The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis.

Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of alternating ß-D-glucuronosyl and N-acetyl-α-D-glucosaminyl residues by exostosins. These enzymes generally possess two glycosyltransferase domains (GT47 and GT64)-each thought to add one type of monosaccharide unit to the backbone. Although previous structures of murine exostosin-like 2 (EXTL2) provide insight into the GT64 domain, the rest of the bi-domain architecture is yet to be characterised; hence, how the two domains co-operate is unknown. Here, we report the structure of human exostosin-like 3 (EXTL3) in apo and UDP-bound forms. We explain the ineffectiveness of EXTL3's GT47 domain to transfer ß-D-glucuronosyl units, and we observe that, in general, the bi-domain architecture would preclude a processive mechanism of backbone extension. We therefore propose that heparan sulfate backbone polymerisation occurs by a simple dissociative mechanism.

Designing interactions by control of protein-ligand complex conformation: tuning arginine-arene interaction geometry for enhanced electrostatic protein-ligand interactions.

We investigated galectin-3 binding to 3-benzamido-2-O-sulfo-galactoside and -thiodigalactoside ligands using a combination of site-specific mutagenesis, X-ray crystallography, computational approaches, and binding thermodynamics measurements. The results reveal a conformational variability in a surface-exposed arginine (R144) side chain in response to different aromatic C3-substituents of bound galactoside-based ligands. Fluorinated C3-benzamido substituents induced a shift in the side-chain conformation of R144 to allow for an entropically favored electrostatic interaction between its guanidine group and the 2-O-sulfate of the ligand. By contrast, binding of ligands with non-fluorinated substituents did not trigger a conformational change of R144. Hence, a sulfate-arginine electrostatic interaction can be tuned by the choice of ligand C3-benzamido structures to favor specific interaction modes and geometries. These results have important general implications for ligand design, as the proper choice of arginine-aromatic interacting partners opens up for ligand-controlled protein conformation that in turn may be systematically exploited in ligand design.

Structural basis for allosteric substrate specificity regulation in anaerobic ribonucleotide reductases.

BACKGROUND: The specificity of ribonucleotide reductases (RNRs) toward their four substrates is governed by the binding of deoxyribonucleoside triphosphates (dNTPs) to the allosteric specificity site. Similar patterns in the kinetics of allosteric regulation have been a strong argument for a common evolutionary origin of the three otherwise widely divergent RNR classes. Recent structural information settled the case for divergent evolution; however, the structural basis for transmission of the allosteric signal is currently poorly understood. A comparative study of the conformational effects of the binding of different effectors has not yet been possible; in addition, only one RNR class has been studied. RESULTS: Our presentation of the structures of a class III anaerobic RNR in complex with four dNTPs allows a full comparison of the protein conformations. Discrimination among the effectors is achieved by two side chains, Gln-114 and Glu-181, from separate monomers. Large conformational changes in the active site (loop 2), in particular Phe-194, are induced by effector binding. The conformational differences observed in the protein when the purine effectors are compared with the pyrimidine effectors are large, while the differences observed within the purine group itself are more subtle. CONCLUSIONS: The subtle differences in base size and hydrogen bonding pattern at the effector site are communicated to major conformational changes in the active site. We propose that the altered overlap of Phe-194 with the substrate base governs hydrogen bonding patterns with main and side chain hydrogen bonding groups in the active site. The relevance for evolution is discussed.

Structure and function of the radical enzyme ribonucleotide reductase.

