of the protein has been found to depend on the degree of exposure of the segments to water. The ordering of water molecules around the protein segments and their tagged potential energies have been found to be anticorrelated with each other. Some of these findings can be verified by suitable experimental studies.     

Hydration structure of human lysozyme investigated by molecular dynamics simulation and cryogenic X-ray crystal structure analyses: on the correlation between crystal water sites,solvent density,and solvent dipole

The hydration structure of human lysozyme was studied with cryogenic X-ray diffraction experiment and molecular dynamics simulations. The crystal structure analysis at a resolution of 1.4 A provided 405 crystal water molecules around the enzyme. In the simulations at 300 K, the crystal structure was immersed in explicit water molecules. We examined correlations between crystal water sites and two physical quantities calculated from the 1-ns simulation trajectories: the solvent density reflecting the time-averaged distribution of water molecules, and the solvent dipole measuring the orientational ordering of water molecules around the enzyme. The local high solvent density sites were consistent with the crystal water sites, and better correlation was observed around surface residues with smaller conformational fluctuations during the simulations. Solvent dipoles around those sites exhibited coherent and persistent ordering, indicating that the hydration water molecules at the crystal water sites were highly oriented through the interactions with hydrophilic residues. Those water molecules restrained the orientational motions of adjoining water molecules and induced a solvent dipole field, which was persistent during the simulations around the enzyme. The coherent ordering was particularly prominent in and around the active site cleft of the enzyme. Because the ordering was significant up to the third to fourth solvent layer region from the enzyme surface, the coherently ordered solvent dipoles likely contributed to the molecular recognition of the enzyme in a long-distance range. The present work may provide a new approach combining computational and the experimental studies to understand protein hydration.     

Restricted dynamics of water around a protein-carbohydrate complex: Computer simulation studies

Water-mediated protein-carbohydrate interaction is a complex phenomenon responsible for different biological processes in cellular environment. One of the unexplored but important issues in this area is the role played by water during the recognition process and also in controlling the microscopic properties of the complex. In this study, we have carried out atomistic molecular dynamics simulations of a protein-carbohydrate complex formed between the hyaluronan binding domain of the murine Cd44 protein and the octasaccharide hyaluronan in explicit water. Efforts have been made to explore the heterogeneous influence of the complex on the dynamic properties of water present in different regions around it. It is revealed from our analyses that the heterogeneous dynamics of water around the complex are coupled with differential time scales of formation and breaking of hydrogen bonds at the interface. Presence of a highly rigid thin layer of motionally restricted water molecules bridging the protein and the carbohydrate in the common region of the complex has been identified. Such water molecules are expected to play a crucial role in controlling properties of the complex. Importantly, it is demonstrated that the formation of the protein-carbohydrate complex affects the transverse and longitudinal degrees of freedom of the interfacial water molecules in a heterogeneous manner.     

De novo ligand design with explicit water molecules: an application to bacterial neuraminidase

Most computer-aided drug design methods ignore the presence of crystallographically-determined water molecules in the binding site of a target protein. In this paper, our de novo ligand design methods are applied to the X-ray crystal structure of bacterial neuraminidase in the presence of some selected water molecules. We have found that, for this particular protein, the complete removal of all bound water molecules leads to difficulties in generating any potential ligands if the unsatisfied hydrogen-bonding sitepoints left by removing these water molecules are to be satisfied by a ligand. As more of the crystallographically determined water molecules are allowed in the binding site, it becomes much easier to generate ligands in larger numbers and with wider chemical diversity. This example shows that, in some cases, bound water molecules can be more accessible for hydrogen bonding to an incoming ligand than the actual protein binding sitepoints associated with them. From the point of view of de novo ligand design, water molecules can thus act as versatile amphiprotic hydrogen-bonding sitepoints and reduce the conformational constraints of a particular binding site.     

