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Background The carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb). Results Here, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by β-lyases. Conclusion The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier. Electronic supplementary material The online version of this article (doi:10.1186/s12858-017-0082-4) contains supplementary material, which is available to authorized users.

We investigated direct interactions between the human immunodeficiency virus (HIV)-trans-activator of transcription (Tat) protein and amyloid β peptide. Amyloid β-Tat complexes are readily formed extracellularly in the brain. In vitro studies showed that in the presence of Tat, the uniform amyloid fibrils turned into double twisted fibrils followed by populations with thick unstructured filaments and aggregated large patches in a dose-dependent manner. The fibers became more rigid and mechanically resistant. Tat attached externally to fibrils, causing their lateral aggregation into thick multifibrilar structures. These present growth in β sheet and enhanced adhesion. The neurotoxic properties of Tat and amyloid β aggregates were strongly synergistic when complexed together in vitro and in animal models. These data suggest that the increased rigidity and mechanical resistance of the amyloid β-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils accounted for the increased damage, likely through pore formation in membranes.

Galectins, highly conserved β-galactoside-binding lectins, have diverse regulatory roles in development and immune homeostasis and can mediate protective functions during microbial infection. In recent years, the role of galectins in viral infection has generated considerable interest. Studies on highly pathogenic viruses have provided invaluable insight into the participation of galectins in various stages of viral infection, including attachment and entry. Detailed mechanistic and structural aspects of these processes remain undetermined. To address some of these gaps in knowledge, we used Zebrafish as a model system to examine the role of galectins in infection by infectious hematopoietic necrosis virus (IHNV), a rhabdovirus that is responsible for significant losses in both farmed and wild salmonid fish. Like other rhabdoviruses, IHNV is characterized by an envelope consisting of trimers of a glycoprotein that display multiple N-linked oligosaccharides and play an integral role in viral infection by mediating the virus attachment and fusion. Zebrafish’s proto-typical galectin Drgal1-L2 and the chimeric-type galectin Drgal3-L1 interact directly with the glycosylated envelope of IHNV, and significantly reduce viral attachment. In this study, we report the structure of the complex of Drgal1-L2 with N-acetyl-d-lactosamine at 2.0 Å resolution. To gain structural insight into the inhibitory effect of these galectins on IHNV attachment to the zebrafish epithelial cells, we modeled Drgal3-L1 based on human galectin-3, as well as, the ectodomain of the IHNV glycoprotein. These models suggest mechanisms for which the binding of these galectins to the IHNV glycoprotein hinders with different potencies the viral attachment required for infection.

HIV can infiltrate the brain and lead to HIV-associated neurocognitive disorders (HAND). The pathophysiology of HAND is poorly understood, and there are no diagnostic biomarkers for it. Previously, an increase in inducible nitric oxide synthase levels and protein tyrosine nitration in the brain were found to correlate with the severity of HAND.1,2 In this study, we analyzed human brains from individuals who had HIV infection without encephalitis and with encephalitis/HAND and compared them to the brains of healthy individuals. We identified the nitrated proteins and determined the sites of modification using affinity enrichment followed by high-resolution and high-mass-accuracy nanoLC–MS/MS. We found that nitrated proteins were predominantly present in the HIV-infected individuals with encephalitis, and, interestingly, the modifications were predominantly located on immunoglobulin variable regions. Our molecular model indicated potential interactions with HIV envelope proteins and changes on the heavy and light chain interface upon the nitration and nitrohydroxylation of these residues. Therefore, our findings suggest a role for these modifications in the immune response, which may have implications in disease pathogenesis.

Although lectins are “hard-wired” in the germline, the presence of tandemly arrayed carbohydrate recognition domains (CRDs), of chimeric structures displaying distinct CRDs, of polymorphic genes resulting in multiple isoforms, and in some cases, of a considerable recognition plasticity of their carbohydrate binding sites, significantly expand the lectin ligand-recognition spectrum and lectin functional diversification. Analysis of structural/functional aspects of galectins and F-lectins—the most recently identified lectin family characterized by a unique CRD sequence motif (a distinctive structural fold) and nominal specificity for l-Fuc—has led to a greater understanding of self/nonself recognition by proteins with tandemly arrayed CRDs. For lectins with a single CRD, however, recognition of self and nonself glycans can only be rationalized in terms of protein oligomerization and ligand clustering and presentation. Spatial and temporal changes in lectin expression, secretion, and local concentrations in extracellular microenvironments, as well as structural diversity and spatial display of their carbohydrate ligands on the host or microbial cell surface, are suggestive of a dynamic interplay of their recognition and effector functions in development and immunity.

The acute motor axonal neuropathy (AMAN) variant of Guillain-Barré syndrome (GBS) is associated with anti-GD1a and anti-GM1 IgG antibodies. The basis of preferential motor nerve injury in this disease is not clear, however, because biochemical studies demonstrate that sensory and motor nerves express similar quantities of GD1a and GM1 gangliosides. To elucidate the pathophysiology of AMAN, we have developed several monoclonal antibodies (mAbs) with GD1a reactivity and reported that one mAb, GD1a-1, preferentially stained motor axons in human and rodent nerves. To understand the basis of this preferential motor axon staining, several derivatives of GD1a were generated by various chemical modifications of N-acetylneuraminic (sialic) acid residues (GD1a NeuAc 1-amide, GD1a NeuAc ethyl ester, GD1a NeuAc 1-alcohol, GD1a NeuAc 1-methyl ester, GD1a NeuAc 7-alcohol, GD1a NeuAc 7-aldehyde) on this ganglioside. Binding of anti-GD1a mAbs and AMAN sera with anti-GD1a Abs to these derivatives was examined. Our results indicate that mAbs with selective motor axon staining had a distinct pattern of reactivity with GD1a-derivatives compared to mAbs that stain both motor and sensory axons. The fine specificity of the anti-GD1a antibodies determines their motor selectivity, which was validated by cloning a new mAb (GD1a-E6) with a chemical and immunocytochemical binding pattern similar to that of GD1a-1 but with two orders of magnitude higher affinity. Control studies indicate that selective binding of mAbs to motor nerves is not due to differences in antibody affinity or ceramide structural specificity. Since GD1a-reactive mAb with preferential motor axon staining showed similar binding to sensory- and motor nerve-derived GD1a in a solid phase assay, we generated computer models of GD1a based on binding patterns of different GD1a-reactive mAbs to different GD1a-derivatives. These modelling studies suggest that critical GD1a epitopes recognized by mAbs are differentially expressed in motor and sensory nerves. The GD1a-derivative binding patterns of AMAN sera resembled those with motor-specific mAbs. On the basis of these findings we postulate that both the fine specificity and ganglioside orientation/exposure in the tissues contribute to target recognition by anti-ganglioside antibodies and this observation provides one explanation for preferential motor axon injury in AMAN.