Ribonucleotide reductases (RNRs) catalyze all new production in nature of deoxyribonucleotides for DNA synthesis by reducing the corresponding ribonucleotides. The reaction involves the action of a radical that is produced differently for different classes of the enzyme. Class I enzymes, which are present in eukaryotes and microorganisms, use an iron center to produce a stable tyrosyl radical that is stored in one of the subunits of the enzyme. The other classes are only present in microorganisms. Class II enzymes use cobalamin for radical generation and class III enzymes, which are found only in anaerobic organisms, use a glycyl radical. The reductase activity is in all three classes contained in enzyme subunits that have similar structures containing active site cysteines. The initiation of the reaction by removal of the 3'-hydrogen of the ribose by a transient cysteinyl radical is a common feature of the different classes of RNR. This cysteine is in all RNRs located on the tip of a finger loop inserted into the center of a special barrel structure. A wealth of structural and functional information on the class I and class III enzymes can now give detailed views on how these enzymes perform their task. The class I enzymes demonstrate a sophisticated pattern as to how the free radical is used in the reaction, in that it is only delivered to the active site at exactly the right moment. RNRs are also allosterically regulated, for which the structural molecular background is now starting to be revealed.

A glycyl radical site in the crystal structure of a class III ribonucleotide reductase.

Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides. Three classes have been identified, all using free-radical chemistry but based on different cofactors. Classes I and II have been shown to be evolutionarily related, whereas the origin of anaerobic class III has remained elusive. The structure of a class III enzyme suggests a common origin for the three classes but shows differences in the active site that can be understood on the basis of the radical-initiation system and source of reductive electrons, as well as a unique protein glycyl radical site. A possible evolutionary relationship between early deoxyribonucleotide metabolism and primary anaerobic metabolism is suggested.

Crystal structures of two self-hydroxylating ribonucleotide reductase protein R2 mutants: structural basis for the oxygen-insertion step of hydroxylation reactions catalyzed by diiron proteins.

The R2 protein of ribonucleotide reductase catalyzes the dioxygen-dependent one-electron oxidation of Tyr122 at a diiron-carboxylate site. Methane monooxygenase and related hydroxylases catalyze hydrocarbon hydroxylation at diiron sites structurally related to the one in R2. In protein R2, the likely reaction site for dioxygen is close to Phe208. The crystal structure of an iron ligand mutant R2, Y122F/E238A, reveals the hydroxylation of Phe208 at the meta, or epsilon-, ring position and the subsequent coordination of this residue to the diiron site. In another mutant, F208Y, the "foreign" residue Tyr208 is hydroxylated to Dopa. The structures of apo and diferrous F208Y presented here suggest that Tyr208 is coordinated to the iron site of F208Y throughout the Dopa generation cycle. Together, the structural data on these two mutants suggest two possible reaction geometries for the hydroxylation reaction catalyzed by these modified R2 diiron sites, geometries which might be relevant for the hydroxylation reaction catalyzed by other diiron sites such as methane monooxygenase. A critical role for residue Glu238 in directing the oxidative power of the reactive intermediate toward oxidation of Tyr122 is proposed.

Mercury concentrations in pond fish in relation to a coal-fired power plant.

Many studies have reported that atmospheric mercury is the primary cause for bioaccumulation in fish from remote lakes. Few data, however, are available on the possible effects of near-field mercury deposition on mercury concentrations in fish from local waters. Mercury concentrations were surveyed in fish from 23 ponds in the vicinity of a 543-megawatt coal-fired power plant located at Dickerson, Maryland. A stratified random sampling design was used to select ponds within zones delineated by concentric arcs mapped at 3, 7, 10, and 15 km from the plant. For each pond, mercury concentrations were measured by atomic absorption spectrometry in sunfish (bluegill or green sunfish) in all ponds, and largemouth bass, which were present in 14 of the ponds. Mean mercury concentrations in the ponds ranged from 0.01 to 0.38 ppm for sunfish and 0.04 to 0.43 ppm for bass. Stepwise multiple regression identified variables related to tissue concentrations. Differences between strata were tested with analysis of covariance, after adjusting the concentrations to account for differences in water quality. The observed pattern of mercury bioaccumulation did not match the pattern predicted by a wet deposition model.

Kinetics of transient radicals in Escherichia coli ribonucleotide reductase. Formation of a new tyrosyl radical in mutant protein R2.

