Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases.

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

Structural basis for ligand reception by anaplastic lymphoma kinase.

The proto-oncogene ALK encodes anaplastic lymphoma kinase, a receptor tyrosine kinase that is expressed primarily in the developing nervous system. After development, ALK activity is associated with learning and memory1 and controls energy expenditure, and inhibition of ALK can prevent diet-induced obesity2. Aberrant ALK signalling causes numerous cancers3. In particular, full-length ALK is an important driver in paediatric neuroblastoma4,5, in which it is either mutated6 or activated by ligand7. Here we report crystal structures of the extracellular glycine-rich domain (GRD) of ALK, which regulates receptor activity by binding to activating peptides8,9. Fusing the ALK GRD to its ligand enabled us to capture a dimeric receptor complex that reveals how ALK responds to its regulatory ligands. We show that repetitive glycines in the GRD form rigid helices that separate the major ligand-binding site from a distal polyglycine extension loop (PXL) that mediates ALK dimerization. The PXL of one receptor acts as a sensor for the complex by interacting with a ligand-bound second receptor. ALK activation can be abolished through PXL mutation or with PXL-targeting antibodies. Together, these results explain how ALK uses its atypical architecture for its regulation, and suggest new therapeutic opportunities for ALK-expressing cancers such as paediatric neuroblastoma.

Dual function of LapB (YciM) in regulating Escherichia coli lipopolysaccharide synthesis.

Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled-making LPS synthesis a promising target for developing new antibiotics. Escherichia coli adaptor protein LapB (YciM) plays an important role in regulating LPS synthesis by promoting degradation of LpxC, a deacetylase that catalyzes the first committed step in LPS synthesis. Under conditions where LPS is abundant, LapB recruits LpxC to the AAA+ protease FtsH for degradation. LapB achieves this by simultaneously interacting with FtsH through its transmembrane helix and LpxC through its cytoplasmic domain. Here, we describe a cryo-EM structure of the complex formed between LpxC and the cytoplasmic domain of LapB (LapBcyto). The structure reveals how LapB exploits both its tetratricopeptide repeat (TPR) motifs and rubredoxin domain to interact with LpxC. Through both in vitro and in vivo analysis, we show that mutations at the LapBcyto/LpxC interface prevent LpxC degradation. Unexpectedly, binding to LapBcyto also inhibits the enzymatic activity of LpxC through allosteric effects reminiscent of LpxC activation by MurA in Pseudomonas aeruginosa. Our findings argue that LapB regulates LPS synthesis in two steps: In the first step, LapB inhibits the activity of LpxC, and in the second step, it commits LpxC to degradation by FtsH.

Syntheses, Characterizations, Crystal Structures, and Protonation Reactions of Dinitrogen Chromium Complexes Supported with Triamidoamine Ligands.

A novel dinitrogen-dichromium complex, [{Cr(LBn)}2(µ-N2)] (1), has been prepared from reaction of CrCl3 with a lithiated triamidoamine ligand (Li3LBn) under dinitrogen. The X-ray crystal structure analysis of 1 revealed that it is composed of two independent dimeric Cr complexes bridged by N2 in the unit cell. The bridged N-N bond lengths (1.188(4) and 1.185(7) Å) were longer than the free dinitrogen molecule. The elongations of N-N bonds in 1 were also supported by the fact that the ν(N-N) stretching vibration at 1772 cm-1 observed in toluene is smaller than the free N2. Complex 1 was identified to be a 5-coordinated high spin Cr(IV) complex by Cr K-edge XANES measurement. The 1H NMR spectrum and temperature dependent magnetic susceptibility of 1 indicated that complex 1 is in the S = 1 ground state, in which two Cr(IV) ions and unpaired electron spins of the bridging N22- ligand are strongly antiferromagnetically coupled. Reaction of complex 1 with 2.3 equiv of Na or K gave chromium complexes with N2 between the Cr ion and the respective alkali metal ion, [{CrNa(LBn)(N2)(Et2O)}2] (2) and [{CrK(LBn)(N2)}4(Et2O)2] (3), respectively. Furthermore, the complexes 2 and 3 reacted with 15-crown-5 and 18-crown-6 to form the respective crown-ether adducts, [CrNa(LBn)(N2)(15-crown-5)] (4) and [CrK(LBn)(N2)(18-crown-6)] (5). The XANES measurements of complexes 2, 3, 4, and 5 revealed that they are high spin Cr(IV) complexes like complex 1. All complexes reacted with a reducing agent and a proton source to form NH3 and/or N2H4. The yields of these products in the presence of K+ were higher than those in the presence of Na+. The electronic structures and binding properties of 1, 2, 3, 4, and 5 were evaluated and discussed based on their DFT calculations.

