Formation Of Covalent Complex Research Articles

Hydrophobic Interactions Contribute to Conformational Stabilization of Endoglycoceramidase II by Mechanism-Based Probes.

Small compound active site interactors receive considerable attention for their ability to positively influence the fold of glycosidases. Endoglycoceramidase II (EGCII) from Rhodococcus sp. is an endo-β-glucosidase releasing the complete glycan from ceramide in glycosphingolipids. Cleavage of the β-glycosidic linkage between glucose and ceramide is also catalyzed by glucocerebrosidase (GBA), the exo-β-glucosidase deficient in Gaucher disease. We demonstrate that established β-glucoside-configured cyclophellitol-type activity-based probes (ABPs) for GBA also are effective, mechanism-based, and irreversible inhibitors of EGCII. The stability of EGCII is markedly enhanced by formation of covalent complexes with cyclophellitol ABPs substituted with hydrophobic moieties, as evidenced by an increased melting temperature, resistance against tryptic digestion, changes in (15)N-(1)H transverse relaxation optimized spectroscopy spectra of the [(15)N]Leu-labeled enzyme, and relative hydrophobicity as determined by 8-anilino-1-naphthalenesulfonic acid fluorescence. The stabilization of EGCII conformation correlates with the shape and hydrophobicity of the substituents of the ABPs. We conclude that the amphipathic active site binders with aliphatic moieties act as a "hydrophobic zipper" on the flexible EGCII protein structure.

A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites both In Vitro and In Vivo by Targeting Type IB Topoisomerase.

Visceral leishmaniasis is a fatal parasitic disease, and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here the development of a novel spirooxindole derivative, N-benzyl-2,2'α-3,3',5',6',7',7α,α'-octahydro-2methoxycarbonyl-spiro[indole-3,3'-pyrrolizidine]-2-one (compound 4c), which inhibits Leishmania donovani topoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in a competitive manner. Unlike camptothecin (CPT), the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, it hinders drug-DNA-enzyme covalent complex formation. Fluorescence studies show that the stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole), with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of compound 4c produced two probable hits for the binding site, one in the small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastigotes of L. donovani and also induces apoptosis-like cell death in the parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites, and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild-type and drug-resistant parasites from infected mouse peritoneal macrophages but has less of an effect on host macrophages. Moreover, compound 4c showed strong antileishmanial efficacies in the BALB/c mouse model of leishmaniasis. This compound potentially can be used as a lead for developing excellent antileishmanial agents against emerging drug-resistant strains of the parasite.

Abstract 2761: Cellular consequences of human tyrosyl-DNA phosphodiesterase I dysregulation

Abstract Tyrosyl-DNA phosphodiesterase I (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that catalyzes the resolution of 3’ and 5’ phospho-DNA adducts. Tdp1 has been implicated in the repair of DNA topoisomerase I (Topo1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 utilizes a two-step catalytic cycle that centers on the formation of an obligatory Tdp1-DNA covalent complex (Tdp1-cc) through its nucleophilic histidine (Hisnuc), resulting in dissociation of the adduct, while its general acid/base histidine (Hisgab) mediates Tdp1 dissociation. A Tdp1Hisgab to Arg (H493R) mutant stabilizes the Tdp1-cc and is associated with autosomal recessive ataxia SCAN1. Alternative substitutions of Hisgab or substitutions of the Hisnuc transforms yeast Tdp1 into a potent toxin via stabilization of Tdp1-cc. We propose that stabilization of this Tdp1-DNA covalent complex is a potential novel therapeutic anti-cancer strategy. As proof-of-concept, we analyzed two catalytic Hisgab (H493R or H493N) mutants and one Hisnuc (H263A) mutant of hTdp1 in HEK293 cells. Doxycycline-induced expression of Tdp1H263A, Tdp1H493R, and Tdp1H493N mutant enzymes induced Tdp1-dependent cytotoxicity without additional genotoxic stress. Utilizing two different immuno-assays, we validated that the observed Tdp1-dependent toxicity correlates with stabilization of their enzyme-DNA covalent complex. Moreover, all of these Tdp1 catalytic mutants show reduced catalytic activity compared to wild type hTdp1, but they do not all show a stabilized Tdp1-cc in this in vitro assay. This indicates a significant difference between in vitro Tdp1 activity and cellular Tdp1 activity, which is most likely due to the difference in substrate; a small oligonucleotide with a 3’phospho-tyrosyl modification versus covalent complex of full length Topo1 with genomic DNA. However, these results confirm our previous yeast studies: Stabilization of the Tdp1-cc converts a DNA repair enzyme into a cellular toxin, which constitutes a potential novel therapeutic strategy to treat cancer. In addition, we are comparing schedule dependent ‘drug’-combinations of our toxic Tdp1 mutant expression with topotecan, etoposide (targets Topo2-cc) and cisplatin to evaluate the potential therapeutic value of this novel Tdp1 targeted strategy. This work is in part supported by the ADDA, UAB ACS-IRG, and DOD OCRP WX81WH-15-1-0198. Citation Format: Selma M. Cuya, Kellie M. Regal, Robert C.A.M. Van Waardenburg. Cellular consequences of human tyrosyl-DNA phosphodiesterase I dysregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2761.

