Conformational behaviour of glycomimetics: NMR and molecular modelling studies of the C-glycoside analogue of the disaccharide methyl beta-D-galactopyranosyl-(1-->3)-beta-D-glucopyranoside

The conformational behaviour of the C-glycoside beta-C-Gal-(1-->3)-beta-Glc-OMe (1) has been studied using a combination of molecular mechanics and NMR spectroscopy (proton-proton coupling constants and nuclear Overhauser effects). It is shown that the C-disaccharide populates two distinctive conformational families in solution, the normal syn-psi conformation, which is the predominating conformation of parent O-glycoside 2, and the anti-psi conformation, which has not been detected for the O-disaccharide.     

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism. Instead, we characterize the cleft-closing transition as a load and lock mechanism. We find that when two cross-cleft hydrogen bonds are disrupted the protein undergoes a rapid cleft opening transition. At the same time, the dynamical behavior of the cleft in the presence of the glutamate ligand is only weakly affected by the S652 peptide bond in its flipped conformation observed in the crystal structure. The residue E705 plays significant role in stabilization of the closed conformation via electrostatic interactions. The presence of the E705-K730 salt bridge seems to correlate strongly withthe cleft opening transition in the MD simulations.     

On the Bioactive Conformation of a Small Peptide and its Set of Thermodynamically Accessible Conformations

Abstract

In order to investigate the relationship between the bioactive conformation of a peptide and its set of thermodynamically accessible structures in solution, the conformational profile of the tetrapeptide Ac-Pro-Ala-Pro-Tyr-OH was characterized by computational methods. Search of the conformational space was performed within the molecular mechanics framework using the AMBER4.0 force field with an effective dielectric constant of 80. Unique structures of the peptide were compared with its bioactive conformation for the protein Streptomyces griseus Protease A, as taken from the crystal structure of the enzyme-peptide complex. The results show that the bound conformation is close to one of the unique conformations characterized in the conformational search of the isolated peptide. Moreover, the lowest energy minimum characterized in the conformational search exhibits large deviations when compared to the bound conformation of the crystal structure.     

Theoretical study of the structure of adenosine deaminase complexes with adenosine analogues: I. Aza-, deaza- and isomeric azadeazaanalogues of adenosine

The conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13 isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme (the 1ADD structure was used) within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of the ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1-deazaadenosine (1ADD) known from the X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of the bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.     

Investigations into the role of oxacarbenium ions in glycosylation reactions by ab initio molecular dynamics

We present a constrained ab initio molecular dynamics method that allows the modeling of the conformational interconversions of glycopyranosyl oxacarbenium ions. The model was successfully tested by estimating the barriers to ring inversion for two 4-substituted tetrahydropyranosyl oxacarbenium ions. The model was further extended to predict the pathways that connect the (4)H(3) half-chair conformation of 2,3,4,6-tetra-O-methyl-d-glucopyranosyl cation to its inverted (5)S(1) conformation and the (4)H(3) half-chair conformation of 2,3,4,6-tetra-O-methyl-d-mannopyranosyl cation to its inverted (3)E conformation. The modeled interconversion pathways reconcile a large body of experimental work on the acid-catalyzed hydrolysis of glycosides and the mechanisms of a number of glucosidases and mannosidases.     

Theoretical Study of the Structure of Adenosine Deaminase Complexes with Adenosine Analogues: I. Aza-, Deaza-, and Isomeric Azadeazaanalogues of Adenosine

Conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13-isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme [the structure of ADA complex with 1-deazaadenosine (1ADD) was used] within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1ADD known from X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.     

Role of substrate conformational features in the stereospecificity of aromatase

Hydroxylation of 19-hydroxyandrost-4-ene-3,17-dione (19OHA) by aromatase occurs at the 19-pro-R hydrogen, suggesting that the C19 group has a preferred conformation in the enzyme active site. X-ray crystallographic studies have led to a postulate that the steroid plays a role in determining this conformation. In an effort to quantitate the steroid's role, we estimated conformational constraints about the C10-C19 bond of 19OHA using molecular mechanics calculations. Rotational barriers less than or equal to 6 kcal/mol and energy differences between conformers less than or equal to 1 kcal/mol were found. We perturbed these conformational constraints by preparing an altered substrate, 19-hydroxyandrosta-4,6-diene-3,17-dione (19OHAD). The stereospecificity of aromatization for 19OHA and 19OHAD was found to be the same. Thus, theoretical and experimental approaches both indicate that conformational constraints intrinsic to 19OHA cannot be a major determinant in the sterospecificity of its oxidation by aromatase.     