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U•A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U•A or U•G base pairs in a background of approximately 109 T•A or C•G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T•A and U•A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases. Uracil (U) belongs in RNA, where it takes the place filled by thymine in DNA. But if uracil appears in DNA in error, it can lead to potentially life-threatening mutations. This can typically occur by chemical modification of cytosine. To counter this threat, cells use the enzyme uracil DNA glycosylase to remove uracil from DNA. The detailed mechanism by which this enzyme polices DNA for stray uracils is now revealed. The DNA helix is not static but in a process rather like molecular 'breathing'; base pairs separate briefly then reform. When a uracil base pops out of the helix it is grabbed by uracil DNA glycosylase and removed. Thymine, differing only in one methyl group from uracil, is similarly grabbed but as it does not quite fit the policing enzyme's active site, it is released to go about its business in the DNA molecule. The enzyme uracil DNA glycosylase does not actively extrude just the uracil base from the DNA helix to facilitate its removal; instead, transient, passive opening of thymine: adenine and uracil: adenine base pairs allows both thymine and uracil to become extrahelical, but only uracil can subsequently fit in the active site.

MICALs (Molecule Interacting with CasL) are conserved multidomain enzymes essential for cytoskeletal reorganization in nerve development, endocytosis, and apoptosis. In these enzymes, a type-2 calponin homology (CH) domain always follows an N-terminal monooxygenase (MO) domain. Although the CH domain is required for MICAL-1 cellular localization and actin-associated function, its contribution to the modulation of MICAL activity towards actin remains unclear. Here, we present the structure of a fragment of MICAL-1 containing the MO and the CH domains—determined by X-ray crystallography and small angle scattering—as well as kinetics experiments designed to probe the contribution of the CH domain to the actin-modification activity. Our results suggest that the CH domain, which is loosely connected to the MO domain by a flexible linker and is far away from the catalytic site, couples F-actin to the enhancement of redox activity of MICALMO-CH by a cooperative mechanism involving a trans interaction between adjacently bound molecules. Binding cooperativity is also observed in other proteins regulating actin assembly/disassembly dynamics, such as ADF/Cofilins.

Galectins are highly conserved lectins that are key to multiple biological functions, including pathogen recognition and regulation of immune responses. We previously reported that CvGal1, a galectin expressed in phagocytic cells (hemocytes) of the eastern oyster (Crassostrea virginica), is hijacked by the parasite Perkinsus marinus to enter the host, where it causes systemic infection and death. Screening of an oyster hemocyte cDNA library revealed a novel galectin, which we designated CvGal2, with four tandemly arrayed carbohydrate recognition domains (CRDs). Phylogentic analysis of the CvGal2 CRDs suggests close relationships with homologous CRDs from CvGal1. Glycan array analysis, however, revealed that, unlike CvGal1 which preferentially binds to the blood group A tetrasaccharide, CvGal2 recognizes both blood group A and B tetrasaccharides and related structures, suggesting that CvGal2 has broader binding specificity. Furthermore, SPR analysis demonstrated significant differences in the binding kinetics of CvGal1 and CvGal2, and structural modeling revealed substantial differences in their interactions with the oligosaccharide ligands. CvGal2 is homogeneously distributed in the hemocyte cytoplasm, is released to the extracellular space, and binds to the hemocyte surface. CvGal2 binds to P. marinus trophozoites in a dose-dependent and β-galactoside-specific manner. Strikingly, negligible binding of CvGal2 was observed for Perkinsus chesapeaki, a sympatric parasite species mostly prevalent in the clams Mya arenaria and Macoma balthica. The differential recognition of Perkinsus species by the oyster galectins is consistent with their relative prevalence in oyster and clam species and supports their role in facilitating parasite entry and infectivity in a host-preferential manner.

The final step of peptidoglycan (PG) biosynthesis in bacteria involves cross-linking of peptide side chains. This step in Mycobacterium tuberculosis is catalyzed by ld- and dd-transpeptidases that generate 3→3 and 4→3 transpeptide linkages, respectively. M. tuberculosis PG is predominantly 3→3 cross-linked, and LdtMt2 is the dominant ld-transpeptidase. There are four additional sequence paralogs of LdtMt2 encoded by the genome of this pathogen, and the reason for this apparent redundancy is unknown. Here, we studied one of the paralogs, LdtMt5, and found it to be structurally and functionally distinct. The structures of apo-LdtMt5 and its meropenem adduct presented here demonstrate that, despite overall architectural similarity to LdtMt2, the LdtMt5 active site has marked differences. The presence of a structurally divergent catalytic site and a proline-rich C-terminal subdomain suggest that this protein may have a distinct role in PG metabolism, perhaps involving other cell wall-anchored proteins. Furthermore, M. tuberculosis lacking a functional copy of LdtMt5 displayed aberrant growth and was more susceptible to killing by crystal violet, osmotic shock, and select carbapenem antibiotics. Therefore, we conclude that LdtMt5 is not a functionally redundant ld-transpeptidase, but rather it serves a unique and important role in maintaining the integrity of the M. tuberculosis cell wall.

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.

GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-{alpha}-D-hexose to GDP and {beta}-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the structure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An important loop (L6, residues 119-125) contains a ligand to the essential Mg{sup 2+} (Gln-123), the catalytic base (His-124), and three anionic residues. This loop is not ordered in the X-ray structure of the free enzyme due to dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selective exchange broadening of the imidazole nitrogen resonances of His-124 (k{sub ex} = 6.6 x 10{sup 4} s{sup -1}). The structure of the enzyme-Mg{sup 2+}-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6 in an open conformation, while the structure of the enzyme-Mg{sup 2+}-GDP product complex showed loop L6 in a closed, 'active' conformation. 1H-15N HMQC spectra show the imidazole N of His-124 to be unprotonated, appropriate for general base catalysis. Substituting Mg{sup 2+} with the more electrophilic metal ions Mn{sup 2+} or Co{sup 2+} decreases the pK{sub a} in the pH versus k{sub cat}more » rate profiles, showing that deprotonation of a metal-bound water is partially rate-limiting. The H124Q mutation, which decreases k{sub cat} 103.4-fold and largely abolishes its pH dependence, is rescued by the Y103F mutation, which increases k{sub cat} 23-fold and restores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shifts the conformational equilibrium to the open form moving loop L6 out of the active site, thus permitting direct access of the specific base hydroxide from the solvent. In the proposed dissociative transition state, which occurs in the closed, active conformation of the enzyme, the partial negative charge of the GDP leaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to the -phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transition state is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizing the transition state.« less