The Catalytic Mechanism of the Marine-Derived Macrocyclase PatGmac.

Cyclic peptides are a class of compounds with high therapeutic potential, possessing bioactivities including antitumor and antiviral (including anti-HIV). Despite their desirability, efficient design and production of these compounds has not been achieved to date. The catalytic mechanism of patellamide macrocyclization by the PatG macrocyclase domain has been computationally investigated by using quantum mechanics/molecular mechanics methodology, specifically ONIOM(M06/6-311++G(2d,2p):ff94//B3LYP/6-31G(d):ff94). The mechanism proposed herein begins with a proton transfer from Ser783 to His 618 and from the latter to Asp548. Nucleophilic attack of Ser783 on the substrate leads to the formation of an acyl-enzyme covalent complex. The leaving group Ala-Tyr-Asp-Gly (AYDG) of the substrate is protonated by the substrate's N terminus, leading to the breakage of the P1-P1' bond. Finally, the substrate's N terminus attacks the P1 residue, decomposing the acyl-enzyme complex forming the macrocycle. The formation and decomposition of the acyl-enzyme complex have the highest activation free energies (21.1 kcal mol(-1) and 19.8 kcal mol(-1) respectively), typical of serine proteases. Understanding the mechanism behind the macrocyclization of patellamides will be important to the application of the enzymes in the pharmaceutical and biotechnological industries.

Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures.

During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.

Conserved motif VIII of murine DNA methyltransferase Dnmt3a is essential for methylation activity.

BackgroundDnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood.ResultsMutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202.ConclusionsWe conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.

Stabilization of reactive species by supramolecular encapsulation.

Molecular containers have attracted the interest of supramolecular chemists since the early beginnings of the field. Cavitands' inner cavities were quickly exploited by Cram and Warmuth to construct covalent containers able to stabilize and assist the characterization of short-lived reactive species such as cyclobutadiene or o-benzyne. Since then, more complex molecular architectures have been prepared able to store and isolate a myriad of fleeting species (i.e. organometallic compounds, cationic species, radical initiators…). In this review we cover selected examples of the stabilization of reactive species by encapsulation in molecular containers from the first reports of covalent containers described by Cram et al. to the most recent examples of containers with self-assembled structure (metal coordination cages and hydrogen bonded capsules). Finally, we briefly review examples reported by Rebek et al. in which elusive reaction intermediates could be detected in the inner cavities of self-folding resorcin[4]arene cavitands by the formation of covalent host-guest complexes. The utilization of encapsulated reactive species in catalysis or synthesis is not covered.