Structure-based design technology contour and its application to the design of Renin inhibitors

It is well-known that the structure-based design approach has had a measurable impact on the drug discovery process in identifying novel and efficacious therapeutic agents for a variety of disease targets. The de novo design approach has inherent potential to generate novel molecules that best fit into a protein binding site when compared to all of the computational methods applied to structure-based design. In its initial attempts, this approach did not achieve much success due to technical hurdles. More recently, the algorithmic advancements in the methodologies and clever strategies developed to design drug-like molecules have improved the success rate. We describe a state-of-the-art structure-based design technology called Contour and provide details of the algorithmic enhancements we have implemented. Contour was designed to create novel drug-like molecules by assembling synthetically viable fragments in the protein binding site using a high-resolution crystal structure of the protein. The technology consists of a sophisticated growth algorithm and a novel scoring function based on a directional model. The growth algorithm generates molecules by dynamically selecting only those fragments from the fragment library that are complementary to the binding site, and assembling them by sampling the conformational space for each attached fragment. The scoring function embodying the essential elements of the binding interactions aids in the rank ordering of grown molecules and helps identify those that have high probability of exhibiting activity against the protein target of interest. The application of Contour to identify inhibitors against human renin enzyme eventually leading to the clinical candidate VTP-27,999 will be discussed here.     

Microcalorimetrics studies of the thermodynamics and binding mechanism between L-tyrosinamide and aptamer

In recent years, several high-resolution structures of aptamer complexes have shed light on the binding mode and recognition principles of aptamer complex interactions. In some cases, however, the aptamer complex binding behavior and mechanism are not clearly understood, especially with the absence of structural information. In this study, it was demonstrated that isothermal titration calorimetry (ITC) and circular dichroism (CD) were useful tools for studying the fundamental binding mechanism between a DNA aptamer and L-tyrosinamide (L-TyrNH2). To gain further insight into this behavior, thermodynamic and conformational measurements under different parameters such as salt concentration, temperature, pH value, analogue of L-TyrNH2, and metal ion were carried out. The thermodynamic signature along with the coupled CD spectral change suggest that this binding behavior is an enthalpy-driven process, and the aptamer has a conformational change from B-form to A-form. The results showed that the interaction is an induced fit binding, and the driving forces in this binding behavior may include electrostatic interactions, hydrophobic effects, hydrogen bonding, and the binding-linked protonation process. The amide group and phenolic hydroxyl group of the L-TyrNH2 play a vital role in this binding mechanism. In addition, it should be noted that Mg(2+) not only improves binding affinity but also helps change the structure of the DNA aptamer.     

Co-operative intra-protein structural response due to protein–protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.     

Thermodynamics of buried water clusters at a protein-ligand binding interface

The structure of the complex of cyclophilin A (CypA) with cyclosporin A (CsA, 1) shows a cluster of four water molecules buried at the binding interface, which is rearranged when CsA is replaced by (5-hydroxynorvaline)-2-cyclosporin (2). The thermodynamic contributions of each bound water molecule in the two complexes are explored with the inhomogeneous fluid solvation theory and molecular dynamics simulations. Water (WTR) 133 in complex 1 contributes little to the binding affinity, while WTR6 and 7 in complex 2 play an essential role in mediating protein-ligand binding with a hydrogen bond network. The calculations reveal that the rearrangement of the water molecules contributes favorably to the binding affinity, even though one of them is displaced going from ligand 1 to 2. Another favorable contribution comes from the larger protein-ligand interactions of ligand 2. However, these favorable contributions are not sufficient to overcome the unfavorable desolvation free energy change and the conformational entropy of the hydroxylpropyl group of ligand 2 in the complex, leading to a lower binding affinity of ligand 2. These physical insights may be useful in the development of improved scoring functions for binding affinity prediction.     

Atomic force microscopy of DNA at high humidity: irreversible conformational switching of supercoiled molecules

Three topologically different double-stranded DNA molecules of the same size (bps) have been imaged in air on mica using amplitude modulation atomic force microscopy (AM AFM) under controlled humidity conditions. At very high relative humidity (>90% RH), localized conformational changes of the DNA were observed, while at lower RH, the molecules remained immobile. The conformational changes occurred irreversibly and were driven principally by superhelical stress stored in the DNA molecules prior to binding to the mica surface. The binding mechanism of the DNA to the mica (surface equilibration versus kinetic trapping) modulated the extent of the conformational changes. In cases where DNA movement was observed, increased kinking of the DNA was seen at high humidity when more surface water was present. Additionally, DNA condensation behavior was also present in localized regions of the molecules. This study illustrates that changes in the tertiary structure of DNA can be induced during AFM imaging at high humidity on mica. We propose that AM AFM in high humidity will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution.     