GumK is a membrane-associated inverting glucuronosyltransferase that is part of the biosynthetic route of xanthan, an industrially important exopolysaccharide produced by Xanthomonas campestris. The enzyme catalyzes the fourth glycosylation step in the pentasaccharide-P-P-polyisoprenyl assembly, an oligosaccharide diphosphate lipid intermediate in xanthan biosynthesis. GumK has marginal homology to other glycosyltransferases (GTs). It belongs to the CAZy family GT 70, for which no structure is currently available, and indirect biochemical evidence suggests that it also belongs to the GT-B structural superfamily. Crystals of recombinant GumK from X. campestris have been grown that diffract to 1.9 A resolution. Knowledge of the crystal structure of GumK will help in understanding xanthan biosynthesis and its regulation and will also allow a subsequent rational approach to enzyme design and engineering. The multiwavelength anomalous diffraction approach will be used to solve the phase problem.

During development, neurons are guided to their targets by short- and long-range attractive and repulsive cues. MICAL, a large multidomain protein, is required for the combined action of semaphorins and plexins in axon guidance. Here, we present the structure of the N-terminal region of MICAL (MICAL fd ) determined by x-ray diffraction to 2.0 Å resolution. The structure shows that MICAL fd is an FAD-containing module structurally similar to aromatic hydroxylases and amine oxidases. In addition, we present biochemical data that show that MICAL fd is a flavoenzyme that in the presence of NADPH reduces molecular oxygen to H 2 O 2 ( K m,NAPDH = 222 μM; k cat = 77 sec -1 ), a molecule with known signaling properties. We propose that the H 2 O 2 produced by this reaction may be one of the signaling molecules involved in axon guidance by MICAL.

The DNA repair enzyme uracil DNA glycosylase has been crystallized with a cationic 1-aza-2‘-deoxyribose-containing DNA that mimics the ultimate transition state of the reaction in which the water nucleophile attacks the anomeric center of the oxacarbenium ion−uracil anion reaction intermediate. Comparison with substrate and product structures, and the previous structure of the intermediate determined by kinetic isotope effects, reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis. This structure provides a rare opportunity to reconstruct the detailed structural transformations that occur along an enzymatic reaction coordinate.

To couple the energy present in the electrochemical proton gradient, established across the mitochondrial membrane by the respiratory chain, to the formation of ATP from ADP and Pi, ATP-synthase goes through a sequence of coordinated conformational changes of its major subunits (alpha, beta). These changes are induced by the rotation of the gamma subunit driven by the translocation of protons through the c subunit of the membrane portion of the enzyme. During this process, the F1-portion of the ATP-synthase adopts at least two major conformations depending on the occupancy of the beta subunits: one with two nucleotides, the other with three. In the two-nucleotide structure, the empty beta subunit adopts an open conformation that is highly different from the other conformations of beta subunits: tight, loose and closed. The three-dimensional structures of the F1-ATPase in each of these two major conformations provide a framework for understanding the mechanism of energy coupling by the enzyme. The energetics associated with two different models of the reaction steps, analysed using molecular dynamics calculations, show that three-nucleotide intermediates do not occur in configurations with an open beta subunit; instead, they are stabilized by completing a jaw-like motion that closes the beta subunit around the nucleotide. Consequently, the energy driven, major conformational change takes place with the beta subunits in the tight, loose and closed conformation.

Galectins, a family of soluble beta-galactosyl-binding lectins, are believed to mediate cell-cell and cell-extracellular matrix interactions during development, inflammation, apoptosis, and tumor metastasis. However, neither the detailed mechanisms of their function(s) nor the identities of their natural ligands have been unequivocally elucidated. Of the several galectins present in the nematode Caenorhabditis elegans, the 16-kDa "proto" type and the 32-kDa "tandem-repeat" type are the best characterized so far, but their carbohydrate specificities have not been examined in detail. Here, we report the carbohydrate-binding specificity of the recombinant C. elegans 16-kDa galectin and the structural analysis of its binding site by homology modeling. Our results indicate that unlike the galectins characterized so far, the C. elegans 16-kDa galectin interacts with most blood group precursor oligosaccharides (type 1, Galbeta1,3GlcNAc, and type 2, Galbeta1,4GlcNAc; Talpha, Galbeta1,3GalNAcalpha; Tbeta, Galbeta1,3GalNAcbeta) and gangliosides containing the Tbeta structure. Homology modeling of the C. elegans 16-kDa galectin CRD revealed that a shorter loop containing residues 66-69, which enables interactions of Glu(67) with both axial and equatorial -OH at C-3 of GlcNAc (in Galbeta1,4GlcNAc) or at C-4 of GalNAc (in Galbeta1,3GalNAc), provides the structural basis for this novel carbohydrate specificity.

Escherichia coli ADP-ribose (ADPR) pyrophosphatase (ADPRase), a Nudix enzyme, catalyzes the Mg(2+)-dependent hydrolysis of ADP-ribose to AMP and ribose 5-phosphate. ADPR hydrolysis experiments conducted in the presence of H(2)(18)O and analyzed by electrospray mass spectrometry showed that the ADPRase-catalyzed reaction takes place through nucleophilic attack at the adenosyl phosphate. The structure of ADPRase in complex with Mg(2+) and a nonhydrolyzable ADPR analogue, alpha,beta-methylene ADP-ribose, reveals an active site water molecule poised for nucleophilic attack on the adenosyl phosphate. This water molecule is activated by two magnesium ions, and its oxygen contacts the target phosphorus (P-O distance of 3.0 A) and forms an angle of 177 degrees with the scissile bond, suggesting an associative mechanism. A third Mg(2+) ion bridges the two phosphates and could stabilize the negative charge of the leaving group, ribose 5-phosphate. The structure of the ternary complex also shows that loop L9 moves fully 10 A from its position in the free enzyme, forming a tighter turn and bringing Glu 162 to its catalytic position. These observations indicate that as part of the catalytic mechanism, the ADPRase cycles between an open (free enzyme) and a closed (substrate-metal complex) conformation. This cycling may be important in preventing nonspecific hydrolysis of other nucleotides.