Acute and Temporal Expression of Tumor Necrosis Factor (TNF)-α-stimulated Gene 6 Product, TSG6, in Mesenchymal Stem Cells Creates Microenvironments Required for Their Successful Transplantation into Muscle Tissue

Previously, we demonstrated that when mesenchymal stem cells (MSCs) from mouse ES cells were transplanted into skeletal muscle, more than 60% of them differentiated into muscles in the crush-injured tibialis anterior muscle in vivo, although MSCs neither differentiated nor settled in the intact muscle. Microenvironments, including the extracellular matrix between the injured and intact muscle, were quite different. In the injured muscle, hyaluronan (HA), heavy chains of inter-α-inhibitor (IαI), CD44, and TNF-α-stimulated gene 6 product (TSG-6) increased 24-48 h after injury, although basement membrane components of differentiated muscle such as perlecan, laminin, and type IV collagen increased gradually 4 days after the crush. We then investigated the microenvironments crucial for cell transplantation, using the lysate of C2C12 myotubules for mimicking injured circumstances in vivo. MSCs settled in the intact muscle when they were transplanted together with the C2C12 lysate or TSG6. MSCs produced and released TSG6 when they were cultured with C2C12 lysates in vitro. MSCs pretreated with the lysate also settled in the intact muscle. Furthermore, MSCs whose TSG6 was knocked down by shRNA, even if transplanted or pretreated with the lysate, could not settle in the muscle. Immunofluorescent staining showed that HA and IαI always co-localized or were distributed closely, suggesting formation of covalent complexes, i.e. the SHAP-HA complex in the presence of TSG6. Thus, TSG6, HA, and IαI were crucial factors for the settlement and probably the subsequent differentiation of MSCs.

Fluorescent photoaffinity probes for mitotic protein kinase Aurora A.

We combined the advantages of the selective inhibitor VX689, the bisubstrate-analogue conjugate approach, and photoreactive amino acids to develop 8 photoaffinity probes for Aurora A. The most efficient compounds possessed one-digit nanomolar KD values in the equilibrium binding assay, inhibited Aurora A at elevated concentrations of ATP in the phosphorylation assay in the presence of TPX2, and formed covalent complexes with the recombinant kinase or Aurora A in HeLa cells upon UV-irradiation. The recognition of the correct target by the probes during formation of the covalent complex in the biochemical assay and in situ was demonstrated by competition experiments using the non-labelled inhibitors VX689 and MLN8237.

High-level QM/MM calculations support the concerted mechanism for Michael addition and covalent complex formation in thymidylate synthase.

Thymidylate synthase (TS) is a promising cancer target, due to its crucial function in thymine synthesis. It performs the reductive methylation of 2'-deoxyuridine-5'-phosphate (dUMP) to thymidine-5'-phosphate (dTMP), using N-5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as a cofactor. After the formation of the dUMP/mTHF/TS noncovalent complex, and subsequent conformational activation, this complex has been proposed to react via nucleophilic attack (Michael addition) by Cys146, followed by methylene-bridge formation to generate the ternary covalent intermediate. Herein, QM/MM (B3LYP-D/6-31+G(d)-CHARMM27) methods are used to model the formation of the ternary covalent intermediate. A two-dimensional potential energy surface reveals that the methylene-bridged intermediate is formed via a concerted mechanism, as indicated by a single transition state on the minimum energy pathway and the absence of a stable enolate intermediate. A range of different QM methods (B3LYP, MP2 and SCS-MP2, and different basis sets) are tested for the calculation of the activation energy barrier for the formation of the methylene-bridged intermediate. We test convergence of the QM/MM results with respect to size of the QM region. Inclusion of Arg166, which interacts with the nucleophilic thiolate, in the QM region is important for reliable results; the MM model apparently does not reproduce energies for distortion of the guanidinium side chain correctly. The spin component scaled-Møller-Plessett perturbation theory (SCS-MP2) approach was shown to be in best agreement (within 1.1 kcal/mol) while the results obtained with MP2 and B3LYP also yielded acceptable values (deviating by less than 3 kcal/mol) compared with the barrier derived from experiment. Our results indicate that using a dispersion-corrected DFT method, or a QM method with an accurate treatment of electron correlation, increases the agreement between the calculated and experimental activation energy barriers, compared with the semiempirical AM1 method. These calculations provide important insight into the reaction mechanism of TS and may be useful in the design of new TS inhibitors.

Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor.

The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of Kd values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.

Inactivation of TEM-1 by avibactam (NXL-104): insights from quantum mechanics/molecular mechanics metadynamics simulations.

