Search

The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I

This upload contains all of the experimental data and analysis files pertaining to the accepted manuscript.

The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones—the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient. For nearly 50 years before its molecular identification, NIS was already the molecule at the center of the single most effective internal radiation cancer therapy ever devised: radioiodide (131I-) treatment for thyroid cancer. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency. To date, the structure of NIS has been unknown. Here, we report three structures of rat NIS, determined by single-particle cryo-electron microscopy (cryo-EM): one with no substrates bound, one with 2 Na+ and 1 I- bound, and one with 1 Na+ and the oxyanion perrhenate bound. Structural analyses, functional characterization, and computational studies reveal the substrate binding sites and residues key for transport activity. Our results yield insights into how NIS selects, couples, and translocates anions—thereby establishing a framework for understanding NIS function—and into how it transports different substrates with different stoichiometries and releases substrates from its substrate-binding cavity into the cytosol.

Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell–derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates., Natural selection may favor traits underlying aging-related diseases if they benefit the young. Wang et al. find that oxidative activation of CaMKII provides physiological benefits critical to the initial and continued success of vertebrates but at the cost of disease, frailty, and shortened lifespan.

Both vertebrates and invertebrates display active innate immune mechanisms for defense against microbial infection, including diversified repertoires of soluble and cell-associated lectins that can effect recognition and binding to potential pathogens, and trigger downstream effector pathways that clear them from the host internal milieu. Galectins are widely distributed and highly conserved lectins that have key regulatory effects on both innate and adaptive immune responses. In addition, galectins can bind to exogenous (“non-self”) carbohydrates on the surface of bacteria, enveloped viruses, parasites, and fungi, and function as recognition receptors and effector factors in innate immunity. Like most invertebrates, eastern oysters (Crassostrea virginica) and softshell clams (Mya arenaria) can effectively respond to most immune challenges through soluble and hemocyte-associated lectins. The protozoan parasite Perkinsus marinus, however, can infect eastern oysters and cause “Dermo” disease, which is highly detrimental to both natural and farmed oyster populations. The sympatric Perkinsus chesapeaki, initially isolated from infected M. arenaria clams, can also be present in oysters, and there is little evidence of pathogenicity in either clams or oysters. In this review, we discuss selected observations from our studies on the mechanisms of Perkinsus recognition that are mediated by galectin-carbohydrate interactions. We identified in the oyster two galectins that we designated CvGal1 and CvGal2, which strongly recognize P. marinus trophozoites. In the clam we also identified galectin sequences, and focused on one (that we named MaGal1) that also recognizes Perkinsus species. Here we describe the biochemical characterization of CvGal1, CvGal2, and MaGal1 with focus on the detailed study of the carbohydrate specificity, and the glycosylated moieties on the surfaces of the oyster hemocytes and the two Perkinsus species (P. marinus and P. chesapeaki). Our goal is to gain further understanding of the biochemical basis for the interactions that lead to recognition and opsonization of the Perkinsus trophozoites by the bivalve hemocytes. These basic studies on the biology of host-parasite interactions may contribute to the development of novel intervention strategies for parasitic diseases of biomedical interest.

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.

Purpose Human immunodeficiency virus-1 (HIV)-associated neurocognitive disorder (HAND) is a neurodegenerative disease for which there is no available neuroprotective therapy. Viral proteins, such as Tat, have been implicated as agents of neurotoxicity via multiple mechanisms, including effects by directly binding to the NMDA receptor. We evaluated the ability of the immune response against Tat to modulate neurotoxicity at glutamate receptors. Methods Neurotoxicity was measured in primary neuronal-glial cultures and in hippocampal slice cultures. We used immunoprecipitation experiments to demonstrate interaction between Tat, NMDA receptor, and anti-Tat antibody. Using known structures of Tat and NMDA receptors, we developed a model of their interactions. Results Antibodies to Tat attenuated Tat-mediated neurotoxicity. Interestingly, Tat immune complexes also blocked neurotoxicity caused by NMDA receptor agonists but not kainate/AMPA receptor agonists. Neither Tat nor antibody alone blocked the excitotoxic effect, nor did an unrelated antigen–antibody complex. The protective effect of the Tat immune complexes was also lost when Tat was modified by nitrosylation or by using a deletion mutant of Tat. Conclusions The ability of viral immune complexes to interact with NMDA receptors and prevent excitotoxicity represents a novel host defense mechanism. Host immune responses may influence host susceptibility to various effects of viral proteins, modulating HIV complications, such as onset of HAND. These observations provide rationale for development of vaccine therapies targeting Tat for prevention of HAND.