Regulation of cellular levels of ADP-ribose is important in preventing nonenzymatic ADP-ribosylation of proteins. The Escherichia coli ADP-ribose pyrophosphatase, a Nudix enzyme, catalyzes the hydrolysis of ADP-ribose to ribose-5-P and AMP, compounds that can be recycled as part of nucleotide metabolism. The structures of the apo enzyme, the active enzyme and the complex with ADP-ribose were determined to 1.9A, 2.7A and 2.3A, respectively. The structures reveal a symmetric homodimer with two equivalent catalytic sites, each formed by residues of both monomers, requiring dimerization through domain swapping for substrate recognition and catalytic activity. The structures also suggest a role for the residues conserved in each Nudix subfamily. The Nudix motif residues, folded as a loop-helix-loop tailored for pyrophosphate hydrolysis, compose the catalytic center; residues conferring substrate specificity occur in regions of the sequence removed from the Nudix motif. This segregation of catalytic and recognition roles provides versatility to the Nudix family.

The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.

Galectin-1, S-type β-galactosyl-binding lectins present in vertebrate and invertebrate species, are dimeric proteins that participate in cellular adhesion, activation, growth regulation, and apoptosis. Two high-resolution crystal structures of B. arenarum galectin-1 in complex with two related carbohydrates, LacNAc and TDG, show that the topologically equivalent hydroxyl groups in the two disaccharides exhibit identical patterns of interaction with the protein. Groups that are not equivalent between the two sugars present in the second moiety of the disaccharide, interact differently with the protein, but use the same number and quality of interactions. The structures show additional protein-carbohydrate interactions not present in previously reported lectin-lactose complexes. These contacts provide an explanation for the enhanced affinity of galectin-1 for TDG and LacNAc relative to lactose. Galectins are in dimer-monomer equilibrium at physiological protein concentrations, suggesting that this equilibrium may be involved in organ-specific regulation of activity. Comparison of B. arenarum with other galectin-1 structures shows that among different galectins there are significant changes in accessible surface area buried upon dimer formation, providing a rationale for the variations observed in the free-energies of dimerization. The structure of the B. arenarum galectin-1 has a large cleft with a strong negative potential that connects the two binding sites at the surface of the protein. Such a striking characteristic suggests that this cleft is probably involved in interactions of the galectin with other intra or extra-cellular proteins. Proteins 2000;40:378–388. © 2000 Wiley-Liss, Inc.

In mammals, two separate but homologous cytosolic quinone reductases have been identified: NAD(P)H:quinone oxidoreductase type 1 (QR1) (EC 1.6.99.2) and quinone reductase type 2 (QR2). Although QR1 and QR2 are nearly 50% identical in protein sequence, they display markedly different catalytic properties and substrate specificities. We report here two crystal structures of QR2: in its native form and bound to menadione (vitamin K(3)), a physiological substrate. Phases were obtained by molecular replacement, using our previously determined rat QR1 structure as the search model. QR2 shares the overall fold of the major catalytic domain of QR1, but lacks the smaller C-terminal domain. The FAD binding sites of QR1 and QR2 are very similar, but their hydride donor binding sites are considerably different. Unexpectedly, we found that QR2 contains a specific metal binding site, which is not present in QR1. Two histidine nitrogens, one cysteine thiol, and a main chain carbonyl group are involved in metal coordination. The metal binding site is solvent-accessible, and is separated from the FAD cofactor by a distance of about 13 A.

During mitochondrial ATP synthesis, F 1 -ATPase—the portion of the ATP synthase that contains the catalytic and regulatory nucleotide binding sites—undergoes a series of concerted conformational changes that couple proton translocation to the synthesis of the high levels of ATP required for cellular function. In the structure of the rat liver F 1 -ATPase, determined to 2.8-Å resolution in the presence of physiological concentrations of nucleotides, all three β subunits contain bound nucleotide and adopt similar conformations. This structure provides the missing configuration of F 1 necessary to define all intermediates in the reaction pathway. Incorporation of this structure suggests a mechanism of ATP synthesis/hydrolysis in which configurations of the enzyme with three bound nucleotides play an essential role.

The chemical mechanism by which the F1 moiety of ATP synthase hydrolyzes and synthesizes ATP remains unknown. For this reason, we have carried out studies with orthovanadate (Vi), a phosphate analog which has the potential of “locking” an ATPase, in its transition state by forming a MgADP·Vi complex, and also the potential, in a photochemical reaction resulting in peptide bond cleavage, of identifying an amino acid very near the γ-phosphate of ATP. Upon incubating purified rat liver F1 with MgADP and Vi for 2 h to promote formation of a MgADP·Vi-F1 complex, the ATPase activity of the enzyme was markedly inhibited in a reversible manner. When the resultant complex was formed in the presence of ultraviolet light inhibition could not be reversed, and SDS-polyacrylamide gel electrophoresis revealed, in addition to the five known subunit bands characteristic of F1 (i.e. α, β, γ, δ, and e), two new electrophoretic species of 17 and 34 kDa. Western blot and N-terminal sequencing analyses identified both bands as arising from the β subunit with the site of peptide bond cleavage occurring at alanine 158, a conserved residue within F1-ATPases and the third residue within the nucleotide binding consensus GX 4GK(T/S) (P-loop). Quantification of the amount of ADP bound within the MgADP·Vi-F1 complex revealed about 1.0 mol/mol F1, while quantification of the peptide cleavage products revealed that no more than one β subunit had been cleaved. Consistent with the cleavage reaction involving oxidation of the methyl group of alanine was the finding that [3H] from NaB[3H]4 incorporates into MgADP·Vi-F1 complex following treatment with ultraviolet light. These novel findings provide information about the transition state involved in the hydrolysis of ATP by a single β subunit within F1-ATPases and implicate alanine 158 as residing very near the γ-phosphate of ATP during catalysis. When considered with earlier studies on myosin and adenylate kinase, these studies also implicate a special role for the third residue within the GX 4GK(T/S) sequence of many other nucleotide-binding proteins.