Conformational proofreading: the impact of conformational changes on the specificity of molecular recognition

To perform recognition, molecules must locate and specifically bind their targets within a noisy biochemical environment with many look-alikes. Molecular recognition processes, especially the induced-fit mechanism, are known to involve conformational changes. This raises a basic question: Does molecular recognition gain any advantage by such conformational changes? By introducing a simple statistical-mechanics approach, we study the effect of conformation and flexibility on the quality of recognition processes. Our model relates specificity to the conformation of the participant molecules and thus suggests a possible answer: Optimal specificity is achieved when the ligand is slightly off target; that is, a conformational mismatch between the ligand and its main target improves the selectivity of the process. This indicates that deformations upon binding serve as a conformational proofreading mechanism, which may be selected for via evolution.     

Conformation of the synthetic DNA poly(amino2dA-dT) duplex in high-salt and aqueous alcohol solutions.

It has previously been demonstrated by other workers that the duplex of a synthetic DNA poly(amino2dA-dT) undergoes a salt-induced conformational isomerization. We show in the present work using circular dichroism that the same isomerization is induced in poly(amino2dA-dT) by various alcohols. The isomerization was originally identified as the B-to-Z and then B-to-A conformational transition of DNA but we demonstrate that the high-salt or alcohol conformation of poly (amino2dA-dT) is the non Z-DNA zig-zag double helix we have previously observed with poly(dA-dT) and called X-DNA. X-DNA is a cesium cation specific conformation of poly(dA-dT) while no similar cation specificity is observed with poly(amino2dA-dT). Thus it appears that the extra amino group attached to A and cesium cations make the same thing; they probably dehydrate the double helix minor groove and relieve its conformational variability. Poly(amino2dA-dT) is exceptionally stable in X-DNA and conditions inducing it are mild, which opens the door to assess its molecular structure.     

Virtual and solution conformations of oligosaccharides

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.     

The length of the reactive center loop modulates the latency transition of plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three beta-sheets and several alpha-helices. Despite the similarity of its structure with those of other serpins, PAI-1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under physiological conditions. For the conformational conversion to occur, the reactive center loop (RCL) of PAI-1 must be mobilized and inserted into the major beta-sheet, A sheet. In an effort to understand how the structural conversion is regulated in this conformationally labile serpin, we modulated the length of the RCL of PAI-1. We show that releasing the constraint on the RCL by extension of the loop facilitates a conformational transition of PAI-1 to a stable state. Biochemical data strongly suggest that the stabilization of the transformed conformation is owing to the insertion of the RCL into A beta-sheet, as in the known latent form. In contrast, reducing the loop length drastically retards the conformational change. The results clearly show that the constraint on the RCL is a factor that regulates the conformational transition of PAI-1.     

A new bacterial polysaccharide (YAS34). I. Characterization of the conformations and conformational transition

This paper concerns the study of the conformational transition of a new exopolysaccharide (YAS34) using experimental techniques such as optical rotation, conductimetric and microcalorimetric measurements as a function of temperature. The behaviors of this polysaccharide in the acid or sodium salt form are compared; a deacetylated sample is also prepared to demonstrate the role of substituents. For the native structure (never heated), a conformational transition is observed but the deacetylated polysaccharide exhibits no ordered conformation. Multidetection size exclusion chromatography (SEC) analyses and conductimetric experiments allowed to determine the nature of each conformation and the molecular dimensions. From these results, it is suggested that the native conformation is a double helix which by heating over T(m) (temperature corresponding to half conformational transition) dissociates into disordered single chains. In the acid and sodium salt forms, by cooling below T(m), an ordered conformation is restored. This conformation seems to be an intramolecular double helix 'hairpin-like turn' (called renatured conformation). Nevertheless an irreversible denaturation is obtained progressively in the sodium salt form when the time of heating over T(m) increases. The conformation of the deacetylated polysaccharide corresponds to that of a single flexible chain (disordered conformation). The conformational transition for the native conformation was studied also in relation to the polyelectrolytic character of the polysaccharide: stability as a function of salt nature and salt and polymer concentrations was investigated for the polymer initially in the sodium and acid forms.     

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.     

Mass-weighted molecular dynamics simulation and conformational analysis of polypeptide.

Atomic motions in protein molecules have been studied by molecular dynamics (MD) simulations; dynamics simulation methods have also been employed in conformational studies of polypeptide molecules. It was found that when atomic masses are weighted, the molecular dynamics method can significantly increase the sampling of dihedral conformation space in such studies, compared to a conventional MD simulation of the same total simulation time length. Herein the theoretical study of molecular conformation sampling by the molecular dynamics-based simulation method in which atomic masses are weighted is reported in detail; moreover, a numerical scheme for analyzing the extensive conformational sampling in the simulation of a tetrapeptide amide molecule is presented. From numerical analyses of the mass-weighted molecular dynamics trajectories of backbone dihedral angles, low-resolution structures covering the entire backbone dihedral conformation space of the molecule were determined, and the distribution of rotationally stable conformations in this space were analyzed quantitatively. The theoretical analyses based on the computer simulation and numerical analytical methods suggest that distinctive regimes in the conformational space of the peptide molecule can be identified.     