Despite major advancements in development of antiretroviral therapy, currently there is no effective treatment to block the impact of HIV-Tat protein which is released extracellularly and can cause activation of lymphocytes, glial cells and neurotoxicity. Because the structure of extracellular Tat is currently unknown, we used atomic force microscopy, circular dichroism and computer simulation to study the structure and aggregation characteristics of HIV-Tat protein. We found that about 8% of the HIV-Tat population was in monomeric state, the rest being in aggregated state. The aggregates ranged in size from dimers, trimers, tetramers to large oligomers (50-mers and larger) and all structures presented a globular shape. Circular dichroism measurements suggested that 20% of the sample's structure is alpha-helical, which likely represents the aggregated state, since monomers were few. The reducing agent dithiothreitol broke down the large aggregates, leading to a population of smaller aggregates, but structures smaller than the monomer were found as well, indicating that the molecule is prone to rupture under reducing conditions. CuSO4 induced dimerization and larger aggregates of HIV-Tat. A change of 14-19 aminoacids in protein structure, including the Cys 31 replacement with Ser led to a larger number of monomers in the sample (11%), a distribution of smaller aggregates, and reduced adherence to mica. Freezing of Tat reduced the aggregates size, increased their adherence capacity and ruptured the molecule. Further studies will investigate the effect of protein concentration, solution ionic strength, temperature variation and time of storage in solution, to characterize the aggregated state and its reversibility.

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.

Galectins constitute a conserved and widely distributed lectin family characterized by their binding affinity for β-galactosides and a unique binding site sequence motif in the carbohydrate recognition domain (CRD). In spite of their structural conservation, galectins display a remarkable functional diversity, by participating in developmental processes, cell adhesion and motility, regulation of immune homeostasis, and recognition of glycans on the surface of viruses, bacteria and protozoan parasites. In contrast with mammals, and other vertebrate and invertebrate taxa, the identification and characterization of bona fide galectins in aquatic mollusks has been relatively recent. Most of the studies have focused on the identification and domain organization of galectin-like transcripts or proteins in diverse tissues and cell types, including hemocytes, and their expression upon environmental or infectious challenge. Lectins from the eastern oyster Crassostrea virginica, however, have been characterized in their molecular, structural and functional aspects and some notable features have become apparent in the galectin repertoire of aquatic mollusks. These including less diversified galectin repertoires and different domain organizations relative to those observed in vertebrates, carbohydrate specificity for blood group oligosaccharides, and up regulation of galectin expression by infectious challenge, a feature that supports their proposed role(s) in innate immune responses. Although galectins from some aquatic mollusks have been shown to recognize microbial pathogens and parasites and promote their phagocytosis, they can also selectively bind to phytoplankton components, suggesting that they also participate in uptake and intracellular digestion of microalgae. In addition, the experimental evidence suggests that the protozoan parasite Perkinsus marinus has co-evolved with the oyster host to be selectively recognized by the oyster hemocyte galectins over algal food or bacterial pathogens, thereby subverting the oyster's innate immune/feeding recognition mechanisms to gain entry into the host cells.

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.