Water binding sites in 2-para- and 2-ortho-fluorophenylethanol: a high-resolution UV experiment and ab initio calculations

The singly hydrated complexes of the flexible prototype molecules 2-para-fluorophenylethanol and 2-ortho-fluorophenylethanol have been investigated by combination of high-resolution resonance-enhanced two-photon ionization spectroscopy in a cold supersonic beam and quantum chemistry ab initio calculations. We have identified the conformational structures of the above complexes, which correspond to water binding to the most stable gauche monomer's conformers in both cases. No structural changes of the host molecules upon the attachment of a single water molecule have been found. For the 2-ortho-fluorophenylethanol-water complex we have observed an additional structure with one of the higher-in-energy gauche conformers of the monomer. This corroborates the assumption that the complexation with water stabilizes the higher-energy conformer of the monomer, precluding it from relaxation to the lowest-energy geometry.     

蛋白质分子与配体结合过程构象变化的研究

蛋白质分子与配体的作用模式主要有直接的环区结合及铰链式结合两种方式。针对这两种不同的作用方式,我们提出采用不同的策略进行结合过程的构象研究。对于直接的环区结合模式,通过建立环区主链构象库,来实现蛋白质环区与配体的准柔性对接,并以链霉抗生物素蛋白体系为例对构象库建立的可行性进行了验证计算。对铰链结合方式,采用分步对接的方法进行计算,并具体应用于HIV蛋白酶与其小分子配体的结合过程。计算结果表明,这两种处理方法分别能较好地模拟不同类型的蛋白质与配体结合的的构象变化。     

Energetic Basis of AGCGA‐Rich DNA Folding into a Tetrahelical Structure

It was recently discovered that, besides well‐known G‐quadruplexes and i‐motifs, DNA may adopt another type of noncanonical structure called AGCGA‐quadruplexes. Here, the folding of the VK2 fragment from the regulatory region of the PLEKHG3 gene is studied and, for the first time, the energetic contributions that stabilize this unique fold are described. Similarly to the B‐DNA, it is stabilized by hydrophobic desolvation and, in contrast to G‐quadruplexes, also by specific binding of water molecules. Compared to B‐DNA, VK2 folding is enthalpically less favorable due to poorer base‐stacking interactions, resulting in substantial conformational flexibility. This entropically favorable conformational “breathing” stabilizes the AGCGA‐quadruplexes. In conclusion, AGCGA‐quadruplexes have a distinguishing thermodynamic fingerprint and the corresponding driving forces enabling their folding are consistent with the observed structural features.     

Molecular dynamics and docking simulations as a proof of high flexibility in <Emphasis Type="Italic">E. coli</Emphasis> FabH and its relevance for accurate inhibitor modeling

Bacterial β-ketoacyl-acyl carrier protein synthase III (FabH) has become an attractive target for the development of new antibacterial agents which can overcome the increased resistance of these pathogens to antibiotics in clinical use. Despite several efforts have been dedicated to find inhibitors for this enzyme, it is not a straightforward task, mainly due its high flexibility which makes difficult the structure-based design of FabH inhibitors. Here, we present for the first time a Molecular Dynamics (MD) study of the E. colil FabH enzyme to explore its conformational space. We compare the flexibility of this enzyme for the unliganded protein and an enzyme-inhibitor complex and find a correspondence between our modeling results and the experimental evidence previously reported for this enzyme. Furthermore, through a 100 ns MD simulation of the unliganded enzyme we extract useful information related to the concerted motions that take place along the principal components of displacement. We also establish a relation between the presence of water molecules in the oxyanion hole with the conformational stability of structural important loops. Representative conformations of the binding pocket along the whole trajectory of the unliganded protein are selected through cluster analysis and we find that they contain a conformational diversity which is not provided by the X-ray structures of ecFabH. As a proof of this last hypothesis, we use a set of 10 FabH inhibitors and show that they cannot be correctly modeled in any available X-ray structure, while by using our set of conformations extracted from the MD simulations, this task can be accomplish. Finally, we show the ability of short MD simulations for the refinement of the docking binding poses and for MM-PBSA calculations to predict stable protein-inhibitor complexes in this enzyme.     