The fast and constant development of drug-resistant bacteria represents a serious medical emergence. To overcome this problem, the development of drugs with new structures and modes of action is urgently needed. In this context, avibactam represents a promising, innovative inhibitor of beta-lactamases with a novel molecular structure compared to previously developed inhibitors, showing a promising inhibitory activity toward a significant number of beta-lactamase enzymes. In this work, we studied, at the atomistic level, the mechanisms of formation of the covalent complex between avibactam and TEM-1, an experimentally well-characterized class A beta-lactamase, using classical and quantum mechanics/molecular mechanics (QM/MM) simulations combined with metadynamics. Our simulations provide a detailed structural and energetic picture of the molecular steps leading to the formation of the avibactam/TEM-1 covalent adduct. In particular, they support a mechanism in which the rate-determining step is the water-assisted Glu166 deprotonation by Ser70. In this mechanistic framework, the predicted activation energy is in good agreement with experimental kinetic measurements. Additionally, our simulations highlight the important role of Lys73 in assisting the Ser70 and Ser130 deprotonations. While based on the specific case of the avibactam/TEM-1, the simple protocol we present here can be immediately extended and applied to the study of covalent complex formation in different enzyme-inhibitor pairs.

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

α1-Protease inhibitor Portland (α1PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α1PDX with those of serpin B8 demonstrated that both the P4-P1 RXXR recognition sequence as well as the P1'-P5' sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298-300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α1PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose substitution in α1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298-300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α1PDX inhibitors of PCs.

Preparation of Covalently Linked Complexes Between DNA andO6-Alkylguanine-DNA Alkyltransferase Using Interstrand Cross-Linked DNA

O(6)-alkylguanine-DNA alkyltransferases (AGT) are responsible for the removal of alkylation at both the O(6) atom of guanine and O(4) atom of thymine. AGT homologues show vast substrate differences with respect to the size of the adduct and which alkylated atoms they can restore. The human AGT (hAGT) has poor capabilities for removal of methylation at the O(4) atom of thymidine, which is not the case in most homologues. No structural data are available to explain this poor hAGT repair. We prepared and characterized O(6)G-butylene-O(4)T (XLGT4) and O(6)G-heptylene-O(4)T (XLGT7) interstrand cross-linked (ICL) DNA as probes for hAGT and the Escherichia coli homologues, OGT and Ada-C, for the formation of DNA-AGT covalent complexes. XLGT7 reacted only with hAGT and did so with a cross-linking efficiency of 25%, while XLGT4 was inert to all AGT tested. The hAGT mediated repair of XLGT7 occurred slowly, on the order of hours as opposed to the repair of O(6)-methyl-2'-deoxyguanosine which requires seconds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the repair reaction revealed the formation of a covalent complex with an observed migration in accordance with a DNA-AGT complex. The identity of this covalent complex, as determined by mass spectrometry, was composed of a heptamethylene bridge between the O(4) atom of thymidine (in an 11-mer DNA strand) to residue Cys145 of hAGT. This procedure can be applied to produce well-defined covalent complexes between AGT with DNA.

Novel composites of multifunctional Fe3O4@Au nanofibers for highly efficient glycoprotein imprinting.

A kind of surface glycoprotein imprinting over magnetic Fe3O4@Au multifunctional nanofibers (NFs) was developed and investigated. Magnetic Fe3O4@Au nanoparticles (NPs) as the core materials were modified consecutively with aniline, 3-aminophenylboronic acid (APBA) and acrylic acid to introduce boronic acids and polymerizable double bonds. With horseradish peroxidase (HRP) as a glycoprotein template, thin protein-imprinted films were fabricated via radical induced graft copolymerization of monomers on the surface of the multifunctional NFs. Experimental results show that the magnetic multifunctional Fe3O4@Au NFs can not only direct the selective occurrence of imprinting polymerization, but also drive glycoprotein templates into the polymer through reversible covalent complex formation. The results show that the imprinted NFs reached saturated adsorption at 0.3 mg mL-1 within 90 min and exhibited significant specific recognition towards the template protein. Moreover, the molecularly imprinted polymers (MIPs) were used to electrochemically detect HRP with good linearity in the range of low concentrations from 0.01 to 0.30 mg mL-1 through molecular recognition of K3[Fe(CN)6] current response. The detection limit of this method was found to be 0.005 mg mL-1 (S/N = 3). The synthetic strategy paves the way for preparation of functional nanomaterials by molecular imprinting technique and direct detection of proteins in a more convenient, simpler and cheaper way.

