A theoretical multiscale treatment of protein-protein electron transfer: The ferredoxin/ferredoxin-NADP(+) reductase and flavodoxin/ferredoxin-NADP(+) reductase systems.

In the photosynthetic electron transfer (ET) chain, two electrons transfer from photosystem I to the flavin-dependent ferredoxin-NADP(+) reductase (FNR) via two sequential independent ferredoxin (Fd) electron carriers. In some algae and cyanobacteria (as Anabaena), under low iron conditions, flavodoxin (Fld) replaces Fd as single electron carrier. Extensive mutational studies have characterized the protein-protein interaction in FNR/Fd and FNR/Fld complexes. Interestingly, even though Fd and Fld share the interaction site on FNR, individual residues on FNR do not participate to the same extent in the interaction with each of the protein partners, pointing to different electron transfer mechanisms. Despite of extensive mutational studies, only FNR/Fd X-ray structures from Anabaena and maize have been solved; structural data for FNR/Fld remains elusive. Here, we present a multiscale modelling approach including coarse-grained and all-atom protein-protein docking, the QM/MM e-Pathway analysis and electronic coupling calculations, allowing for a molecular and electronic comprehensive analysis of the ET process in both complexes. Our results, consistent with experimental mutational data, reveal the ET in FNR/Fd proceeding through a bridge-mediated mechanism in a dominant protein-protein complex, where transfer of the electron is facilitated by Fd loop-residues 40-49. In FNR/Fld, however, we observe a direct transfer between redox cofactors and less complex specificity than in Fd; more than one orientation in the encounter complex can be efficient in ET.

An alternative mechanism for the methylation of phosphoethanolamine catalyzed by Plasmodium falciparum phosphoethanolamine methyltransferase.

The phosphobase methylation pathway catalyzed by the phosphoethanolamine methyltransferase in Plasmodium falciparum (PfPMT), the malaria parasite, offers an attractive target for anti-parasitic drug development. PfPMT methylates phosphoethanolamine (pEA) to phosphocholine for use in membrane biogenesis. Quantum mechanics and molecular mechanics (QM/MM) calculations tested the proposed reaction mechanism for methylation of pEA involving the previously identified Tyr-19-His-132 dyad, which indicated an energetically unfavorable mechanism. Instead, the QM/MM calculations suggested an alternative mechanism involving Asp-128. The reaction coordinate involves the stepwise transfer of a proton to Asp-128 via a bridging water molecule followed by a typical Sn2-type methyl transfer from S-adenosylmethionine to pEA. Functional analysis of the D128A, D128E, D128Q, and D128N PfPMT mutants shows a loss of activity with pEA but not with the final substrate of the methylation pathway. X-ray crystal structures of the PfPMT-D128A mutant in complex with S-adenosylhomocysteine and either pEA or phosphocholine reveal how mutation of Asp-128 disrupts a hydrogen bond network in the active site. The combined QM/MM, biochemical, and structural studies identify a key role for Asp-128 in the initial step of the phosphobase methylation pathway in Plasmodium and provide molecular insight on the evolution of multiple activities in the active site of the PMT.

Structure and mechanisms of transport of human Asc1/CD98hc amino acid transporter.

Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4-3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.

G protein-specific mechanisms in the serotonin 5-HT2A receptor regulate psychosis-related effects and memory deficits.

G protein-coupled receptors (GPCRs) are sophisticated signaling machines able to simultaneously elicit multiple intracellular signaling pathways upon activation. Complete (in)activation of all pathways can be counterproductive for specific therapeutic applications. This is the case for the serotonin 2 A receptor (5-HT2AR), a prominent target for the treatment of schizophrenia. In this study, we elucidate the complex 5-HT2AR coupling signature in response to different signaling probes, and its physiological consequences by combining computational modeling, in vitro and in vivo experiments with human postmortem brain studies. We show how chemical modification of the endogenous agonist serotonin dramatically impacts the G protein coupling profile of the 5-HT2AR and the associated behavioral responses. Importantly, among these responses, we demonstrate that memory deficits are regulated by Gαq protein activation, whereas psychosis-related behavior is modulated through Gαi1 stimulation. These findings emphasize the complexity of GPCR pharmacology and physiology and open the path to designing improved therapeutics for the treatment of stchizophrenia.