ガレクチンは、恒温脊椎動物に広く見出され、細胞間や細胞-細胞外マトリクス間の相互作用などの種々の生物現象に関わると考えられるが、細胞外での安定性をはじめ、作用機作の詳細は今なお不明である。さらに、変温脊椎動物や無脊椎動物でもガレクチンが存在するが、それらの性質や内在性のリガンドとなる糖鎖、生理的機能やガレクチン家系における進化系譜など、ほとんど明らかではない。両生類のガレクチンは、ヒキガエルの Bufo arenarum、カエルの Rana tigerina と R. catesbeiana、アフリカツメガエルの Xenopus laevis、アホロートルの Ambystoma mexicanum などから単離されているが、詳細に調べられているのは X. laevis と B. arenarum のみである。私たちは最近、ヒキガエル (B. arenarum) 卵巣ガレクチンが、一次構造をはじめ、三次構造や糖特異性に関して、祖先型にあたる X. laevis のガレクチンよりも、むしろウシのガレクチン-1に近いことを示した。この私たちの知見はいくつかの疑問をなげかけている。すなわち、Xenopus 属 (古カエル亜目) のようないわゆる「原始的な」グループに対して、Bufo 属 (新カエル亜目) のような「近代的な」両生類の現存種内で、どれだけ広範にガレクチン-1に似たレクチンが卵母細胞に存在するのか?これらのレクチンが存在するか否かは、その種の生活史や環境要因の結果なのか? あるいは新カエル亜目に存在するガレクチン-1様のレクチンが、その構造や特異性を脊椎動物を通して進化的に系統保存されてきたことは、このレクチンの生理機能が極めて重要であることを示しているのか?といった問題である。X. laevis ガレクチンは、主として成体の皮膚に限られるのに対して、B. arenarum ガレクチンは、卵母細胞や受精後の各段階に存在することから、これらのカエルは系統的には近いが、それぞれのガレクチンは基本的には全く異なる生理機能、たとえば X. laevis では生体防御機構、B. arenarum では発生過程ではたらくことが考えられる。恒温脊椎動物のガレクチン-1は、胞胚期の最も初期に発現し、子宮内膜上への栄養芽胚の着床に関与すると言われている。一方、B. arenarum のガレクチン活性は、卵母細胞から受精卵、胞胚前の発生段階にかけて見つかり、Bufo 属の卵のまわりを被って保護しているゼリー層との接着や、胚発生における細胞間や細胞と細胞間マトリクス間の相互作用に関与していることが考えられる。ガレクチンを系統的に調べたり、その機能を考えるうえで、両生類は非常に良いモデルといえるだろう。

Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the three dimensional structure of no member of this super-family has been elucidated. To gain structural insight, the known structures of several other nucleotides binding proteins can be used as a framework for modeling these domains. We have modeled both nucleotide binding domains of the protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) using the two similar domains of mitochondrial F1-ATPase. The models obtained, provide useful insights into the putative functions of these domains and their possible interaction as well as a rationale for the basis of Cystic Fibrosis causing mutations. First, the two nucleotide binding domains (folds) of CFTR are each predicted to span a 240-250 amino acid sequence rather than the 150-160 amino acid sequence originally proposed. Second, the first nucleotide binding fold, is predicted to catalyze significant rates of ATP hydrolysis as a catalytic base (E504) resides near the y phosphate of ATP. This prediction has been verified experimentally [Ko, Y.H., and Pedersen, P.L. (1995) J. Biol. Chem. 268, 24330-24338], providing support for the model. In contrast, the second nucleotide binding fold is predicted at best to be a weak ATPase as the glutamic acid residue is replaced with a glutamine. Third, F508, which when deleted causes approximately 70% of all cases of cystic fibrosis, is predicted to lie in a cleft near the nucleotide binding pocket. All other disease causing mutations within the two nucleotide binding domains of CFTR either reside near the Walker A and Walker B consensus motifs in the heart of the nucleotide binding pocket, or in the C motif which lies outside but near the nucleotide binding pocket. Finally, the two nucleotide binding domains of CFTR are predicted to interact, and in one of the two predicted orientations, F508 resides near the interface. This is the first report where both nucleotide binding domains of an ABC transporter and their putative domain-domain interactions have been modeled in three dimensions. The methods and the template used in this work can be used to analyze the structures and function of the nucleotide binding domains of all other members of the ABC transporter super-family.

The galectin CvGal1 from the eastern oyster (Crassostrea virginica), which possesses four tandemly arrayed carbohydrate recognition domains, was previously shown to display stronger binding to galactosamine and N-acetylgalactosamine relative to d-galactose. CvGal1 expressed by phagocytic cells is “hijacked” by the parasite Perkinsus marinus to enter the host, where it proliferates and causes systemic infection and death. In this study, a detailed glycan array analysis revealed that CvGal1 preferentially recognizes type 2 blood group A oligosaccharides. Homology modeling of the protein and its oligosaccharide ligands supported this preference over type 1 blood group A and B oligosaccharides. The CvGal ligand models were further validated by binding, inhibition, and competitive binding studies of CvGal1 and ABH-specific monoclonal antibodies with intact and deglycosylated glycoproteins, hemocyte extracts, and intact hemocytes and by surface plasmon resonance analysis. A parallel glycomic study carried out on oyster hemocytes (Kurz, S., Jin, C., Hykollari, A., Gregorich, D., Giomarelli, B., Vasta, G. R., Wilson, I. B. H., and Paschinger, K. (2013) J. Biol. Chem. 288,) determined the structures of oligosaccharides recognized by CvGal1. Proteomic analysis of the hemocyte glycoproteins identified β-integrin and dominin as CvGal1 “self”-ligands. Despite strong CvGal1 binding to P. marinus trophozoites, no binding of ABH blood group antibodies was observed. Thus, parasite glycans structurally distinct from the blood group A oligosaccharides on the hemocyte surface may function as potentially effective ligands for CvGal1. We hypothesize that carbohydrate-based mimicry resulting from the host/parasite co-evolution facilitates CvGal1-mediated cross-linking to β-integrin, located on the hemocyte surface, leading to cell activation, phagocytosis, and host infection.

