Effect of the Crystal Environment on Side-Chain Conformational Dynamics in Cyanovirin-N Investigated through Crystal and Solution Molecular Dynamics Simulations.

Side chains in protein crystal structures are essential for understanding biochemical processes such as catalysis and molecular recognition. However, crystal packing could influence side-chain conformation and dynamics, thus complicating functional interpretations of available experimental structures. Here we investigate the effect of crystal packing on side-chain conformational dynamics with crystal and solution molecular dynamics simulations using Cyanovirin-N as a model system. Side-chain ensembles for solvent-exposed residues obtained from simulation largely reflect the conformations observed in the X-ray structure. This agreement is most striking for crystal-contacting residues during crystal simulation. Given the high level of correspondence between our simulations and the X-ray data, we compare side-chain ensembles in solution and crystal simulations. We observe large decreases in conformational entropy in the crystal for several long, polar and contacting residues on the protein surface. Such cases agree well with the average loss in conformational entropy per residue upon protein folding and are accompanied by a change in side-chain conformation. This finding supports the application of surface engineering to facilitate crystallization. Our simulation-based approach demonstrated here with Cyanovirin-N establishes a framework for quantitatively comparing side-chain ensembles in solution and in the crystal across a larger set of proteins to elucidate the effect of the crystal environment on protein conformations.

Mechanisms of allosteric activation and inhibition of the deoxyribonucleoside triphosphate triphosphohydrolase from Enterococcus faecalis.

EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.

Network visualization of conformational sampling during molecular dynamics simulation.

Effective data reduction methods are necessary for uncovering the inherent conformational relationships present in large molecular dynamics (MD) trajectories. Clustering algorithms provide a means to interpret the conformational sampling of molecules during simulation by grouping trajectory snapshots into a few subgroups, or clusters, but the relationships between the individual clusters may not be readily understood. Here we show that network analysis can be used to visualize the dominant conformational states explored during simulation as well as the connectivity between them, providing a more coherent description of conformational space than traditional clustering techniques alone. We compare the results of network visualization against 11 clustering algorithms and principal component conformer plots. Several MD simulations of proteins undergoing different conformational changes demonstrate the effectiveness of networks in reaching functional conclusions.

Characterization of the deoxynucleotide triphosphate triphosphohydrolase (dNTPase) activity of the EF1143 protein from Enterococcus faecalis and crystal structure of the activator-substrate complex.

The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn(2+). In contrast, with Mg(2+) hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed a tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the α-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the α3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.

Crystal molecular dynamics simulations to speed up MM/PB(GB)SA evaluation of binding free energies of di-mannose deoxy analogs with P51G-m4-Cyanovirin-N.

Complexes of two Cyanovirin-N (CVN) mutants, m4-CVN and P51G-m4-CVN, with deoxy di-mannose analogs were employed as models to generate conformational ensembles using explicit water Molecular Dynamics (MD) simulations in solution and in crystal environment. The results were utilized for evaluation of binding free energies with the molecular mechanics Poisson-Boltzmann (or Generalized Born) surface area, MM/PB(GB)SA, methods. The calculations provided the ranking of deoxy di-mannose ligands affinity in agreement with available qualitative experimental evidences. This confirms the importance of the hydrogen-bond network between di-mannose 3'- and 4'-hydroxyl groups and the protein binding site B(M) as a basis of the CVN activity as an effective HIV fusion inhibitor. Comparison of binding free energies averaged over snapshots from the solution and crystal simulations showed high promises in the use of the crystal matrix for acceleration of the conformational ensemble generation, the most time consuming step in MM/PB(GB)SA approach. Correlation between energy values based on solution versus crystal ensembles is 0.95 for both MM/PBSA and MM/GBSA methods.

Solution and crystal molecular dynamics simulation study of m4-cyanovirin-N mutants complexed with di-mannose.

Cyanovirin-N (CVN) is a highly potent anti-HIV carbohydrate-binding agent that establishes its microbicide activity through interaction with mannose-rich glycoprotein gp120 on the virion surface. The m4-CVN and P51G-m4-CVN mutants represent simple models for studying the high-affinity binding site, B(M). A recently determined 1.35 A high-resolution structure of P51G-m4-CVN provided details on the di-mannose binding mechanism, and suggested that the Arg-76 and Glu-41 residues are critical components of high mannose specificity and affinity. We performed molecular-dynamics simulations in solution and a crystal environment to study the role of Arg-76. Network analysis and clustering were used to characterize the dynamics of Arg-76. The results of our explicit solvent solution and crystal simulations showed a significant correlation with conformations of Arg-76 proposed from x-ray crystallographic studies. However, the crystal simulation showed that the crystal environment strongly biases conformational sampling of the Arg-76 residue. The solution simulations demonstrated no conformational preferences for Arg-76, which would support its critical role as the residue that locks the ligand in the bound state. Instead, a comparative analysis of trajectories from >50 ns of simulation for two mutants revealed the existence of a very stable eight-hydrogen-bond network between the di-mannose ligand and predominantly main-chain atoms. This network may play a key role in the specific recognition and strong binding of mannose oligomers in CVN and its homologs.