GDP-mannose mannosyl hydrolase (GDPMH) from E. coli catalyzes the hydrolysis of GDP-α- d -sugars to GDP and β- d -sugars by nucleophilic substitution with inversion at the anomeric C1 of the sugar, with general base catalysis by His-124. The 1.3 A X-ray structure of the GDPMH-Mg2+-GDP complex was used to model the complete substrate, GDP-mannose into the active site. The substrate is linked to the enzyme by 12 hydrogen bonds, as well as by the essential Mg2+. In addition, His-124 was found to participate in a hydrogen bonded triad: His-124-NδH⋯Tyr-127-OH⋯Pro-120(C O). The contributions of these hydrogen bonds to substrate binding and to catalysis were investigated by site-directed mutagenesis. The hydrogen bonded triad detected in the X-ray structure was found to contribute little to catalysis since the Y127F mutation of the central residue shows only 2-fold decreases in both kcat and Km. The GDP leaving group is activated by the essential Mg2+ which contributes at least 105-fold to kcat, and by nine hydrogen bonds, including those from Tyr-103, Arg-37, Arg-52, and Arg-65 (via an intervening water), each of which contribute factors to kcat ranging from 24- to 309-fold. Both Arg-37 and Tyr-103 bind the β-phosphate of the leaving GDP and are only 5.0 A apart. Accordingly, the R37Q/Y103F double mutant shows partially additive effects of the two single mutants on kcat, indicating cooperativity of Arg-37 and Tyr-103 in promoting catalysis. The extensive activation of the GDP leaving group suggests a mechanism with dissociative character with a cationic oxocarbenium-like transition state and a half-chair conformation of the sugar ring, as found with glycosidase enzymes. Accordingly, Asp-22 which contributes 102.1- to 102.6-fold to kcat, is positioned to both stabilize a developing cationic center at C1 and to accept a hydrogen bond from the C2–OH of the mannosyl group, and His-88, which contributes 102.3-fold to kcat, is positioned to accept a hydrogen bond from the C3–OH of the mannose facilitating its distortion to a half-chair conformation. Also, the fluorinated substrate GDP-2-fluoro-α- d -mannose, for which the oxocarbenium ion-like transition state centered at C1 would be destabilized by electron withdrawal, shows a 16-fold lower kcat and a 2.5-fold greater Km than does GDP-α- d -mannose. The product of the contributions to catalysis of Arg-37 and Tyr-103 (taking their cooperativity into account), Arg-52, Arg-65, Mg2+, Asp-22, His-124, and His-88 is ≥1019, which exceeds the 1012-fold rate acceleration produced by GDPMH by a factor ≥107. Hence, additional pairs or groups of catalytic residues must act cooperatively to promote catalysis.

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.

Background: NAD(P)H:quinone acceptor oxidoreductase (QR1) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. Remarkably, the same enzyme activates cancer prodrugs that become cytotoxic only after two-electron reduction. QR1's ability to bioactivate quinones and its elevated expression in many human solid tumors makes this protein an excellent target for enzyme-directed drug development. Until now, structural analysis of the mode of binding of chemotherapeutic compounds to QR1 was based on model building using the structures of complexes with simple substrates; no structure of complexes of QR1 with chemotherapeutic prodrugs had been reported. Results: Here we report the high-resolution crystal structures of complexes of QR1 with three chemotherapeutic prodrugs: RH1, a water-soluble homolog of dimethylaziridinylbenzoquinone; EO9, an aziridinylindolequinone; and ARH019, another aziridinylindolequinone. The structures, determined to resolutions of 2.0 A, 2.5 A, and 1.86 A, respectively, were refined to R values below 21% with excellent geometry. Conclusions: The structures show that compounds can bind to QR1 in more than one orientation. Surprisingly, the two aziridinylindolequinones bind to the enzyme in different orientations. The results presented here reveal two new factors that must be taken into account in the design of prodrugs targeted for activation by QR1: the enzyme binding site is highly plastic and changes to accommodate binding of different substrates, and homologous drugs with different substituents may bind to QR1 in different orientations. These structural insights provide important clues for the optimization of chemotherapeutic compounds that utilize this reductive bioactivation pathway.

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.

The most commonly quoted mechanism of the coupling between the electrochemical proton gradient and the formation of ATP from ADP and Pi assumes that all states of the F1 portion of the ATP synthase have β subunits in “tight,” “loose,” and “open” conformations. Models based on this assumption are inconsistent with some of the available experimental evidence. A mechanism that includes an additional β subunit conformation, “closed,” observed in the rat liver structure overcomes these difficulties.

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.

