Structure of a legume lectin with an ordered N-linked carbohydrate in complex with lactose.

The three-dimensional structure of the lactose complex of the Erythrina corallodendron lectin (EcorL), a dimer of N-glycosylated subunits, was determined crystallographically and refined at 2.0 angstrom resolution to an R value of 0.19. The tertiary structure of the subunit is similar to that of other legume lectins, but interference by the bulky N-linked heptasaccharide, which is exceptionally well ordered in the crystal, forces the EcorL dimer into a drastically different quaternary structure. Only the galactose moiety of the lactose ligand resides within the combining site. The galactose moiety is oriented differently from ligands in the mannose-glucose specific legume lectins and is held by hydrophobic interactions with Ala88, Tyr106, Phe131, and Ala218 and by seven hydrogen bonds, four of which are to the conserved Asp89, Asn133, and NH of Gly107. The specificity of legume lectins toward the different C-4 epimers appears to be associated with extensive variations in the outline of the variable parts of the binding sites.

Combining experimental information from crystal and solution studies: joint X-ray and NMR refinement.

Joint refinement of macromolecules against crystallographic and nuclear magnetic resonance (NMR) observations is presented as a way of combining experimental information from the two methods. The model of interleukin-1 beta derived by the joint x-ray and NMR refinement is shown to be consistent with the experimental observations of both methods and to have crystallographic R value and geometrical parameters that are of the same quality as or better than those of models obtained by conventional crystallographic studies. The few NMR observations that are violated by the model serve as an indicator for genuine differences between the crystal and solution structures. The joint x-ray-NMR refinement can resolve structural ambiguities encountered in studies of multidomain proteins, in which low- to medium-resolution diffraction data can be complemented by higher resolution NMR data obtained for the individual domains.

Lectin-carbohydrate interactions: different folds, common recognition principles.

Lectins can be found in many organisms and are involved in a multitude of cellular processes that depend on specific recognition of complex carbohydrates. The stereochemical principles underlying the recognition process have been the subject of extensive biochemical and structural studies. When examined from the viewpoint of the bound sugar, the structural information accumulated so far on lectins and other proteins that are specific to galactose and glucose (or mannose), provides suggestive evidence for distinct ligand-dependent distribution of hydrogen-bond partners in the combining site.

Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases.

The tyrosine phosphatase RPTP gamma is a candidate tumor suppressor gene since it is located on human chromosome 3p14.2-p21 in a region frequently deleted in certain types of renal and lung carcinomas. In order to evaluate its oncogenic potential and to explore its normal in vivo functions, we have isolated cDNAs and deduced the complete sequences of both human and murine RPTP gamma. The murine RPTP gamma gene has been localized to chromosome 14 to a region syntenic to the location of the human gene. Northern (RNA) blot analysis reveals the presence of two major transcripts of 5.5 and 8.5 kb in a variety of murine tissues. In situ hybridization analysis reveals that RPTP gamma mRNA is expressed in specific regions of the brain and that the localization of RPTP gamma changes during brain development. RPTP gamma is composed of a putative extracellular domain, a single transmembrane domain, and a cytoplasmic portion with two tandem catalytic tyrosine phosphatase domains. The extracellular domain contains a stretch of 266 amino acids with striking homology to the zinc-containing enzyme carbonic anhydrase (CAH), indicating that RPTP gamma and RPTP beta (HPTP zeta) represent a subfamily of receptor tyrosine phosphatases. We have constructed a model for the CAH-like domain of RPTP gamma based upon the crystal structure of CAH. It appears that 11 of the 19 residues that form the active site of CAH are conserved in RPTP gamma. Yet only one of the three His residues that ligate the zinc atom and are required for catalytic activity is conserved. On the basis of this model we propose that the CAH-like domain of RPTP gamma may have a function other than catalysis of hydration of metabolic CO2.

The ACT domain family.

A novel ligand-binding domain, named the 'ACT domain', was recently identified by a PSI-BLAST search. The archetypical ACT domain is the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH), which folds with a ferredoxin-like betaalphabetabetaalphabeta topology. A pair of ACT domains form an eight-stranded antiparallel sheet with two molecules of the allosteric inhibitor serine bound in the interface. The ACT domain is found in a variety of contexts and is proposed to be a conserved regulatory ligand binding fold. Rat phenylalanine hydroxylase has a regulatory domain with a similar fold, but different ligand-binding mode. Putative ACT domains in some proteins of unknown structure (e.g. acetohydroxyacid synthase regulatory subunits) may also fold like the 3PGDH regulatory domain. The regulatory domain of threonine deaminase, although not a member of the ACT sequence family, is similar in structure to the paired 3PGDH regulatory domains. Repeats of ACT-like domains can create nonequivalent ligand-binding sites with the potential for complex regulatory patterns. The structures and mechanisms of such systems have only begun to be examined.