Capturing and analyzing the excited-state structure of a Cu(I) phenanthroline complex by time-resolved diffraction and theoretical calculations.

Time-resolved crystallography and density functional theory calculations are used to analyze the geometric and electronic changes that occur upon photoexcitation of [Cu(I)(dmp)(dppe)](+) in crystalline [Cu(I)(dmp)(dppe)][PF(6)] [dmp = 2,9-dimethyl-1,10-phenanthroline; dppe = 1,2-bis(diphenylphosphino)ethane]. In the pump-probe experiment, laser and X-ray pulses are synchronized to capture an image of the instantaneous molecular distortions in the transient triplet state. Parallel theoretical calculations, with the phenyl groups replaced by methyl groups, yield information on the distortion of the isolated cation and the change in electron density upon excitation. The experimental distortions are significantly less than the calculated values and are different for the two independent molecules in the asymmetric unit; these findings are attributed to the constraining influence of the crystal matrix. The calculations indicate that the electron transfer upon excitation is mostly from the dmpe ligand to the dmp ligand, while the Cu atomic charge changes by only approximately +0.1e, although the charge distribution on Cu is significantly affected. As found for homoleptic [Cu(I)(dmp)(2)](+), the change in the population of the Cu atom is close to the calculated difference between the corresponding Cu(II) and Cu(I) complexes. Charge density difference maps confirm these conclusions and show a large rearrangement of the electron density on the Cu atom upon excitation.

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli.

ZnuA is the periplasmic Zn(2+)-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn(2+)-bound, and Co(2+)-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn(2+) with Co(2+) results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn(2+) periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn(2+) (estimated K (d) < 20 nM), Co(2+), Ni(2+), Cu(2+), Cu(+), and Cd(2+), but not Mn(2+). Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn(2+) substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer.

Crystal structure of an apo form of Shigella flexneri ArsH protein with an NADPH-dependent FMN reductase activity.

The arsH gene or its homologs are a frequent part of the arsenic resistance system in bacteria and eukaryotes. Although a specific biological function of the gene product is unknown, the ArsH protein was annotated as a member of the NADPH-dependent FMN reductase family based on a conserved (T/S)XRXXSX(T/S) fingerprint motif common for FMN binding proteins. Presented here are the first crystal structure of an ArsH protein from Shigella flexneri refined at 1.7 A resolution and results of enzymatic activity assays that revealed a strong NADPH-dependent FMN reductase and low azoreductase activities. The ArsH apo protein has an alpha/beta/alpha-fold typical for FMN binding proteins. The asymmetric unit consists of four monomers, which form a tetramer. Buried surface analysis suggests that this tetramer is likely to be the relevant biological assembly. Dynamic light scattering experiments are consistent with this hypothesis and show that ArsH in solution at room temperature does exist predominantly in the tetrameric form.

The 2.2 A resolution crystal structure of Bacillus cereus Nif3-family protein YqfO reveals a conserved dimetal-binding motif and a regulatory domain.

YqfO of Bacillus cereus is a member of the widespread Nif3 family of proteins, which has been highlighted as an important target for structural genomics. The N- and C-terminal domains are conserved across the family and contain a dimetal-binding motif in a putative active site. YqfO contains an insert in the middle of the protein, present in a minority of bacterial family members. The structure of YqfO was determined at a resolution of 2.2 A and reveals conservation of the putative active site. It also reveals the previously unknown structure of the insert, which despite extremely limited sequence conservation, bears great similarity to PII, CutA, and a number of other trimeric regulatory proteins. Our results suggest that this domain acts as a signal sensor to regulate the still-unknown catalytic activity of the more-conserved domains.

Shedding light on the structure of a photoinduced transient excimer by time-resolved diffraction.

Time-resolved single-crystal diffraction performed with synchrotron radiation shows that the 53(1) micros phosphorescent state, generated in the crystalline phase of trimeric {[3,5-(CF3)(2)Pyrazolate]Cu}(3) molecules by exposure to 355 nm of light at 17 K, is due to the formation of an excimer rather than the shortening of the intramolecular Cu...Cu distances within the trimeric units, or the formation of a continuous chain of interacting molecules. One of the intermolecular Cu...Cu distances contracts by 0.56 Angstroms from 4.018(1) to 3.46(1) Angstroms;, whereas the interplanar spacing of the trimers is reduced by 0.65 Angstroms; from 3.952(1) to 3.33(1) Angstroms. Density-functional theory calculations support the formation of a Cu...Cu bond through the intermetallic transfer of a Cu 3d electron to a molecular orbital with a large 4p contribution on the reacting Cu atoms.