Electron transfer in proteins: theory, applications and future perspectives.

The study of electron transfer (ET) by means of computational techniques has experienced a great development in the last few decades. In particular, understanding the atomic details of its mechanism in complex biological systems is currently possible with a large range of different in silico modelling tools. We review here some theories and representative major contributions to this development. We also underline some of our group's main inputs, focusing on long range and protein-protein electron transfer, and analyse future perspectives. At the end of the article, we emphasize the importance of the basic electron transfer knowledge in the frame of medical and bioengineering applications: mitochondrial therapeutic targets, bioengineering for clean energy, and biosensors.

Atomic detail of chemical transformation at the transition state of an enzymatic reaction.

Transition path sampling (TPS) has been applied to the chemical step of human purine nucleoside phosphorylase (PNP). The transition path ensemble provides insight into the detailed mechanistic dynamics and atomic motion involved in transition state passage. The reaction mechanism involves early loss of the ribosidic bond to form a transition state with substantial ribooxacarbenium ion character, followed by dynamic motion from the enzyme and a conformational change in the ribosyl group leading to migration of the anomeric carbon toward phosphate, to form the product ribose 1-phosphate. Calculations of the commitment probability along reactive paths demonstrated the presence of a broad energy barrier at the transition state. TPS identified (i) compression of the O4'...O5' vibrational motion, (ii) optimized leaving group interactions, and (iii) activation of the phosphate nucleophile as the reaction proceeds through the transition state region. Dynamic motions on the femtosecond timescale provide the simultaneous optimization of these effects and coincide with transition state formation.

Monte Carlo simulations using PELE to identify a protein-protein inhibitor binding site and pose.

In silico binding site location and pose prediction for a molecule targeted at a large protein surface is a challenging task. We report a blind test with two peptidomimetic molecules that bind the flu virus hemagglutinin (HA) surface antigen, JNJ7918 and JNJ4796 (recently disclosed in van Dongen et al., Science, 2019, 363). Tests with a series of conventional approaches such as rigid (receptor) docking against available X-ray crystal structures or against an ensemble of structures generated by quick methodologies (NMA, homology modeling) gave mixed results, due to the shallowness and flexibility of the binding site and the sheer size of the target. However, tests with our Monte Carlo platform PELE in two protocols involving either exploration of the whole protein surface (global exploration), or the latter followed by refinement of best solutions (local exploration) yielded remarkably good results by locating the actual binding site and generating binding modes that recovered all native contacts found in the X-ray structures. Thus, the Monte Carlo scheme of PELE seems promising as a quick methodology to overcome the challenge of identifying entirely unknown binding sites and modes for protein-protein disruptors.

Δ9 -Tetrahydrocannabinolic acid alleviates collagen-induced arthritis: Role of PPARγ and CB1 receptors.

BACKGROUND AND PURPOSE: Δ9 -Tetrahydrocannabinolic acid (Δ9 -THCA-A), the precursor of Δ9 -THC, is a non-psychotropic phytocannabinoid that shows PPARγ agonist activity. Here, we investigated the ability of Δ9 -THCA-A to modulate the classic cannabinoid CB1 and CB2 receptors and evaluated its anti-arthritis activity in vitro and in vivo. EXPERIMENTAL APPROACH: Cannabinoid receptors binding and intrinsic activity, as well as their downstream signalling, were analysed in vitro and in silico. The anti-arthritis properties of Δ9 -THCA-A were studied in human chondrocytes and in the murine model of collagen-induced arthritis (CIA). Plasma disease biomarkers were identified by LC-MS/MS based on proteomic and elisa assays. KEY RESULTS: Functional and docking analyses showed that Δ9 -THCA-A can act as an orthosteric CB1 receptor agonist and also as a positive allosteric modulator in the presence of CP-55,940. Also, Δ9 -THCA-A seemed to be an inverse agonist for CB2 receptors. In vivo, Δ9 -THCA-A reduced arthritis in CIA mice, preventing the infiltration of inflammatory cells, synovium hyperplasia, and cartilage damage. Furthermore, Δ9 -THCA-A inhibited expression of inflammatory and catabolic genes on knee joints. The anti-arthritic effect of Δ9 -THCA-A was blocked by either SR141716 or T0070907. Analysis of plasma biomarkers, and determination of cytokines and anti-collagen antibodies confirmed that Δ9 -THCA-A mediated its activity mainly through PPARγ and CB1 receptor pathways. CONCLUSION AND IMPLICATIONS: Δ9 -THCA-A modulates CB1 receptors through the orthosteric and allosteric binding sites. In addition, Δ9 -THCA-A exerts anti-arthritis activity through CB1 receptors and PPARγ pathways, highlighting its potential for the treatment of chronic inflammatory diseases such as rheumatoid arthritis.