An excitatory scorpion toxin with a distinctive feature: an additional alpha helix at the C terminus and its implications for interaction with insect sodium channels.

BACKGROUND: Scorpion neurotoxins, which bind and modulate sodium channels, have been divided into two groups, the alpha and beta toxins, according to their activities. The beta-toxin class includes the groups of excitatory and depressant toxins, which differ in their mode of action and are highly specific against insects. The three-dimensional structures of several alpha and beta toxins have been determined at high resolution, but no detailed 3D structure of an excitatory toxin has been presented so far. RESULTS: The crystal structure of an anti-insect excitatory toxin from the scorpion Buthotus judaicus, Bj-xtrIT, has been determined at 2.1 A resolution and refined to an R factor of 0.209. The first 59 residues form a closely packed module, structurally similar to the conserved alpha and beta toxins ('long toxins') affecting sodium channels. The last 17 residues form a C-terminal extension not previously seen in scorpion toxins. It comprises a short alpha helix anchored to the N-terminal module by a disulfide bridge and is followed by a highly mobile stretch of seven residues, of which only four are seen in the electron-density map. This mobile peptide covers part of a conserved hydrophobic surface that is thought to be essential for interaction with the channel in several long toxins. CONCLUSIONS: Replacement of the last seven residues by a single glycine abolishes the activity of Bj-xtrIT, strongly suggesting that these residues are intimately involved in the interaction with the channel. Taken together with the partial shielding of the conserved hydrophobic surface and the proximity of the C terminus to an adjacent surface rich in charged residues, it seems likely that the bioactive surface of Bj-xtrIT is formed by residues surrounding the C terminus. The 3D structure and a recently developed expression system for Bj-xtrIT pave the way for identifying the structural determinants involved in the bioactivity and anti-insect specificity of excitatory toxins.

Mutagenic analysis of Vav reveals that an intact SH3 domain is required for transformation.

The vav proto-oncogene encodes a protein with multiple modulae domains that enable it to function as a mediator, linking tyrosine signaling to downstream events in hematopoietic cells. Circumstantial evidence suggests that protein-protein interactions exerted by two of these domains, the Src homology 2 (SH2) and the Src homology 3 (SH3), play an important role in the regulation of Vav activity. To study the relevance of the SH3 domain for the function of vav as a transforming gene, we have created several mutations in the SH3 domain located at its carboxy region. Substitution of the non-conserved aspartic acid 797 (to asparagine, D797N) retained the transforming potential of the vav oncogene, whereas substitutions of five highly conserved amino-acids: alanine 789 (to asparagine, A789N), leucine 801 (to arginine, L801R), tryptophan 821 (to arginine, W821R), glycine 830 (to valine, G830V) and valine 837 (to glutamic acid, V837E) greatly reduced its transforming potential. The mutant proteins resemble Vav in many biochemical properties; however, while the transforming mutant protein (D797N) associates with several unidentified proteins in a manner similar to that of Vav, the non-transforming mutant Vav proteins react very poorly with these proteins. Among the known Vav-interacting proteins, hnRNP-K associates with all mutant proteins except A789N and V837E whereas binding of Zyxin to any of the mutant proteins is not affected. Taken together, our results clearly demonstrate that the SH3 domain has a positive effect on vav activity and is needed for vav transformation. The vavSH3C associating protein(s) that are crucial for its activity as a transforming gene have probably not yet been identified.

Structure of human oxyhaemoglobin at 2.1 A resolution.

The structure of human oxyhaemoglobin was determined by single crystal X-ray analysis at 2.1 A resolution. Data were collected on an Arndt-Wonacott camera at -2 degrees C. The structure was refined to an R factor of 0.223 by the Jack-Levitt method, starting from Baldwin's model of human carbon monoxide haemoglobin. The active sites in the alpha and beta subunit are distinct. The iron atoms are 0.16(8) A and 0.00(8) A from the mean plane of the porphyrin carbons and nitrogens (0.12(8) A and -0.11(8) A from the mean plane of the porphyrin nitrogens) in the alpha and beta subunit, respectively, in correlation with the orientation of HisF8 relative to the porphyrin nitrogens. The haem group appears to be nearly planar in the alpha subunit but ruffled in the beta subunit. The Fe-O(1)-O(2) angles are 153(7) degrees and 159(12) degrees in the alpha and beta subunit, respectively. The oxygen molecule forms a hydrogen bond to N epsilon of HisE7 in the alpha, but either none or a weak one in the beta subunit. The following bond lengths were found: Fe-N epsilon (HisF8) = 1.94(9) A (alpha) and 2.07(9) A (beta); Fe-O(1) = 1.66(8) A (alpha) and 1.87(13) A (beta); Fe-Nporph (mean = 1.99(5) A (alpha) and 1.96(6) A (beta). These dimensions agree with the values obtained in oxymyoglobin and model compounds. The C-terminal residues, ArgHC3(141 alpha) and HisHC3(146 beta), are relatively delocalized, and their positions do not enable them to form the intersubunit salt bridges in which they are involved in deoxyhaemoglobin. The penultimate tyrosine residues, TyrHC2 140 alpha and 145 beta, are relatively localized and maintain the hydrogen bonds to the carbonyl oxygens of ValFG5 (93 alpha and 98 beta), with only minor variations compared to their geometry in deoxyhaemoglobin. TyrHC2(145 beta), however, alternates between a major and a minor site, in conjunction with CysF9(93 beta), both sharing the internal pocket between the F and H helices while in the major conformation. This suggests that the role of the penultimate tyrosines in the allosteric mechanism may differ from that previously proposed by Perutz. The overall quaternary structure of oxyhaemoglobin is identical, within experimental error, to that of carbon monoxide haemoglobin, and thus confirms the applicability of the allosteric mechanisms proposed by Perutz and Baldwin & Chothia to the process of oxygen binding.