The F1 moiety of rat liver ATP synthase has a molecular mass of 370,000, exhibits the unique substructure α3β3γδ∊, and fully restores ATP synthesis to F1-depleted membranes. Here we provide new information about rat liver F1 as it relates to the relationship of its unique substructure to its nucleotide binding properties, enzymatic states, and crystalline form. Seven types of experiments were performed in a comprehensive study. First, the capacity of F1 to bind [3H]ADP, the substrate for ATP synthesis and [32P]AMP-PNP (5′-adenylyl-β,γ-imidodiphosphate), a nonhydrolyzable ATP analog, was quantified. Second, double-label experiments were performed to establish whether ADP and AMP-PNP bind to the same or different sites. Third, total nucleotide binding was assessed by the luciferin-luciferase assay. Fourth, F1 was subfractionated into an αγ and a βδ∊ fraction, both of which were subjected to nucleotide binding assays. Fifth, the nucleotide binding capacity of F1 was quantified after undergoing ATP hydrolysis. Sixth, the intensity of the fluorescence probe pyrene maleimide bound at α subunits was monitored before and after F1 experienced ATP hydrolysis. Finally, the catalytic activity and nucleotide content of F1 obtained from crystals being used in x-ray crystallographic studies was determined. The picture of rat liver F1 that emerges is one of an enzyme molecule that 1) loads nucleotide readily at five sites; 2) requires for catalysis both the αγ and the βδ∊ fractions; 3) directs the reversible binding of ATP and ADP to different regions of the enzyme's substructure; 4) induces inhibition of ATP hydrolysis only after ADP fills at least five sites; and 5) exists in several distinct forms, one an active, symmetrical form, obtained in the presence of ATP and high Pi and on which an x-ray map at 3.6 A has been reported (Bianchet, M., Ysern, X., Hullihen, J., Pedersen, P. L., and Amzel, L. M.(1991) J. Biol. Chem. 266, 21197-21201). These results are discussed within the context of a multistate model for rat liver F1 and also discussed relative to those reported for bovine heart F1, which has been crystallized with inhibitors in an asymmetrical form and has a propensity for binding nucleotides more tightly.

The plasma of the striped bass Morone saxatilis contains a fucose-specific lectin (MsaFBP32) that consists of two F-type carbohydrate recognition domains (CRDs) in tandem. The crystal structure of the complex of MsaFBP32 with l -fucose reported here shows a cylindrical 81-A-long and 60-A-wide trimer divided into two globular halves: one containing N-terminal CRDs (N-CRDs) and the other containing C-terminal CRDs (C-CRDs). The resulting binding surfaces at the opposite ends of the cylindrical trimer have the potential to cross-link cell surface or humoral carbohydrate ligands. The N-CRDs and C-CRDs of MsaFBP32 exhibit significant structural differences, suggesting that they recognize different glycans. Analysis of the carbohydrate binding sites provides the structural basis for the observed specificity of MsaFBP32 for simple carbohydrates and suggests that the N-CRD recognizes more complex fucosylated oligosaccharides and with a relatively higher avidity than the C-CRD. Modeling of MsaFBP32 complexed with fucosylated glycans that are widely distributed in prokaryotes and eukaryotes rationalizes the observation that binary tandem CRD F-type lectins function as opsonins by cross-linking “non-self” carbohydrate ligands and “self” carbohydrate ligands, such as sugar structures displayed by microbial pathogens and glycans on the surface of phagocytic cells from the host.

Recent work has focused on obtaining a better understanding of the three-dimensional structural relationships between the α and β subunits of the F 1 moiety and the location of nucleotide binding domains within these subunits. Four types of approach are currently being pursued: X-ray crystallographic, chemical, molecular biological and biochemical. Here we briefly review some of the major conclusions of these studies, and point out some of the problems that must be resolved before an adequate model that relates structure to function in the ATP synthase molecule can be formulated.

Quinone reductases type 1 (QR1) are FAD‐containing enzymes that catalyze the reduction of many quinones, including menadione (Vit K3), to hydroquinones using reducing equivalents provided by NAD(P)H. The reaction proceeds with a ping‐pong mechanism in which the NAD(P)H and the substrate occupy alternatively overlapping regions of the same binding site and participate in a double hydride transfer: one from NAD(P)H to the FAD of the enzyme, and one from the FADH2 of the enzyme to the quinone substrate. The main function of QR1 is probably the detoxification of dietary quinones but it may also contribute to the reduction of vitamin K for its involvement in blood coagulation. In addition, the same reaction that QR1 uses in the detoxification of quinones, activates some compounds making them cytotoxic. Since QR1 is elevated in many tumors, this property has encouraged the development of chemotherapeutic compounds that become cytotoxic after reduction by QR1. The structures of QR1 alone, and in complexes with substrates, inhibitors, and chemotherapeutic prodrugs, combined with biochemical and mechanistic studies have provided invaluable insight into the mechanism of the enzyme as well as suggestions for the improvements of the chemotherapeutic prodrugs. Similar information is beginning to accumulate about another related enzyme, QR2.

The bacteriophage lambda O protein binds to the lambda replication origin (orilambda) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to orilambda is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production of crystals of the origin-binding domain of lambda O that diffract to 2.5 A is reported. Anomalous dispersion methods will be used to solve this structure.

DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone-acceptor) oxidoreductase, is involved in the reductive activation process of several cytotoxic antitumor quinones and nitrobenzenes. It has been observed in our and other laboratories that the rat enzyme is significantly more effective in activating these drugs than the human and mouse enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal prodrugs for activation by DT-diaphorase in human tumors. In this study, using site-directed mutagenesis to replace residues in the rat enzyme with the human sequences and residues in the human enzyme with the rat sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsible for the catalytic differences between the rat and the human (and mouse) enzymes. With an exchange of a single amino acid, the rat mutant Y104Q behaved like the wild-type human enzyme, and the human mutant Q104Y behaved like the wild-type rat enzyme in their ability to reductively activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The study also confirms the conclusion of the x-ray structural analysis of rat enzyme that residue 130 (Thr in the rat enzyme and Ala in the human and mouse enzymes) is positioned near the binding region of the nicotinamide portion of NAD(P)H. This structural information is very important for designing suitable drugs and approaches for human cancer chemotherapy mediated by DT-diaphorase.

GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-alpha-d-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP.Tris(+) complex [Gabelli, S. B., et al. (2004) Structure 12, 927-935], the GDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essential Mg(2+). As determined by the effects of site-specific mutants on k(cat), these components contribute factors of 24-, 100-, 309-, 24-, and/=10(5)-fold, respectively, to catalysis. Both R37 and Y103 bind the beta-phosphate of GDP and are only 5.0 A apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additive effects of the two single mutants on k(cat), indicating cooperativity of R37 and Y103 in promoting catalysis, and antagonistic effects on K(m). Second, the conserved residue, D22, is positioned to accept a hydrogen bond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an alpha-d-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased k(cat) by factors of 10(2.1) and 10(2.6), respectively, for the hydrolysis of GDP-alpha-d-mannose, and showed smaller effects on K(m), suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinated substrate, GDP-2F-alpha-d-mannose, for which a cationic oxocarbenium transition state would be destabilized by electron withdrawal, exhibited a 16-fold decrease in k(cat) and a smaller, 2.5-fold increase in K(m). The D22A and D22N mutations further decreased the k(cat) with GDP-2F-alpha-d-mannose to values similar to those found with GDP-alpha-d-mannose, and decreased the K(m) of the fluorinated substrate. The choice of histidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to the greater basicity of histidine, since the pK(a) of E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 10(2.2)-fold decrease in k(cat) and a 4.0-fold increase in K(m) at pH 9.3. Similarly, the catalytic triad detected in the X-ray structure (H124- - -Y127- - -P120) is unnecessary for orienting H124, since the Y127F mutation had only 2-fold effects on k(cat) and K(m) with either H124 or E124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessary to preserve electroneutrality in the active complex.

Nudix hydrolases catalyze the hydrolysis of n ucleoside d iphosphates linked to other moieties, X , and contain the sequence motif or Nudix box, GX 5 EX 7 REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate at which nucleophilic substitution occurs, and in the number of required divalent cations. While most proceed by associative nucleophilic substitutions by water at specific internal phosphorus atoms of a diphosphate or polyphosphate chain, members of the GDP-mannose hydrolase sub-family catalyze dissociative nucleophilic substitutions, by water, at carbon. The site of substitution is likely determined by the positions of the general base and the entering water. The rate accelerations or catalytic powers of Nudix hydrolases range from 10 9 - to 10 12 -fold. The reactions are accelerated 10 3 –10 5 -fold by general base catalysis by a glutamate residue within, or beyond the Nudix box, or by a histidine beyond the Nudix box. Lewis acid catalysis, which contributes ⩾10 3 –10 5 -fold to the rate acceleration, is provided by one, two, or three divalent cations. One divalent cation is coordinated by two or three conserved residues of the Nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand from beyond the Nudix box. Some Nudix enzymes require one (MutT) or two additional divalent cations (Ap 4 AP), to neutralize the charge of the polyphosphate chain, to help orient the attacking hydroxide or oxide nucleophile, and/or to facilitate the departure of the anionic leaving group. Additional catalysis (10–10 3 -fold) is provided by the cationic side chains of lysine and arginine residues and by H-bond donation by tyrosine residues, to orient the general base, or to promote the departure of the leaving group. The overall rate accelerations can be explained by both independent and cooperative effects of these catalytic components.

Gene Dr1184 from Deinococcus radiodurans codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA). Nudix enzymes with the same specificity have been found in yeast, humans, and mice. The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily. The structure of the complex of the enzyme with Mg 2+ , its activating cation, reveals the position of the catalytic site. A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding. Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data.

Anguilla anguilla agglutinin (AAA), a fucolectin found in the serum of European eel, participates in the recognition of bacterial liposaccharides by the animal innate immunity system. Because AAA specifically recognizes fucosylated terminals of H and Lewis (a) blood groups, it has been used extensively as a reagent in blood typing and histochemistry. AAA contains a newly discovered carbohydrate recognition domain present in proteins of organisms ranging from bacteria to vertebrates. The crystal structure of the complex of AAA with alpha-L-fucose characterizes the novel fold of this entire lectin family, identifying the residues that provide the structural determinants of oligosaccharide specificity. Modification of these residues explains how the different isoforms in serum can provide a diverse pathogen-specific recognition.

Sre1, the fission yeast homologue of the mammalian sterol regulatory element binding protein, SREBP, is a hypoxic transcription factor required for sterol homeostasis and low oxygen growth. The level of the N-terminal transcription factor domain of Sre1, Sre1N, is regulated by Ofd1-Nro1 in an oxygen-dependent manner. Ofd1 is a putative prolyl hydroxylase of the 2-oxyoglutarate-Fe(II) dioxygenase protein superfamily. Unlike the prolyl hydroxylase PHD which hydroxylates proline residues in the hypoxia-inducible factor HIF, Ofd1 utilizes its N-terminal dioxygenase domain to control the interaction between itself and its inhibitor Nro1: in hypoxia, Nro1 binds to Ofd1 and this inhibits its C-terminal degradation domain (CTDD) function on Sre1N destabilization. As shown previously, Ofd1CTDD by itself is sufficient to promote Sre1N degradation and there's direct interaction between Nro1 and Ofd1CTDD. However, the molecular mechanism by which Ofd1CTDD accelerates Sre1N degradation remains unclear. Here we report the crystal structure of Ofd1CTDD at 2.0 A resolution. Interestingly, Ofd1CTDD has a double-stranded beta helix (DBSH) fold like that of the 2-oxyoglutarate-Fe(II) dioxygenase protein superfamily but lacks the iron-binding motif characteristic of the superfamily. This structure may help elucidate how Ofd1CTDD promotes Sre1N turnover and how Ofd1-Nro1 as a whole functions as an oxygen-sensor.

We report the characterization of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) as a mechanism-based inhibitor of NQO1. Inactivation of NQO1 by ES936 was time- and concentration-dependent and required the presence of a pyridine nucleotide cofactor consistent with a need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studies by the low partition ratio (1.40 +/- 0.03). The orientation of ES936 in the active site of NQO1 was examined by X-ray crystallography and found to be opposite to that observed for other indolequinones acting as substrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenol leaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr 127 and Tyr 129 residues in the active site. To determine if ES936 was covalently modifying NQO1, ES936-treated protein was analyzed by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS). The control NQO1 protein had a mass of 30864 +/- 6 Da (n = 20, theoretical, 30868.6 Da) which increased by 217 Da after ES936 treatment (31081 +/- 7 Da, n = 20) in the presence of NADH. The shift in mass was consistent with adduction of NQO1 by the reactive iminium derived from ES936 (M + 218 Da). Chymotryptic digestion of the protein followed by LC/MS analysis located a tetrapeptide spanning amino acids 126-129 which was adducted with the reactive iminium species derived from ES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129 residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and may be a useful tool in defining the role of NQO1 in cellular systems and in vivo.