Atomistic simulations shed new light on the activation mechanisms of RORγ and classify it as Type III nuclear hormone receptor regarding ligand-binding paths.

The molecular recognition of the RORγ nuclear hormone receptor (NHR) ligand-binding domain (LBD) has been extensively studied with numerous X-ray crystal structures. However, the picture afforded by these complexes is static and does not fully explain the functional behavior of the LBD. In particular, the apo structure of the LBD seems to be in a fully active state, with no obvious differences to the agonist-bound structure. Further, several atypical in vivo inverse agonists have surprisingly been found to co-crystallize with the LBD in agonist mode (with co-activator), leading to a disconnection between molecular recognition and functional activity. Moreover, the experimental structures give no clues on how RORγ LBD binders access the interior of the LBD. To address all these points, we probe here, with a variety of simulation techniques, the fine structural balance of the RORγ LBD in its apo vs. holo form, the differences in flexibility and stability of the LBD in complex with agonists vs. inverse agonists and how binders diffuse in and out of the LBD in unbiased simulations. Our data conclusively point to the stability afforded by the so-called "agonist lock" between H479 and Y502 and the precise location of Helix 12 (H12) for the competence of the LBD to bind co-activator proteins. We observe the "water trapping" mechanism suggested previously for the atypical inverse agonists and discover a different behavior for the latter when co-activator is present or absent, which might help explain their conflicting data. Additionally, we unveil the same entry/exit path for agonists and inverse agonist into and out of the LBD for RORγ, suggesting it belongs to the type III NHR sub-family.

Remote mutations and active site dynamics correlate with catalytic properties of purine nucleoside phosphorylase.

It has been found that with mutation of two surface residues (Lys(22) --> Glu and His(104) --> Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O5'...ImmG:N4' and H257:Ndelta...ImmG:O5' consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O4'...ImmG:O5'...H257:Ndelta became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP.

Insight into why pyrrolidinyl peptide nucleic acid binding to DNA is more stable than the DNA x DNA duplex.

Molecular dynamics (MD) simulations and experimental measurements of the stability of a novel pyrrolidinyl PNA binding to DNA (PNA x DNA) in both parallel and antiparallel configurations were carried out. For comparison, simulations were also performed for the DNA x DNA duplex. The conformations of the three simulated systems were found to retain well-defined base pairing and base stacking as their starting B-like structure. A large gas-phase energy repulsion of the two negatively charged sugar-phosphate backbones of the DNA strands was found to reduce the stability of the DNA x DNA duplex significantly compared with that of the PNA x DNA complexes, especially in the antiparallel binding configuration. In addition, the antiparallel PNA x DNA was observed to be less solvated than that of the other two systems. The simulated binding free energies and the experimental melting temperatures for the three investigated systems are in good agreement, indicating that the antiparallel PNA x DNA is the most stable duplex.

Petrosamine, a potent anticholinesterase pyridoacridine alkaloid from a Thai marine sponge Petrosia n. sp.

Two pyridoacridine alkaloids, including a known petrosamine and a new 2-bromoamphimedine were isolated from a Thai marine sponge Petrosia n. sp. The alkaloids were characterized on the basis of 1D and 2D NMR, MS, and IR spectroscopy. Only petrosamine showed strong acetylcholinesterase inhibitory activity approximately six times higher than that of the reference galanthamine. A computational docking study of petrosamine with the enzyme from the electric eel Torpedo californica (TcAChE) showed the major contribution to the petrosamine-TcAChE interaction to be arising from the quaternary ammonium group of petrosamine.