The Tetracycline Repressor-A Paradigm for a Biological Switch

The excessive use of antibiotics has enabled bacteria to develop resistance through a variety of mechanisms. The most common bacteriostatic action of the broad-spectrum antibiotic tetracycline (Tc) is by the inactivation of the bacterial ribosome so that the protein biosynthesis is interrupted and the bacteria die. The most common mechanism of resistance in gram-negative bacteria against Tc is associated with the membrane-intrinsic protein TetA, which exports invaded Tc out of the bacterial cell before it can attack its target, the ribosome. The expression of TetA is tightly regulated by the homodimeric Tet repressor (TetR)(2), which binds specifically with two helix-turn-helix motifs of operator DNA (tetO; K(ass) approximately 10(11) M(-1)) located upstream from the tetA gene on a plasmid or transposon. When Tc diffuses into the cell it chelates Mg(2+) and the complex [MgTc](+) binds to (TetR)(2) to form the induced complex (TetR small middle dot[MgTc](+))(2). This process is associated with conformational changes, which sharply reduce the affinity of (TetR)(2) to tetO, so that expression of TetA can take place, thus conferring resistance to the bacteria cells against Tc. Crystallographic studies show sequence-specific protein-nucleic acid interactions in the (TetR)(2) small middle dottetO complex and how the binding of two [MgTc](+) to (TetR)(2) enforces conformational changes that are stabilized by cooperative binding of two chains of eight water molecules each so that the formed (TetR small middle dot[MgTc](+))(2) is no longer able to recognize and bind to tetO. Since the switching mechanisms of the TetR/[MgTc](+) system is so tight, it has proven very useful in the regulation of eukaryotic gene expression and may also be applicable in gene therapy.     

The CK2 alpha/CK2 beta interface of human protein kinase CK2 harbors a binding pocket for small molecules

The Ser/Thr kinase CK2 (previously called casein kinase 2) is composed of two catalytic chains (CK2 alpha) attached to a dimer of noncatalytic subunits (CK2 beta). CK2 is involved in suppression of apoptosis, cell survival, and tumorigenesis. To investigate these activities and possibly affect them, selective CK2 inhibitors are required. An often-used CK2 inhibitor is 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In a complex structure with human CK2 alpha, DRB binds to the canonical ATP cleft, but additionally it occupies an allosteric site that can be alternatively filled by glycerol. Inhibition kinetic studies corroborate the dual binding mode of the inhibitor. Structural comparisons reveal a surprising conformational plasticity of human CK2 alpha around both DRB binding sites. After local rearrangement, the allosteric site serves as a CK2 beta interface. This opens the potential to construct molecules interfering with the CK2 alpha/CK2 beta interaction.     

Analytical assays based on detecting conformational changes of single molecules.

One common strategy for the detection of biomolecules is labeling either the target itself or an antibody that binds to it. Herein, a different approach, based on detecting the conformational change of a probe molecule induced by binding of the target is discussed. That is, what is being detected is not the presence of the target or the probe, but the conformational change of the probe. Recently, a single-molecule sensor has been developed that exploits this mechanism to detect hybridization of a single DNA oligomer to a DNA probe, as well as specific binding of a single protein to a DNA probe. Biomolecular recognition often involves large conformational changes of the molecules involved, and therefore this strategy may be applicable to other assays.     

Structural basis of telomeric RNA quadruplex--acridine ligand recognition

Human telomeric DNA is now known to be transcribed into noncoding RNA sequences, termed TERRA. These sequences, which are believed to play roles in the regulation of telomere function, can form higher-order quadruplex structures and may themselves be the target of therapeutic intervention. The crystal structure of a TERRA quadruplex-acridine small-molecule complex at a resolution of 2.60 ?, is reported here and contrasts remarkably with the structure of the analogous DNA quadruplex complex. The bimolecular RNA complex has a parallel-stranded topology with propeller-like UUA loops. These loops are held in particular conformations by multiple hydrogen bonds involving the O2' hydroxyl groups of the ribonucleotide sugars and play an active role in binding the acridine molecules to the RNA quadruplex. By contrast, the analogous DNA quadruplex complex has simpler 1:1 acridine binding, with no loop involvement. There are significant loop conformational changes in the RNA quadruplex compared to the native TERRA quadruplex (Collie, G. W.; Haider, S. M.; Neidle, S.; Parkinson, G. N. Nucleic Acids Res. 2010, 38, 5569 - 5580), which have implications for the future design of small molecules targeting TERRA quadruplexes, and RNA quadruplexes more generally.