Reconstitution of the tyrosyl radical in ribonucleotide reductase protein R2 requires oxidation of a diferrous site by oxygen. The reaction involves one externally supplied electron in addition to the three electrons provided by oxidation of the Tyr-122 side chain and formation of the mu-oxo-bridged diferric site. Reconstitution of R2 protein Y122F, lacking the internal pathway involving Tyr-122, earlier identified two radical intermediates at Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a novel internal transfer pathway (Sahlin, M., Lassmann, G., Pötsch, S., Sjöberg, B. -M., and Gräslund, A. (1995) J. Biol. Chem. 270, 12361-12372). Here, we report the construction of the double mutant W107Y/Y122F and its three-dimensional structure and demonstrate that the tyrosine Tyr-107 can harbor a transient, neutral radical (Tyr-107(.)). The Tyr-107(.) signal exhibits the hyperfine structure of a quintet with coupling constants of 1.3 mT for one beta-methylene proton and 0.75 mT for each of the 3 and 5 hydrogens of the phenyl ring. Rapid freeze quench kinetics of EPR-visible intermediates reveal a preferred radical transfer pathway via Trp-111, Glu-204, and Fe-2, followed by a proton coupled electron transfer through the pi-interaction of the aromatic rings of Trp-(Tyr-)107 and Trp-111. The kinetic pattern observed in W107Y/Y122F is considerably changed as compared with Y122F: the Trp-111(.) EPR signal has vanished, and the Tyr-107(.) has the same formation rate as does Trp-111(.) in Y122F. According to the proposed consecutive reaction, Trp-111(.) becomes very short lived and is no longer detectable because of the faster formation of Tyr-107(.). We conclude that the phenyl rings of Trp-111 and Tyr-107 form a better stacking complex so that the proton-coupled electron transfer is facilitated compared with the single mutant. Comparison with the formation kinetics of the stable tyrosyl radical in wild type R2 suggests that these protein-linked radicals are substitutes for the missing Tyr-122. However, in contrast to Tyr-122(.) these radicals lack a direct connection to the radical transfer pathway utilized during catalysis.

Crystal structure of reduced protein R2 of ribonucleotide reductase: the structural basis for oxygen activation at a dinuclear iron site.

BACKGROUND: Ribonucleotide reductases (RNRs) catalyze the formation of the deoxyribonucleotides that are essential for DNA synthesis. The R2 subunit of Escherichia coli RNR is a homodimer containing one dinuclear iron centre per monomer. A tyrosyl radical is essential for catalysis, and is formed via a reaction in which the reduced, diferrous form of the iron centre activates dioxygen. To help understand the mechanism of oxygen activation, we examined the structure of the diferrous form of R2. RESULTS: The crystal structures of reduced forms of both wild type R2 and a mutant of R2 (Ser211--> Ala) have been determined at 1.7 A and 2.2 A resolution, respectively. The diferrous iron centre was compared to the previously determined structure of the oxidized, diferric form of R2. In both forms of R2 the iron centre is coordinated by the same carboxylate dominated ligand sphere, but in the reduced form there are clear conformational changes in three of the carboxylate ligands and the bridging mu-oxo group and two water molecules are lost. In the reduced form of R2 the coordination number decreases from six to four for both ferrous ions, explaining their high reactivity towards dioxygen. The structure of the mutant Ser211--> Ala, known to have impaired reduction kinetics, shows a large conformational change in one of the neighbouring helices although the iron coordination is very similar to the wild type protein. CONCLUSIONS: Carboxylate shifts are often important for carboxylate coordinated metal clusters; they allow the metals to achieve different coordination modes in redox reactions. In the case of reduced R2 these carboxylate shifts allow the formation of accessible reaction sites for dioxygen. The Ser211--> Ala mutant displays a conformational change in the helix containing the mutation, explaining its altered reduction kinetics.

Glycyl-tRNA synthetase.