Transition Path Sampling Study of the Reaction Catalyzed by Purine Nucleoside Phosphorylase.

The Transition Path Sampling (TPS) method is a powerful technique for studying rare events in complex systems, that allows description of reactive events in atomic detail without prior knowledge of reaction coordinates and transition states. We have applied TPS in combination with a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) method to study the enzyme human purine nucleoside phosphorylase (hPNP). This enzyme catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to generate the corresponding purine base and (deoxy)ribose 1-phosphate. Hundreds of reactive trajectories were generated. Analysis of this transition path ensembles provides insight into the detailed mechanistic dynamics of reaction in the enzyme. Our studies have indicated a reaction mechanism involving the cleavage of the N-ribosidic bond to form transition states with substantial ribooxacarbenium ion character, that is then followed by conformational changes in the enzyme and the ribosyl group leading to migration of the anomeric carbon of the ribosyl group toward phosphate to form the product ribose 1-phosphate. This latter process is crucial in PNP, because several strong H-bonds form between active site residues in order to capture and align the phosphate nucleophile. Calculations of the commitment probability along reactive paths demonstrated the presence of a broad energy barrier at the transition state. Analysis of these transition state structures showed that bond-breaking and bond-forming distances are not a good choice for the reaction coordinate, but that the pseudorotational phase of the ribose ring is also a significant variable.

Insight into analysis of interactions of saquinavir with HIV-1 protease in comparison between the wild-type and G48V and G48V/L90M mutants based on QM and QM/MM calculations.

Saquinavir (SQV) was the first HIV-1 PR inhibitor licensed for clinical use and widely used for acquired immunodeficiency syndrome (AIDS) therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations. The two most important HIV-1 PR mutants are G48V and G48V/L90M. Inhibition studies of SQV on these mutants demonstrated 13.5- and 419-fold reductions of susceptibility, respectively. In this study, an analysis of energetic binding affinity between saquinavir and the HIV-1 PR wild-type and these two mutants has been performed in detail based on density functional theory and the hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. We have found that the interaction of SQV with flap residue 48 of the mutants is significantly perturbed, as shown by the reduced stability of binding between SQV and residue 48 for the G48V and G48V/L90M mutants over the wild-type. This was associated with conformational changes of the inhibitor and the enzyme, leading to the loss of hydrogen bonding between the binding subsite P2 and the backbone carbonyl of residue 48. Moreover, the G48V/L90M mutations cause the repositioning of the residues close to residues 48 and 90, at important locations as a part of the flap and catalytic regions, respectively. The repositioning of these residues consequently perturbed the binding affinity of SQV in the pocket as indicated by the decreasing interaction energies. In addition to the loss of inhibitor/enzyme binding, it is interesting to observe that the mutation leads significantly to an increase of the stability of the enzyme.

Binding energy analysis for wild-type and Y181C mutant HIV-1 RT/8-Cl TIBO complex structures: quantum chemical calculations based on the ONIOM method.

Two-layered and three-layered ONIOM calculations were performed to compare the binding energies of 8-Cl TIBO inhibitor when bound into the human immunodeficiency virus reverse transcriptase binding pocket and a Y181C variant. Both consisted of 20 residues within a radius of 15 A. A combination of different methods [MP2/6-31G(d), B3LYP/6-31G(d,p), and PM3] were performed to take advantage of ONIOM's layering strategy analysis. The obtained results clearly indicate that the Y181C mutation reduces the binding affinity and stability of the inhibitor by approximately 8-9 kcal/mol as obtained from different combined MO:MO methods. Analyses regarding the energetic components of the interaction and deformation energies for 8-Cl TIBO inhibitor upon binding were also examined extensively. Additional calculations involving the interaction energies between 8-Cl TIBO with individual residues surrounding the binding pocket were performed at MP2/6-31G(d,p) and B3LYP/6-31G(d,p) levels of theory to gain more insight into the energetic differences of wild-type and Y181C mutant type at the atomistic level.