Free-energy landscapes of the coupled conformational transition and inclusion processes of altro-cyclodextrins

Abstract

Mono-altro-cyclodextrin (altro-CD) may undergo a conformational change of its altropyranose unit when encapsulating guest molecules of different sizes. This conformational transition is found to be coupled to the inclusion processes. In the present contribution, the possible conformational transition pathways in the four (self-)inclusion processes of altro-α and -β-CDs with moieties of variant shapes are explored from the insights of free-energy calculations. The two-dimensional free-energy landscapes characterising the coupled (self-)inclusion and isomerisation processes are determined, and the lowest free-energy pathways (LFEP) connecting the minima of the landscapes are located. The conformational statistics of the altropyranose units along the LFEPs reveal different transition pathways in the four (self-)inclusion processes. It can be concluded that when accommodating a free bulky guest molecule, the altropyranose unit will adjust its conformation to match the guest. However, such induced fit effect in the self-complexation of altro-CD derivatives will be weakened. The conformation of the altropyranose unit changes accompanying the self-complexation, but always adopts the ⁴C₁ one in the self-inclusion complex, irrespective of the shape of the guest moieties. The present results help determine the transition states of the (self-)inclusion processes of CDs and further improve the understanding of the mechanical properties of CD-based molecular shuttles.     

Molecular Dynamic Simulation Reveals Damaging Impact of RAC1 F28L Mutation in the Switch I Region

Ras-related C3 botulinum toxin substrate 1 (RAC1) is a plasma membrane-associated small GTPase which cycles between the active GTP-bound and inactive GDP-bound states. There is wide range of evidences indicating its active participation in inducing cancer-associated phenotypes. RAC1 F28L mutation (RAC^F28L) is a fast recycling mutation which has been implicated in several cancer associated cases. In this work we have performed molecular docking and molecular dynamics simulation (~0.3 μs) to investigate the conformational changes occurring in the mutant protein. The RMSD, RMSF and NHbonds results strongly suggested that the loss of native conformation in the Switch I region in RAC1 mutant protein could be the reason behind its oncogenic transformation. The overall results suggested that the mutant protein attained compact conformation as compared to the native. The major impact of mutation was observed in the Switch I region which might be the crucial reason behind the loss of interaction between the guanine ring and F28 residue.     

Guest-dependent conformation of 18-crown-6 tetracarboxylic acid: relation to chiral separation of racemic amino acids

(+)-18-crown-6 tetracarboxylic acid (18C6H(4)) has been used as a chiral selector for various amines and amino acids. To further clarify the structural scaffold of 18C6H(4) for chiral separation, single crystal X-ray analysis of its glycine(+) (1), H3O+ (2), H5O2+ (3), NH4+ (4), and 2CH3NH3+ (5) complexes was performed and the guest-dependent conformation of 18C6H(4) was investigated. The crown ether ring of 18C6H4 in 3, 4, and 5 took a symmetrical C2 or C2-like conformation, whereas that in 1 and 2 took an asymmetric C1 conformation, which is commonly observed in complexes with various optically active amino acids. The overall survey of the present and related complexes suggests that the molecular conformation of 18C6H4 is freely changeable within an allowable range, depending on the molecular shape and interaction mode with the cationic guest. On the basis of the present results, we propose the allowable conformational variation of 18C6H4 and a possible transition pathway from its primary conformation to the conformation suitable for chiral separation of racemic amines and amino acids.     

Acid destabilization of the solution conformation of Bcl-xL does not drive its pH-dependent insertion into membranes

Regulation of programmed cell death by Bcl-xL is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH-dependence similar to the translocation domain of diphtheria toxin, where an acid-induced molten globule conformation in the absence of lipid vesicles mediates the change from solution to membrane conformations. By contrast, Bcl-xL deltaTM in the absence of lipid vesicles exhibits no gross conformational changes upon acidification as observed by near- and far-UV circular dichroism spectropolarimetry. Additionally, no significant local conformational changes upon acidification were observed by heteronuclear NMR spectroscopy of Bcl-xL deltaTM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl-xL deltaTM (deltaG(o)) was determined to be 15.8 kcal x mol(-1). Surprisingly, under conditions that favor a membrane conformation (pH 4.9), deltaG(o) was 14.6 kcal x mol(-1). These results differ from those obtained with many other membrane-insertable proteins where acid-induced destabilization is important. Therefore, other contributions must be necessary to destabilize the solution conformation Bcl-xL and favor the membrane conformation at pH 4.9. Such contributions might include the presence of a negatively charged membrane or an electrostatic potential across the membrane. Thus, for proteins that adopt both solution and membrane conformations, an obligatory molten globule intermediate may not be necessary. The absence of a molten globule intermediate might have evolved to protect Bcl-xL from intracellular proteases as it undergoes this conformational change essential for its activity.