Preparations of trans- and cis-µ-1,2-Peroxodiiron(III) Complexes.

The iron(II) complex with cis,cis-1,3,5-tris(benzylamino)cyclohexane (Bn3CY) (1) has been synthesized and characterized, which reacted with dioxygen to form the peroxo complex 2 in acetone at -60 °C. On the basis of spectroscopic measurements for 2, it was confirmed that the peroxo complex 2 has a trans-µ-1,2 fashion. Additionally, the peroxo complex 2 was reacted with benzoate anion as a bridging agent to give a peroxo complex 3. The results of resonance Raman and 1H-NMR studies supported that the peroxo complex 3 is a cis-µ-1,2-peroxodiiron(III) complex. These spectral features were interpreted by using DFT calculations.

Synthesis and Physico-Chemical Properties of Homoleptic Copper(I) Complexes with Asymmetric Ligands as a DSSC Dye.

To develop low-cost and efficient dye-sensitized solar cells (DSSCs), we designed and prepared three homoleptic Cu(I) complexes with asymmetric ligands, M1, M2, and Y3, which have the advantages of heteroleptic-type complexes and compensate for their synthetic challenges. The three copper(I) complexes were characterized by elemental analysis, UV-vis absorption spectroscopy, and electrochemical measurements. Their absorption spectra and orbital energies were evaluated and are discussed in the context of TD-DFT calculations. The complexes have high VOC values (0.48, 0.60, and 0.66 V for M1, M2, and Y3, respectively) which are similar to previously reported copper(I) dyes with symmetric ligands, although their energy conversion efficiencies are relatively low (0.17, 0.64, and 2.66%, respectively).

A Link Between Alzheimer's and Type II Diabetes Mellitus? Ca+2 -Mediated Signal Control and Protein Localization.

We propose protein localization dependent signal activation (PLDSA) as a model to describe pre-existing protein partitioning between the cytosol, and membrane surface, as a means to modulate signal activation, specificity, and robustness. We apply PLDSA to explain possible molecular links between type II diabetes mellitus (T2DM) and Alzheimer's disease (AD) by describing Ca+2 -mediated interactions between the Src non-receptor tyrosine kinase and p52Shc adaptor protein. We suggest that these interactions may serve as a contributing factor to disease development and progression. In particular, we propose that signaling response is regulated, in part, by Ca+2 -mediated partitioning of lipid-bound and soluble forms of Src and p52shc. Thus, protein-protein interactions that drive signaling in response to extracellular ligand binding are also mediated by partitioning of signaling proteins between membrane-bound and soluble populations. We propose that PLDSA effects may explain, in part, the evolutionary basis of promiscuous protein interaction domains and their importance in cellular function.

Dinitrogen-Molybdenum Complex Induces Dinitrogen Cleavage by One-Electron Oxidation.

Reported here is the N2 cleavage of a one-electron oxidation reaction using trans-[Mo(depe)2 (N2 )2 ] (1) (depe=Et2 PCH2 CH2 PEt2 ), which is a classical molybdenum(0)-dinitrogen complex supported by two bidentate phosphine ligands. The molybdenum(IV) terminal nitride complex [Mo(depe)2 N][BArf4 ] (2) (BArf4 =B(3,5-(CF3 )2 C6 H3 )4 ) is synthesized by the one-electron oxidation of 1 upon addition of a mild oxidant, [Cp2 Fe][BArf4 ] (Cp=C5 H5 ), and proceeds by N2 cleavage from a MoII -N=N-MoII structure. In addition, the electrochemical oxidation reaction for 1 also cleaved the N2 ligand to give 2. The dimeric Mo complex with a bridging N2 is detected by in situ resonance Raman and in situ UV-vis spectroscopies during the electrochemical oxidation reaction for 1. Density-functional theory (DFT) calculations reveal that the unstable monomeric oxidized MoI species is converted into 2 via the dimeric structure involving a zigzag transition state.

Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to FeII-O2 Complex.

Nitric oxide synthase (NOS) is a cytochrome P450-type mono-oxygenase that catalyzes the oxidation of l-arginine (Arg) to nitric oxide (NO) through a reaction intermediate N-hydroxy-l-arginine (NHA). The mechanism underlying the reaction catalyzed by NOS from Deinococcus radiodurans was investigated using pulse radiolysis. Radiolytically generated hydrated electrons reduced the heme iron of NOS within 2 µs. Subsequently, ferrous heme reacted with O2 to form a ferrous-dioxygen intermediate with a second-order rate constant of 2.8 × 108 M-1 s-1. In the tetrahydrofolate (H4F)-bound enzyme, the ferrous-dioxygen intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 103 s-1. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of H4F, this step did not proceed, suggesting that H4F was reduced with the ferrous-dioxygen intermediate to form a second intermediate. The intermediate further converted to the original ferric form with a first-order rate constant of 4 s-1. A similar intermediate could be detected after pulse radiolysis in the presence of NHA, although the intermediate decayed more slowly (0.5 s-1). These data suggested that a common catalytically active intermediate involved in the substrate oxidation of both Arg and NHA may be formed during catalysis. In addition, we investigated the solvent isotope effects on the kinetics of the intermediate after pulse radiolysis. Our experiments revealed dramatic kinetic solvent isotope effects on the conversion of the intermediate to the ferric form, of 10.5 and 2.5 for Arg and NHA, respectively, whereas the faster phases were not affected. These data suggest that the proton transfer in DrNOS is the rate-limiting reaction of the intermediate with the substrates.

Nuclear Resonance Vibrational Spectroscopy Definition of O2 Intermediates in an Extradiol Dioxygenase: Correlation to Crystallography and Reactivity.

The extradiol dioxygenases are a large subclass of mononuclear nonheme Fe enzymes that catalyze the oxidative cleavage of catechols distal to their OH groups. These enzymes are important in bioremediation, and there has been significant interest in understanding how they activate O2. The extradiol dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) provides an opportunity to study this process, as two O2 intermediates have been trapped and crystallographically defined using the slow substrate 4-nitrocatechol (4NC): a side-on Fe-O2-4NC species and a Fe-O2-4NC peroxy bridged species. Also with 4NC, two solution intermediates have been trapped in the H200N variant, where H200 provides a second-sphere hydrogen bond in the wild-type enzyme. While the electronic structure of these solution intermediates has been defined previously as FeIII-superoxo-catecholate and FeIII-peroxy-semiquinone, their geometric structures are unknown. Nuclear resonance vibrational spectroscopy (NRVS) is an important tool for structural definition of nonheme Fe-O2 intermediates, as all normal modes with Fe displacement have intensity in the NRVS spectrum. In this study, NRVS is used to define the geometric structure of the H200N-4NC solution intermediates in HPCD as an end-on FeIII-superoxo-catecholate and an end-on FeIII-hydroperoxo-semiquinone. Parallel calculations are performed to define the electronic structures and protonation states of the crystallographically defined wild-type HPCD-4NC intermediates, where the side-on intermediate is found to be a FeIII-hydroperoxo-semiquinone. The assignment of this crystallographic intermediate is validated by correlation to the NRVS data through computational removal of H200. While the side-on hydroperoxo semiquinone intermediate is computationally found to be nonreactive in peroxide bridge formation, it is isoenergetic with a superoxo catecholate species that is competent in performing this reaction. This study provides insight into the relative reactivities of FeIII-superoxo and FeIII-hydroperoxo intermediates in nonheme Fe enzymes and into the role H200 plays in facilitating extradiol catalysis.

Co(III) Complexes with N2S3-Type Ligands as Structural/Functional Models for the Isocyanide Hydrolysis Reaction Catalyzed by Nitrile Hydratase.