Glycyl-tRNA synthetase, a class II aminoacyl-tRNA synthetase, catalyzes the synthesis of glycyl-tRNA, which is required to insert glycine into proteins. In a side reaction the enzyme also synthesizes dinuceloside polyphosphates, which probably participate in regulation of cell functions. Glycine is the smallest amino acid occurring in natural proteins, probably established as a protein component very early in evolution. Besides the amino and the carboxyl groups there is no functional group in the molecule. Alanine, the amino acid which is structurally most similar to glycine, possesses an additional methyl group as 'side chain'. Glycyl-tRNA synthetase is one of the few synthetases which exhibit different oligomeric structures in different organisms (alpha 2 beta 2 and alpha 2). The alpha 2 beta 2 enzymes exhibit similarities to PheRS (also an alpha 2 beta 2 enzyme). The alpha 2 forms belong to the subclass IIa enzymes with regard to sequence homologies. In eukaryotes the polypeptide is weakly associated with multienzyme complexes consisting of aminoacyl-tRNA synthetases. In the aminoacylation reaction a 'half-of-the-sites' mechanism as found for GlyRS from Bombyx mori is probably used by all glycyl-tRNA synthetases under in vivo conditions. Essentially, tRNAGly is recognized by GlyRS through standard identity elements in the anticodon region and in the acceptor stem. The last three facts may indicate that GlyRS is an enzyme which still possesses properties of a primordial aminoacyl-tRNA synthetase. Nine genes of glycyl-tRNA synthetases from six organisms have been sequenced. They encode synthetase subunits of chain lengths ranging from 300-700 amino acids. One crystal structure, that of the alpha 2 enzyme from Thermus thermophilus, has also been determined. The two subunits each possess three domains: the active site resembling that of aspartyl and seryl enzymes, a C-terminal anticodon recognition domain, and one domain which almost certainly interacts with the acceptor stem of tRNAGly. Antibodies against glycyl-RNA synthetase occur in the sera of patients suffering from polymyositis and interstitial lung disease.

Crystal structure of glycyl-tRNA synthetase from Thermus thermophilus.

The sequence and crystal structure at 2.75 A resolution of the homodimeric glycyl-tRNA synthetase from Thermus thermophilus, the first representative of the last unknown class II synthetase subgroup, have been determined. The three class II synthetase sequence motifs are present but the structure was essential for identification of motif 1, which does not possess the proline previously believed to be an essential class II invariant. Nevertheless, crucial contacts with the active site of the other monomer involving motif 1 are conserved and a more comprehensive description of class II now becomes possible. Each monomer consists of an active site strongly resembling that of the aspartyl and seryl enzymes, a C-terminal anticodon recognition domain of 100 residues and a third domain unusually inserted between motifs 1 and 2 almost certainly interacting with the acceptor arm of tRNA(Gly). The C-terminal domain has a novel five-stranded parallel-antiparallel beta-sheet structure with three surrounding helices. The active site residues most probably responsible for substrate recognition, in particular in the Gly binding pocket, can be identified by inference from aspartyl-tRNA synthetase due to the conserved nature of the class II active site.

Crystallisation of the glycyl-tRNA synthetase from Thermus thermophilus and initial crystallographic data.

The glycyl-tRNA synthetase from Thermus thermophilus is a dimer of molecular mass 115 kDa, which has been crystallised using the vapour diffusion method from 5 to 7% polyethylene glycol 6000, 0.8 to 1.4 M NaCl at protein concentrations of 2 to 8 mg/ml. Nucleation is carried out at 4 degrees C and crystals are subsequently transferred to 15 degrees C to maximise growth. Crystals are truncated rhombohedra measuring on average 0.4 mm x 0.4 mm x 0.2 mm, which appear within a few days and reach full size in one to two months. GlyRS crystallises in two closely related space groups, P2(1)2(1)2(1) and C2,2,2(1), both with the same cell a = 125 A, b = 254 A, c = 104 A. Crystal packing in P2(1)2(1)2(1) is strongly C-centred. The crystals have VM = 3.6 A3/Da and a solvent content of 61%, with one dimer in the asymmetric unit in C2,2,2(1) and two dimers in P2(1)2(1)2(1). The best native data extend to 2.9 A in C2,2,2(1) and are 90.6% complete with an R-factor between symmetry-related reflections of 10.0%. The structure has been solved by multiple isomorphous replacement and model building is in progress.