Inhibition of Naja kaouthia venom activities by plant polyphenols.

Plant polyphenols from the aqueous extracts of Pentace burmanica, Pithecellobium dulce, Areca catechu and Quercus infectoria were tested for their inhibitory activities against Naja kaouthia (NK) venom by in vitro neutralization method. The first three extracts could completely inhibit the lethality of the venom at 4 LD50 concentration and the venom necrotizing activity at the minimum necrotizing dose while also inhibited up to 90% of the acetylcholinesterase activity of NK venom at much lower tannin concentrations than that of Quercus infectoria. The ED50 of plant tannins in inhibiting NK venom activities varied according to condensed tannins and their content in the extracts. Molecular docking of the complexes between alpha-cobratoxin and either hydrolysable or condensed tannins at their lowest energetic conformations were proposed. The anti-venom activities of these plant polyphenols by selectively blocking the nicotinic acetylcholine receptor and non-selectively by precipitation of the venom proteins were suggested.

Tryptophan-free human PNP reveals catalytic site interactions.

Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The (13)C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.

Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain.

8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze the release of 8-vinyladenine from 8vdA-10. Molecular dynamics simulations implicate a role for Arg180 in oxacarbenium ion destabilization and the lack of catalysis. However, 8vdA-10 is an active site analogue and inhibits RTA with a Ki value of 2.4 microM. Adenine is also released from the second adenosine in the modified tetraloop, demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analogue defines the specificities of RTA for the two adenylate depurination sites in a RNA substrate with a GAGA tetraloop. The rate of nonenzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.

Insights into saquinavir resistance in the G48V HIV-1 protease: quantum calculations and molecular dynamic simulations.

The spread of acquired immune deficiency syndrome has increasingly become a great concern owing largely to the failure of chemotherapies. The G48V is considered the key signature residue mutation of HIV-1 protease developing with saquinavir therapy. Molecular dynamics simulations of the wild-type and the G48V HIV-1 protease complexed with saquinavir were carried out to explore structure and interactions of the drug resistance. The molecular dynamics results combined with the quantum-based and molecular mechanics Poisson-Boltzmann surface area calculations indicated a monoprotonation took place on D25, one of the triad active site residues. The inhibitor binding of the triad residues and its interaction energy in the mutant were similar to those in the wild-type. The overall structure of both complexes is almost identical. However, the steric conflict of the substituted valine results in the conformational change of the P2 subsite and the disruption of hydrogen bonding between the -NH of the P2 subsite and the backbone -CO of the mutated residue. The magnitude of interaction energy changes was comparable to the experimental K(i) data. The designing for a new drug should consider a reduction of steric repulsion on P2 to enhance the activity toward this mutant strain.

Structural flexibility of non-nucleoside HIV-1 reverse transcriptase inhibitor: 9-Cl TIBO as explained by potential energy surface and (13)C and (1)H NMR calculations, based on ab initio and density functional study.

The conformational analysis of the HIV-1 reverse transcriptase inhibitor, (+)-(s)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)imidazol[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione or 9-Cl TIBO, has been investigated using high level of calculations, ab initio, and DFT theory. The potential energy surface as the function of two important rotatable dihedral angles of the 9-Cl TIBO side chain was generated by the Hartree-Fock method at the 3-21G basis set. Eight pronounced local minima were found to exist within an energy difference of less than 10 kJ/mol. The energy barriers between the different local minima are lower than 15 kJ/mol. A second derivative (frequency) analysis showed that all conformers are stable at this level of theory. These structures were used as starting points for full geometry optimizations at the HF/6-31G and B3LYP/6-31G levels of theory to obtain the absolute geometries and structural information. The comparisons of calculated conformers with the bound conformer in the X-ray structure were sequentially considered. Additionally, to obtain some structural information and to correlate between calculated structures and the structure in solution, NMR chemical shift calculations were also performed on all eight local minimum structures at B3LYP/6-311++G level, using the GIAO approach. The calculated (1)H NMR and (13)C NMR chemical shifts for the lowest energetic conformer give the greatest correspondence with the experimental results.