Structures of the Erythrina corallodendron lectin and of its complexes with mono- and disaccharides.

The structures of the Erythrina corallodendron lectin (EcorL) and of its complexes with galactose, N-acetylgalactosamine, lactose and N-acetyllactosamine were determined at a resolution of 1.9 to 1.95 A. The final R-values of the five models are in the range 0.169 to 0.181. The unusual, non-canonical, dimer interface of EcorL is made of beta-strands from the two monomers, which face one another in a "hand-shake" mode. The galactose molecule in the primary binding site is bound in an identical way in all four complexes. Features of the electrostatic potential of the galactose molecule match those of the potential in the combining site, thus probably pointing to the contribution of the electrostatic energy to determining the orientation of the ligand. No conformational change occurs in the protein upon binding the ligand. Subtle variations in the binding mode of the second monosaccharide (glucose in the complex with lactose and N-acetylglucosamine in the complex with N-acetyllactosamine) were observed. The mobility of Gln219 is lower in the complexes with the disaccharides than in the complexes with the monosaccharides, indicating further recruitment of this residue to ligand binding through more extensive hydrogen bonding in the former complexes. Water molecules that have been located in the combining sites of the five structures undergo rearrangement in response to binding of the different ligands. The new structural information is in qualitative agreement with thermodynamic data on the binding to EcorL.

Crystallization and preliminary X-ray diffraction studies of soybean agglutinin.

Soybean agglutinin crystallizes in the monoclinic space group C2 with unit cell dimensions a = 118.6 A, b = 88.9 A, c = 165.9 A, beta = 103.0 degrees and one tetramer of 120,000 Mr per asymmetric unit. The crystals are suitable for high-resolution work.

The crystal structure of human deoxyhaemoglobin at 1.74 A resolution.

The structure of human deoxyhaemoglobin was refined at 1.74 A resolution using data collected on film at room temperature from a synchrotron X-ray source. The crystallographic R-factor is 16.0%. The estimated error in atomic positions is 0.1 A overall, 0.14 A for main-chain atoms of internal segments, and 0.05 A for the iron atoms. The effects of intermolecular contacts on the structure were investigated; such contacts cause only highly localized distortions, as judged from the degree of molecular asymmetry that they induce. The geometry of the iron-nitrogen complex closely resembles that of the deoxymyoglobin structure of Takano (1977) and of the 5-co-ordinated model compounds of Hoard (1975) and Jameson et al. (1980). The distance of the iron from the mean plane of N(porphyrin) is 0.40(5) A and 0.36(5) A, respectively, at the alpha and beta haems, in contrast to the corresponding distance of +0.12(8) A and -0.11(8) A in oxyhaemoglobin ( Shaanan , 1983); the Fe-N epsilon (F8) bond length is 2.12(4) A and the Fe-N(porphyrin) bond length is 2.06(2) A; the last is also in good agreement with extended X-ray fluorescence spectroscopy measurements on deoxyhaemoglobin ( Eisenberger et al., 1978; Perutz et al., 1982). The haems are domed toward the proximal side; the separation between the mean planes of N(porphyrin) and C(porphyrin) being 0.16(6) A and 0.10(6) A, respectively at the alpha and beta haems. At the alpha haems, the normals to the mean pyrrole planes are tilted uniformly toward the haem centre, by about three degrees relative to the haem normal, and there is a folding of about four degrees of the haem about an axis running between the methene carbons that are between the pyrrole rings bearing like-type side-chains. At the beta haems, there is no such folding, and only pyrroles II and IV (those eclipsed by His F8) are appreciably tilted, by about eight degrees. The independence of these parameters from restraints imposed on the model was verified by unrestrained refinement of the entire molecule starting from a structure with modified haem geometry.

Crystallization and preliminary X-ray diffraction studies of the lectin from Erythrina corallodendron.