Introduction Quinones are ubiquitous dietary compounds. Reduction of quinones by one-electron reduction systems (cytochrome P450 reductases) results in the formation of reactive semiquinones that can be re-oxidized to quinones by molecular oxygen with the formation of superoxide radical (02-*). Superoxide can lead to the formation of other reactive oxygen species, including the highly mutagenic and carcinogenic hydroxyl radical. In addition, the presence of quinones and their one-electron reduction products can result in the depletion of reduced thiol species. Cytosolic quinone reductase (QR) 1 provides a pathway that obviates all these deleterious reactions. Reduction of quinones via the obligatory two-electron reduction carried out by QR results in the formation of hydroquinones that can be glucuronidated and readily excreted. QR1 [NAD(P)H : (quinone acceptor) oxidoreductase; EC 1.6.99.21 is a widely distributed, predominantly cytosolic, FAD-containing flavoprotein that promotes the obligatory two-electron reduction of many quinones and quinoneimines. (The enzyme has also been referred to as menadione reductase, DT-diaphorase and vitamin-K reductase). It uses NADH or NADPH as reductants with equal facility and is potently inhibited by dicoumarol and similar anticoagulants. Furthermore, QR activity is elevated in many animal tissues and cell lines by addition of xenobiotics. The discovery, purification, molecular characteristics, induction and other properties of this enzyme have been presented previously [ 1,2]. The purification and properties of another mammalian cytosolic FAD-dependent flavoprotein was described in the early 1960's by Liao et al. [3]. This enzyme was able to catalyse the oxidation of reduced N-ribosyland N-alkylnicotinamides by menadione and other quinones. Recently, the determination of the sequence indicated that this enzyme is highly homologous with QR1 [4,5], leading to the conclusion that it is

The structure of the nucleotide-free F 1 -ATPase from a thermoalkaliphilic bacterium presented in this issue of Structure (Stocker et al., 2007) reveals the structural interactions that prevent the enzyme from operating naturally in the hydrolytic direction. The data provide new insights into the mechanism of the F o F 1 -ATP synthase.

Isothermal titration calorimetry (ITC) measurements of the binding 1-beta carbohydrate-substituted galactopyranoside derivatives to galectin-1 from bovine spleen, a dimer with one binding site per subunit, were performed at 283-285 and 298 K. The disaccharides were lactose, methyl beta-lactoside, lactulose, 4-O-beta-D-galactopyranosyl-D-mannopyranoside, 3-O-beta-D-galactopyranosyl-D-arabinose, 2'-O-methyllactose, lacto-N-biose, N-acetyllactosamine, and thiodigalactopyranoside. The site binding enthalpies, DeltaHb, are the same at both temperatures and range from -42.2 +/- 3.3 kJ mol-1 for thiodigalactopyranoside to -24.5 +/- 0.5 kJ mol-1 for lacto-N-biose, and the site binding constants range from 4.86 +/- 0.78 x 10(3) M-1 for methyl beta-lactoside at 297.8 K to 6.54 +/- 0.97 x 10(4) M-1 for N-acetyllactosamine at 281.3 K. The binding reactions are enthalpically driven, exhibit enthalpy-entropy compensation, and, with the exception of N-acetyllactosamine, follow a van't Hoff dependence of the binding constant on temperature. The number of contacts at distances4.0 A between the disaccharide and galectin was determined from the energy-minimized conformation of the complex derived from the X-ray crystallographic structure of the galectin-N-acetyllactosamine complex determined by Liao et al. [Liao, D. I., Kapadia, G., Ahmed, H., Vasta, G. R., and Herzberg, O. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1428-1432]. The binding enthalpies calculated from changes in the solvent-accessible surface areas of the galectin binding site upon binding of the disaccharide were in close agreement with the experimental values for lactose, lactulose, lacto-N-biose, and N-acetyllactosamine, all of which exhibit binding enthalpies-36 kJ mol-1. Differential scanning calorimetry measurements on solutions of galectin and its disaccharide complexes show that the galectin dimer does not dissociate upon denaturation in contrast to the legume lectins. At the denaturation temperature, the galectin in the absence of sugar exists as a tetramer, and the extent of this association is substantially reduced in the presence of a disaccharide.

International audience; The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded beta-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.

One of the most challenging problems in biology today is to understand the molecular mechanism by which mitochondria synthesize ATP. The process is catalyzed by an unusually complex molecule called the ATP synthase, the substructure of which is comprised of a number of different polypeptide chains. Here, we briefly review progress made to date in obtaining nucleotide binding and structural information about the F1 moiety of the rat liver ATP synthase complex. Also, implications of this information for the function of the enzyme in synthesizing ATP is discussed.

SummaryNudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the committed step of folate synthesis in bacteria. That this dihydroneopterin hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed in vivo: knockout of this gene in E. coli leads to a marked reduction in folate synthesis that is completely restored by a plasmid carrying the gene. We also determined the crystal structure of this enzyme using data to 1.8 Å resolution and studied the kinetics of the reaction. These results provide insight into the structural bases for catalysis and substrate specificity in this enzyme and allow the definition of the dihydroneopterin triphosphate pyrophosphatase family of Nudix enzymes.

SummaryFission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 Å resolution shows an all-α-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

The F1 moiety of the mitochondrial ATP synthase is composed of five different subunits with stoichiometry alpha 3 beta 3 gamma delta epsilon and exhibits the capacity to synthesize ATP from ADP and Pi. We have previously crystallized rat liver F1 and described its structure at 9-A resolution (Amzel, L. M., McKinney, M., Narayanan, P., and Pedersen, P. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5852-5856). Here we present an x-ray map of this complex enzyme at 3.6 A, which provides a much more informative description of its quaternary structure. The overall dimensions of the F1 molecule are 120 A x 120 A x 74 A. The enzyme exhibits 3-fold symmetry relating the three copies of each of the two major subunits, alpha and beta. As the alpha subunits (but not the beta subunits) contain cysteine residues, it has been possible to identify the alpha subunits by heavy atom labeling with mersalyl and to relate their positions in the F1 molecule to the beta subunits. Significantly, the alpha and beta subunits each exist as trimeric arrays which are organized in two slightly offset, interdigitated layers along the 3-fold axis. In one trimeric layer the alpha subunits are located close to the axis with homologous subunits interacting with each other; in the other trimeric layer the beta subunits are far from the axis, and they interact only with alpha subunits and not with one another. At one end of the structure, part of the interface between each alpha and beta subunit encloses a space or "pocket" that is accessible to the solvent; at the other end the interfaces between the subunits are more open and exposed. The present work represents the highest resolution map reported to date for the F1 moiety of an ATP synthase complex.