It has been before reported that, in addition to hydration of nitriles, the Fe-type nitrile hydratase (NHase) also catalyzes the hydrolysis of tert-butylisocyanide ( tBuNC). In order to investigate the unique isocyanide hydrolysis by NHase, we prepared three related Co(III) model complexes, PPh4[Co(L)] (1), PPh4[Co(L-O3)] (2), and PPh4[Co(L-O4)] (3), where L is bis( N-(2-mercapto-2-methylpropionyl)aminopropyl)sulfide. The suffixes L-O3 and L-O4 indicate ligands with a sulfenate and a sulfinate and with two sulfinates, respectively, instead of the two thiolates of L. The X-ray analyses of 1 and 3 reveal trigonal bipyramidal and square pyramidal structures, respectively. Complex 2, however, has five-coordinate trigonal-bipyramidal geometry with η2-type S-O coordination by a sulfenyl group. Addition of tBuNC to 1, 2, and 3 induces an absorption spectral change as a result of formation of an octahedral Co(III) complex. This interpretation is also supported by the crystal structures of PPh4[Co(L-O4)( tBuNC)] (4) and (PPh4)2[Co(L-O4)(CN)] (5). A water molecule interacts with 3 but cannot be activated as reported previously, as demonstrated by the lack of absorption spectral change in the pH range of 5.5-10.2. Interestingly, the coordinated tBuNC is hydrolyzed by 2 and 3 at pH 10.2 to produce tBuNH2 and CO molecule, but 1 does not react. These findings provide strong evidence that hydrolysis of tBuNC by NHase proceeds not by activation of the coordinated water molecule but by coordination of the substrate. The mechanism of the hydrolysis reaction of tBuNC is explained with support provided by DFT calculations; a positively polarized C atom of tBuNC on the Co(III) center is nucleophilically attacked by a hydroxide anion activated through an interaction of the sulfenyl/sulfinyl oxygen with the nucleophile.

Dinitrogen Fixation by Vanadium Complexes with a Triamidoamine Ligand.

Dinitrogen-divanadium complexes with triamidoamine ligands, 1-3, were synthesized and characterized by resonance Raman, UV-vis, and NMR spectroscopy and elemental and X-ray structure analyses. X-ray structure analyses reveal that all three of the complexes have a dimeric structure with a µ-N2 ligand (N-N bond length 1.200-1.221 Å). Resonance Raman and NMR spectra of 1-3 in solution show that these complexes maintain a dimeric structure in benzene and toluene solutions. 15N NMR spectra of 1 and 3 have peaks assignable to µ-N2 ligands at 33.4 and 27.6 ppm, respectively, but 2 does not have a similar peak under the same conditions. In 51V NMR spectra, the peaks of vanadium ions were observed at -173.3, -143.8, and -240.2 ppm, respectively, which are in a higher magnetic field region in comparison to those of dinitrogen-divanadium complexes reported previously. The structure and electronic properties of 1 are supported by DFT calculations. Additionally, all complexes react with excess amounts of M[C10H8] (M = Na, K) and the proton sources HOTf, HCl, and [LutH]OTf (Lut = 2,6-dimethylpyridine) to produce ammonia without hydrazine. The ammonia produced was evaluated as an ammonium salt by 1H and 15N NMR spectroscopy. The yield of NH3 produced in the reaction of 1 with Na[C10H8] and HOTf under N2 was 151% (per V atom).

Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues.

In Escherichia coli, the [2Fe-2S] transcriptional factor, SoxR, functions as a sensor of oxidative stress. The transcriptional activity in SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. We previously proposed that superoxide (O2•-) has a direct role as a signal for E. coli SoxR and that the sensitivity of the E. coli SoxR response to O2•- is 10-fold higher than that of Pseudomonas aeruginosa SoxR. The difference between the two homologues reflects interspecies differences in the regulatory role of O2•- activation. To investigate the determinants of SoxR's sensitivity to O2•-, we substituted several amino acids that are not conserved among enteric bacteria SoxR homologues and investigated the interaction of SoxR with O2•- using pulse radiolysis. The substitution of E. coli SoxR Lys residues 89 and 92 with Ala residues (K89AK92A), located close to [2Fe-2S] clusters, dramatically affected this protein's reaction with O2•-. The second-order rate constant of the reaction was 3.3 × 107 M-1 s-1, which was 10 times smaller than that of wild-type SoxR. Conversely, the corresponding substitution of Ala90 with Lys in P. aeruginosa SoxR increased the rate approximately 10-fold. In contrast, introductions of the Arg127Ser128Asp129 → Leu127Gln128Ala129 substitution into E. coli SoxR, and the corresponding substitution (Leu125Gln126Ala127 → Arg125Ser126Asp127) in P. aeruginosa SoxR, did not affect the reaction rates. In addition, the Lys mutation in E. coli SoxR (K89AK92A) showed a defect in vivo transcriptional activity by measuring ß-galactosidase expression in response to paraquat. Our findings clearly support the idea Lys is critical to the response to O2•- and further transcriptional activity of SoxR.

Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src.

Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-ß-D-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation.

Overproduction and biophysical characterization of human HSP70 proteins.

Heat shock proteins (HSP) perform vital cellular functions and modulate cell response pathways to physical and chemical stressors. A key feature of HSP function is the ability to interact with a broad array of protein binding partners as a means to potentiate downstream response pathways or facilitate protein folding. These binding interactions are driven by ATP-dependent conformational rearrangements in HSP proteins. The HSP70 family is evolutionarily conserved and is associated with diabetes and cancer progression and the etiopathogenesis of hepatic, cardiovascular, and neurological disorders in humans. However, functional characterization of human HSP70s has been stymied by difficulties in obtaining large quantities of purified protein. Studies of purified human HSP70 proteins are essential for downstream investigations of protein-protein interactions and in the rational design of novel family-specific therapeutics. Within this work, we present optimized protocols for the heterologous overexpression and purification of either the nucleotide binding domain (NBD) or the nucleotide and substrate binding domains of human HSPA9, HSPA8, and HSPA5 in either Escherichia coli or Saccharomyces cerevisiae. We also include initial biophysical characterization of HSPA9 and HSPA8. This work provides the basis for future biochemical studies of human HSP70 protein function and structure.

N2 activation by an iron complex with a strong electron-donating iminophosphorane ligand.

A new tridentate cyclopentane-bridged iminophosphorane ligand, N-(2-diisopropylphosphinophenyl)-P,P-diisopropyl-P-(2-(2,6-diisopropylphenylamido)cyclopent-1-enyl)phosphoranimine (NpNPiPr), was synthesized and used in the preparation of a diiron dinitrogen complex. The reaction of the iron complex FeBr(NpNPiPr) with KC8 under dinitrogen yielded the dinuclear dinitrogen Fe complex [Fe(NpNPiPr)]2(µ-N2), which was characterized by X-ray analysis and resonance Raman and NMR spectroscopies. The X-ray analysis revealed a diiron complex bridged by the dinitrogen molecule, with each metal center coordinated by an NpNPiPr ligand and dinitrogen in a trigonal-monopyramidal geometry. The N­N bond length is 1.184(6) Å, and resonance Raman spectra indicate that the N­N stretching mode ν(14N2/15N2) is 1755/1700 cm­1. The magnetic moment of [Fe(NpNPiPr)]2(µ-N2) in benzene-d6 solution, as measured by 1H NMR spectroscopy by the Evans method, is 6.91µB (S = 3). The Mössbauer spectrum at 78 K showed δ = 0.73 mm/s and ΔEQ = 1.83 mm/s. These findings suggest that the iron ions are divalent with a high-spin configuration and that the N2 molecule has (N═N)2­ character. Density functional theory calculations performed on [Fe(NpNPiPr)]2(µ-N2) also suggested that the iron is in a high-spin divalent state and that the coordinated dinitrogen molecule is effectively activated by π back-donation from the two iron ions (dπ) to the dinitrogen molecule (πx* and πy*). This is supported by cooperation between a large negative charge on the iminophosphorane ligand and strong electron donation and effective orbital overlap between the iron dπ orbitals and N2 π* orbitals supplied by the phosphine ligand.

Folding mechanism of the metastable serpin α1-antitrypsin.

The misfolding of serpins is linked to several genetic disorders including emphysema, thrombosis, and dementia. During folding, inhibitory serpins are kinetically trapped in a metastable state in which a stretch of residues near the C terminus of the molecule are exposed to solvent as a flexible loop (the reactive center loop). When they inhibit target proteases, serpins transition to a stable state in which the reactive center loop forms part of a six-stranded ß-sheet. Here, we use hydrogen-deuterium exchange mass spectrometry to monitor region-specific folding of the canonical serpin human α(1)-antitrypsin (α(1)-AT). We find large differences in the folding kinetics of different regions. A key region in the metastable → stable transition, ß-strand 5A, shows a lag phase of nearly 350 s. In contrast, the "B-C barrel" region shows no lag phase and the incorporation of the C-terminal residues into ß-sheets B and C is largely complete before the center of ß-sheet A begins to fold. We propose this as the mechanism for trapping α(1)-AT in a metastable form. Additionally, this separation of timescales in the folding of different regions suggests a mechanism by which α(1)-AT avoids polymerization during folding.