Voltage-gated sodium channels (NaV) are integral membrane proteins, part of a macromolecular complex that is central to signaling in the heart and other excitable tissues. Regulation of the essential functions of the channel are complex and may differ among tissue-specific isoforms nevertheless, mechanistic understanding of the molecular regulation of the channel is beginning to emerge. We and others have demonstrated the importance of the carboxyl terminus (CT) in the regulation of the channel. The CT is a hot spot for mutations that produce inherited cardiac arrhythmias, myotonias, epilepsy and autism. In the case of NaV1.5, the cardiac channel, mutations of critical structural motifs in the CT (including an EF hand-like motif and an IQ motif) result in disease conditions such as Brugada and LQT syndromes. Also, altered NaV channel trafficking and function with consequent intracellular Na+ overload contributes to the development of dilated cardiomyopathy. The structure of the CT of the Nav1.5 channel in complex with calmodulin (CaM), determined to 2.9 A resolution, shows that many of the mutations associated with disease states occur at CTNav1.5-CaM interfaces. Based on this structure a mechanism for the transition to the non-inactivated state of the channel is proposed.

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.

With multi-drug resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug-targets are becoming an urgent need. Traditional β-lactam antibiotics that disrupt the D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on β-lactam insensitive L,D-transpeptidases for biosynthesis and maintenance of their peptidoglycan layer. This reliance plays a major role in drug-resistance and persistence of Mycobacterium tuberculosis (Mtb) infections. The crystal structure at 1.7 Å resolution of the Mtb L,D-transpeptidase LdtMt2 containing a bound peptidoglycan fragment, reported here, provides information about catalytic site organization as well as substrate recognition by the enzyme. Based on our structural, kinetic, and calorimetric data, we propose a catalytic mechanism for LdtMt2 in which both acyl-acceptor and acyl-donor substrates reach the catalytic site from the same, rather than different, entrances. Together, this information provides vital insights for the development of novel drugs targeting this validated yet unexploited enzyme.

Protein–carbohydrate interactions mediated by lectins have been recognized as key components of innate immunity in vertebrates and invertebrates, not only for recognition of potential pathogens, but also for participating in downstream effector functions, such as their agglutination, immobilization, and complement-mediated opsonization and killing. More recently, lectins have been identified as critical regulators of mammalian adaptive immune responses. Fish are endowed with virtually all components of the mammalian adaptive immunity, and are equipped with a complex lectin repertoire. In this review, we discuss evidence suggesting that: (a) lectin repertoires in teleost fish are highly diversified, and include not only representatives of the lectin families described in mammals, but also members of lectin families described for the first time in fish species; (b) the tissue-specific expression and localization of the diverse lectin repertoires and their molecular partners is consistent with their distinct biological roles in innate and adaptive immunity; (c) although some lectins may bind endogenous ligands, others bind sugars on the surface of potential pathogens; (d) in addition to pathogen recognition and opsonization, some lectins display additional effector roles, such as complement activation and regulation of immune functions; (e) some lectins that recognize exogenous ligands mediate processes unrelated to immunity: they may act as anti-freeze proteins or prevent polyspermia during fertilization.

Methamphetamine (METH) abuse has reached epidemic proportions, and it has become increasingly recognized that abusers suffer from a wide range of neurocognitive deficits. Much previous work has focused on the deleterious effects of METH on mature neurons, but little is known about the effects of METH on neural progenitor cells (NPCs). It is now well established that new neurons are continuously generated from NPCs in the adult hippocampus, and accumulating evidence suggests important roles for these neurons in hippocampal-dependent cognitive functions. In a rat hippocampal NPC culture system, we find that METH results in a dose-dependent reduction of NPC proliferation, and higher concentrations of METH impair NPC survival. NPC differentiation, however, is not affected by METH, suggesting cell-stage specificity of the effects of METH. We demonstrate that the effects of METH on NPCs are, in part, mediated through oxidative and nitrosative stress. Further, we identify seventeen NPC proteins that are post-translationally modified via 3-nitrotyrosination in response to METH, using mass spectrometric approaches. One such protein was pyruvate kinase isoform M2 (PKM2), an important mediator of cellular energetics and proliferation. We identify sites of PKM2 that undergo nitrotyrosination, and demonstrate that nitration of the protein impairs its activity. Thus, METH abuse may result in impaired adult hippocampal neurogenesis, and effects on NPCs may be mediated by protein nitration. Our study has implications for the development of novel therapeutic approaches for METH-abusing individuals with neurologic dysfunction and may be applicable to other neurodegenerative diseases in which hippocampal neurogenesis is impaired.

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.