On the refinement of time-resolved diffraction data: comparison of the random-distribution and cluster-formation models and analysis of the light-induced increase in the atomic displacement parameters.

Expressions for the random-distribution and cluster-formation models for light-induced changes in crystals studied by time-resolved diffraction are presented. The two models can be distinguished on the basis of differences in the predicted intensities. The light-induced increase in the atomic displacement parameters is analyzed with both simulated and experimental data sets.

The structure of short-lived excited states of molecular complexes by time-resolved X-ray diffraction.

Experimental and computational methods for time-resolved (TR) diffraction now allow the determination of geometry changes on molecular excitation. The first results indicate significant changes in the interatomic distances and molecular shape on photo-excitation, but also a dependence of the induced changes on the molecular environment. Though the use of high-brightness synchrotron sources is essential, it limits the time resolution to the width of the synchrotron pulse which is currently 70-100 ps. The experiments discussed fall into two categories: (i) picosecond powder diffraction experiments on the molecular excitation to a singlet state, and (ii) microsecond experiments on the excited states of inorganic complexes. Both involve reversible processes for which a stroboscopic technique can be applied.

A very large Rh-Rh bond shortening on excitation of the [Rh2(1,8-diisocyano-p-menthane)4]2+ ion by time-resolved synchrotron X-ray diffraction.

A very large Rh-Rh contraction of approximately 0.85 A occurs on excitation of the [Rh(2)(1,8-diisocyano-p-menthane)(4)](2+) ion to its triplet state.

Geometry changes of a Cu(I) phenanthroline complex on photoexcitation in a confining medium by time-resolved X-ray diffraction.

Using a stroboscopic technique, in which the molecule is repeatedly excited and the structural change is probed more than 5000 times per second immediately after excitation, we performed a 16 K time-resolved single-crystal study of the microsecond lifetime triplet state of the Cu(I)phenanthroline derivative[Cu(I)(dmp)(dppe)][PF6] (dppe = 1,2-bis(diphenylphosphino)ethane). The geometry changes on excitation differ for the two symmetry-independent molecules, but are in the same direction as calculated for an isolated reference molecule, although the flattening distortion in the crystal is significantly smaller, implying that the reorganization energy is greatly affected by the confining medium.

Reaction of 2-polyfluoroalkylchromones with 1,3,3-trimethyl-3,4-dihydroisoquinolines and methylketimines as a direct route to zwitterionic axially chiral 6,7-dihydrobenzo[a]quinolizinium derivatives and 2,6-diaryl-4-polyfluoroalkylpyridines.

[reaction: see text] 2-Polyfluoroalkylchromones react with 1,3,3-trimethyl-3,4-dihydroisoquinolines to give zwitterionic axially chiral 6,7-dihydrobenzo[a]quinolizinium derivatives in high yields. In addition, performing this reaction with aromatic methylketimines is a simple and convenient synthesis of 2,6-diaryl-4-polyfluoroalkylpyridines.

Crystal structure and magnetic properties of an ionic C60 complex with decamethylcobaltocene: (Cp*2Co)2C60(C6H4Cl2, C6H5CN)2. Singlet-triplet transitions in the C60(2-) anion.

The C(60) complex with decamethylcobaltocene, (Cp(2)Co)(2)C(60)(C(6)H(4)Cl(2), C(6)H(5)CN)(2) (1) (C(6)H(4)Cl(2) = 1,2-dichlorobenzene; C(6)H(5)CN = benzonitrile), has been obtained as single crystals by the diffusion method. The IR and UV-vis-NIR spectra show the presence of the C(60)(2)(-) and the Cp(2)Co(+) ions, which form a three-dimensional framework with channels accommodating solvent molecules. EPR and SQUID measurements show that C(60)(2)(-) has a diamagnetic singlet (S = 0) state in the 2-140 K range. The appearance of a broad EPR signal and the increase in magnetic susceptibility of 1 above 140 K are assigned to a thermal population of a close lying triplet (S = 1) state. The singlet-triplet energy gap for C(60)(2)(-) in solid 1 is estimated to be 730+/-10 cm(-)(1).

Unusual reaction of 2-(trifluoromethyl)-1,2-dihydro-3lambda(6)-thieno- [2,3-c]chromen-3,3,4-triones with hydrazine as a new route to 3-hydrazinopyridazine derivatives.

Oxidation of 2-(trifluoromethyl)-1,2-dihydro-4H-thieno[2,3-c]chromen-4-ones 2 with H(2)O(2) in AcOH gives 2-(trifluoromethyl)-1,2-dihydrothieno[2,3-c]chromen-3,3,4-triones 3, which are transformed into 3-hydrazinopyridazine derivatives 4 in high yields by treatment with hydrazine hydrate in ethanol.