Crystal structures of ß-1,4-galactosyltransferase 7 enzyme reveal conformational changes and substrate binding.

The ß-1,4-galactosyltransferase 7 (ß4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human ß4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type ß4GalT7 and D211N ß4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N ß4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an SN2 type catalytic mechanism for the ß4GalT7 enzyme.

Toward the accurate calculation of pKa values in water and acetonitrile.

We present a simple approach for the calculation of accurate pKa values in water and acetonitrile based on the straightforward calculation of the gas-phase absolute free energies of the acid and conjugate base with use of only a continuum solvation model to obtain the corresponding solution-phase free energies. Most of the error in such an approach arises from inaccurate differential solvation free energies of the acid and conjugate base which is removed in our approach using a correction based on the realization that the gas-phase acidities have only a small systematic error relative to the dominant systematic error in the differential solvation. The methodology is outlined in the context of the calculation of a set of neutral acids with water as the solvent for a reasonably accurate electronic structure level of theory (DFT), basis set, and implicit solvation model. It is then applied to the comparison of results for three different hybrid density functionals to illustrate the insensitivity to the functional. Finally, the approach is applied to the comparison of results for sets of neutral acids and protonated amine cationic acids in both aqueous (water) and nonaqueous (acetonitrile) solvents. The methodology is shown to generally predict the pKa values for all the cases investigated to within 1 pH unit so long as the differential solvation error is larger than the systematic error in the gas-phase acidity calculations. Such an approach is rather general and does not have additional complications that would arise in a cluster-continuum method, thus giving it strength as a simple high-throughput means to calculate absolute pKa values. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

A novel square-planar Ni(II) complex with an amino-carboxamido-dithiolato-type ligand as an active-site model of NiSOD.

To understand the role of the unique equatorial coordination environment at the active center in nickel superoxide dismutase (NiSOD), we prepared a novel Ni(II) complex with an amino-carboxamido-dithiolato-type square-planar ligand (1, [Ni(2+)(L1)](-)) as a model of the NiSOD active site. Complex 1 has a low-spin square-planar structure in all solvents. Interestingly, the absorption wavelength and ν(C═O) stretching vibrations of 1 are affected by solvents. This provides an indication that the carbonyl oxygens participate in hydrogen-bonding interactions with solvents. These interactions are reflected in the redox potentials; the peak potential of an anodic wave (Epa) values of Ni(II)/Ni(III) waves for 1 are shifted to a positive region for solvents with higher acceptor numbers. This indicates that the disproportionation of superoxide anion by NiSOD may be regulated by hydrogen-bonding interactions between the carboxamido carbonyl and electrophilic molecules through fine-tuning of the redox potential for optimal SOD activity. Interestingly, the Epa value of the Ni(III)/Ni(II) couple in 1 in water (+0.303 V vs normal hydrogen electrode (NHE)) is similar to that of NiSOD (+0.290 V vs NHE). We also investigated the superoxide-reducing and -oxidizing reactions of 1. First, 1 reacts with superoxide to yield the superoxide-bound Ni(II) species (UV-vis: 425, 525, and ∼650 nm; electron paramagnetic resonance (EPR) (4 K): g// = 2.21, g⊥ = 2.01; resonance Raman: ν((16)O-(16)O)/ν((18)O-(18)O) = 1020/986 cm(-1)), which is then oxidized to Ni(III) state only in the presence of both a proton and 1-methylimidazole, as evidenced by EPR spectra. Second, EPR spectra indicate that the oxidized complex of 1 with 1-methylimidazole at the axial site can be reduced by reaction with superoxide. The Ni(III) complex with 1-methylimidazole at the axial site does not participate in any direct interaction with azide anion (pKa 4.65) added as mimic of superoxide (pKa 4.88). According to these data, we propose the superoxide disproportionation mechanism in superoxide-reducing and -oxidizing steps of NiSOD in both Ni(II) and Ni(III) states.