The lectin from Erythrina corallodendron, specific for N-acetyllactosamine, crystallizes in the hexagonal space group P6(1) (P6(5)) with unit cell dimensions a = b = 136.3 A, c = 83.2 A and one dimer of Mr 60,000 in the asymmetric unit. The crystals are suitable for high-resolution work.

Chemical characteristics of dimer interfaces in the legume lectin family.

The Erythrina corallodendron lectin (EcorL) crystallizes in monoclinic and hexagonal crystal forms. Comparison of the newly determined hexagonal form (PDB code 1fyu) with the monoclinic form shows that the dimeric structure of EcorL reflects the inherent biological structure of the protein and is not an artifact of the crystal packing. To further understand the factors determining the dimerization modes of legume lectins, EcorL, concanavalin A (ConA), and Griffonia simplicifolia (GS4) were taken as representatives of the three unique dimers found in the family. Six virtual homodimers were generated. The hydropathy, amino acid composition, and solvation energy were calculated for all nine homodimers. Each of the three native dimers has a distinct chemical composition. EcorL has a dominant hydrophobic component, and ConA has a strong polar component, but in GS4 the three components contribute equally to the interface. This distribution pattern at the interface is unique to the native dimers and distinct from the partition observed in the virtual dimers. Amino acid composition of other members of the family that dimerize like EcorL or ConA maintain the same pattern of amino acids distribution observed in EcorL and ConA. However, lectins that dimerize like GS4 do not show a particularly distinct distribution. In all cases, the calculated solvation energy of the native dimer was lower than that of the virtual dimers, suggesting that the observed mode of dimerization is the most stable organization for the given sequence and tertiary structure. The dimerization type cannot be predicted by sequence analysis.

In vitro phosphorylation of acetylcholinesterase at non-consensus protein kinase A sites enhances the rate of acetylcholine hydrolysis.

Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro. This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzyme's affinity for this substrate and for various active and peripheral site inhibitors. Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo. Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation. Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites. In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents. Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.

Poorly conserved ORFs in the genome of the archaea Halobacterium sp. NRC-1 correspond to expressed proteins.

MOTIVATION: A large fraction of open reading frames (ORFs) identified as 'hypothetical' proteins correspond to either 'conserved hypothetical' proteins, representing sequences homologous to ORFs of unknown function from other organisms, or to hypothetical proteins lacking any significant sequence similarity to other ORFs in the databases. Elucidating the functions and three-dimensional structures of such orphan ORFs, termed ORFans or poorly conserved ORFs (PCOs), is essential for understanding biodiversity. However, it has been claimed that many ORFans may not encode for expressed proteins. RESULTS: A genome-wide experimental study of 'paralogous PCOs' in the halophilic archaea Halobacterium sp. NRC-1 was conducted. Paralogous PCOs are ORFs with at least one homolog in the same organism, but with no clear homologs in other organisms. The results reveal that mRNA is synthesized for a majority of the Halobacterium sp. NRC-1 paralogous PCO families, including those comprising relatively short proteins, strongly suggesting that these Halobacterium sp. NRC-1 paralogous PCOs correspond to true, expressed proteins. Hence, further computational and experimental studies aimed at characterizing PCOs in this and other organisms are merited. Such efforts could shed light on PCOs' functions and origins, thereby serving to elucidate the vast diversity observed in the genetic material.

Rescue of a mutant G-protein by substrate-assisted catalysis.

Signaling by guanine-nucleotide-binding proteins (G-proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G-proteins remains controversial. We have employed substrate-assisted catalysis as a novel approach to study catalysis by G-proteins. In these studies, we have used diaminobenzophenone-phosphonoamidate-GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase-deficient [Leu227]G(s alpha) mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild-type enzyme at the same rate. On the other hand, modification of G(s alpha) by cholera toxin, which catalyses ADP-ribosylation of Arg201 of G(s alpha), decreased the rates of hydrolysis of both GTP and its analog by 95%. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]G(s alpha) mutant and to the essential role of Gln227 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild-type enzyme, favors a model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the rate-limiting step of the GTPase reaction.

The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli.

We used partially purified NhaR and a highly purified His-tagged NhaR derivative to identify the cis-regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction. Gel retardation assay with DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR-binding motif. Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA. The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G-92, G-60, G-29 and A-24 form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a DeltanhaR strain; accordingly, these bases were protected in vitro by the purified His-tagged NhaR. 100 mM Na+, but not K+, removed the protection of G-60 conferred by His-tagged NhaR in vitro. Exposure of intact cells to 100 mM Na+, but not K+, exposed G-60. The maximal effect of Na+ in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5. In contrast to G-60, G-92 was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction. We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR-nhaA contact points.

The putative bioactive surface of insect-selective scorpion excitatory neurotoxins.

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.