Identification of plasma proteinase complexes with serpin-3 in Manduca sexta

Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimicrobial peptide production in insect innate immune responses. Serpins in plasma regulate such cascades by selective inhibition of proteinases, in reactions which result in the formation of covalent serpin-proteinase complexes. We carried out experiments to identify plasma proteinases that are inhibited by Manduca sexta serpin-3, an immune-inducible serpin known to regulate proPO activation. Immunoaffinity chromatography, using antiserum to serpin-3, yielded serpin-3 complexes with proteinases identified by immunoblot analysis as prophenoloxidase-activating proteinase (PAP)-1, PAP-2, PAP-3, and hemolymph proteinase 8 (HP8). HP8 can cleave and activate the Toll ligand, Spätzle, leading to synthesis of antimicrobial peptides. Analysis by mass spectrometry of tryptic peptides derived from the serpin-3 complexes confirmed the presence of PAP-1, PAP-3, and HP8. Purified recombinant serpin-3 and active HP8 formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.

The Catalytic Machinery of Rhomboid Proteases: Combined MD and QM Simulations.

Rhomboid proteases are a ubiquitous family of intramembrane serine proteases in prokaryotic and eukaryotic organisms that cleave membrane proteins in their transmembrane region. Their catalytic activity is centered at a His-Ser catalytic dyad. We applied molecular dynamics and quantum mechanics calculations in order to clarify the protonation state of the catalytic residues of E. coli GlpG rhomboid protease and how it is affected by the immersion of the enzyme in the membrane. We identified (Nε)H150(dpr)_H254(dpr)_S201(pr) as the protonation (and H150 tautomeric) state of free GlpG in both lipid-solubilized and membrane environments. We used our MD-QM/SCRF(VS) computational protocol to rationalize and predict the trend of pKa change caused by the decrease of water exposure of the active site of GlpG due to ligand binding. The catalytic diad of lipid-solubilized GlpG exists as an H254(+)_S201(-) ion pair at the Michaelis complex stage, with Ser201 ready for nucleophilic attack on the substrate. Therefore, deprotonation of S201 does not contribute to the activation barrier of covalent tetrahedral complex formation. In contrast, both catalytic residues, H254 and S201, are neutral in the Michaelis complex of GlpG in the membrane. Therefore, S201 deprotonation by H254 general base catalysis should contribute to the activation barrier of the covalent tetrahedral complex formation.

Identification of the cysteine residue responsible for disulfide linkage of Na+ channel α and β2 subunits.

Voltage-gated Na(+) channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked β subunit (β1 or β3), and one disulfide-linked β subunit (β2 or β4). The final step in Na(+) channel biosynthesis in central neurons is concomitant α-β2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding β2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant β2 cDNA constructs to investigate the cysteine residue(s) responsible for α-β2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and β2 subunits. Loss of α-β2 covalent complex formation disrupts the targeting of β2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal β2 subcellular localization in vivo. WT β2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant β2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-β2 covalent association via a single, extracellular disulfide bond is required for β2 targeting to specialized neuronal subcellular domains and for β2 association with the neuronal cytoskeleton within those domains.

Putting a Stop to l,d-Transpeptidases

In this issue of Structure, Lecoq et al. investigated the structural and dynamic basis for the unexpected inhibition of peptidoglycan-crosslinking l,d-transpeptidases by carbapenem antibiotics. In addition to defining a neutral thiol-imidazole catalytic triad, their studies revealed extensive induced motions upon formation of a long-live covalent drug-enzyme complex.

MxTiSe2 (M=Cr, Mn, Cu) electronic structure study by methods of resonance X-ray photoemission spectroscopy and X-ray absorption spectroscopy

Intercalation of Cu and Mn between the layers of TiSe2 leads to the charge transfer from the doped atom to Ti atoms, electronic states of which form the conduction band of TiSe2. This situation drastically differs from another one being observed during intercalation of other transition metals into TiSe2 that leads to the formation of covalent complexes consisting of intercalated atom and nearest neighborhood of the host lattice. Substitution of Ti by Cr in the host lattice positions does not show both charge transfer nor formation of covalent complexes.