Packing analysis of carbohydrates and polysaccharides. IV. A new method for detailed crystal structure refinement of polysaccharides and its application to V-amylose

A method is described for predicting and solving crystal structures of linear homopolysaccharides. The method is based on the refinement of the structure with respect to either stereochemical constraints or x-ray diffraction intensities. In the refinement process, all conformational and packing features of the molecule, such as bond lengths, bond angles, conformational angles, nonbonded contacts, hydrogen bonds, etc., can be allowed to vary until the structure reaches both a conformation and crystalline packing that are in minimum disagreement with the stereochemical restraints and the diffraction data. In this fashion, both packing and conformational features of the structure can be simultaneously refined, and not separately as has been the custom in the past. The refinement procedure is based on a method of constrained optimization which possesses improved characteristics of reaching a solution and avoiding false minima, in comparison with least squares methods. The procedure is, in addition, capable of easily finding molecules of solvent of crystallization. The method was applied to further refining the previously solved crystal structure of V-amylose. The results indicated that contrary to the previously found six-fold molecular symmetry in the P2₁2₁2₁ space group, the V-amylose molecule exhibits only two-fold symmetry with the asymmetric unit consisting of three glucose residues in one-half turn of the helix. The three residues are nonequivalent principally due to unequal rotational positions of the hydroxymethyl groups. The crystal structure of V-amylose predicted from stereochemical refinement was identical in all details with that obtained from refining against X-ray data. The excellent agreement with the diffraction data was indicated by the crystallographic disagreement index R = 0.25.     

Crystal structures of two engineered thiol trypsins

We have determined the three-dimensional structures of engineered rat trypsins which mimic the active sites of two classes of cysteine proteases. The catalytic serine was replaced with cysteine (S195C) to test the ability of sulfur to function as a nucleophile in a serine protease environment. This variant mimics the cysteine trypsin class of thiol proteases. An additional mutation of the active site aspartate to an asparagine (D102N) created the catalytic triad of the papain-type cysteine proteases. Rat trypsins S195C and D102N,S195C were solved to 2.5 and 2.0 A, respectively. The refined structures were analyzed to determine the structural basis for the 10(6)-fold loss of activity of trypsin S195C and the 10(8)-fold loss of activity of trypsin D102N,S195C, relative to rat trypsin. The active site thiols were found in a reduced state in contrast to the oxidized thiols found in previous thiol protease structures. These are the first reported structures of serine proteases with the catalytic centers of sulfhydryl proteases. Structure analysis revealed only subtle global changes in enzyme conformation. The substrate binding pocket is unaltered, and active site amino acid 102 forms hydrogen bonds to H57 and S214 as well as to the backbone amides of A56 and H57. In trypsin S195C, D102 is a hydrogen-bond acceptor for H57 which allows the other imidazole nitrogen to function as a base during catalysis. In trypsin D102N,S195C, the asparagine at position 102 is a hydrogen-bond donor to H57 which places a proton on the imidazole nitrogen proximal to the nucleophile. This tautomer of H57 is unable to act as a base in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of trisammonium bisperoxotetrafluoroniobate(V) and analogous tantalate(V)

(NH₄)₃[Nb(O₂)₂F₄] (I) and (NH₄)₃[Ta(O₂)₂F₄] (II) are isostructural, and belong to the cubic Fm3m space group with four molecules in the unit cell. The unit cell parameters are a = 9.4442(4) (I) and a = 9.4512(4) Å (II). The structures were solved by the Patterson method and were refined by the least-squares method to the conventional R factors of 0.036 for 86 reflections (I) and 0.043 for 103 reflections (II) (in both structures having I ? 2σ(I)). The disordered distributions of fluorine and peroxo oxygens with partially occupied sites are observed. The disordered NH₄⁺ tetrahedra appear in the structures.The metal atoms exhibit an octahedral coordination with two corners of a polyhedron at the centre of the peroxo bonds. Inter-atomic distances are NbF, 1.95(2), NbO, 1.94, TaF, 1.91(4) and TaO, 2.07 Å.The structures (I, II) are composed of [M^υ(O₂)₂-F₄]^3? octahedra and two symmetrically-independent ammonium cations connected by NH?O and NH?F hydrogen bonds. These two structures are compared with the structure of (NH₄)₃Ti(O₂)F₅].     

Crystal structures of cephaibols.

The crystal structures of the peptaibol antibiotics cephaibol A, cephaibol B and cephaibol C have been determined at ca. 0.9 A resolution. All three adopt a helical conformation with a sharp bend (of about 55 degrees) at the central hydroxyproline. All isovalines were found to possess the D configuration, superposition of all four models (there are two independent molecules in the cephaibol B structure) shows that the N-terminal helix is rigid and the C-terminus is flexible. There are differences in the hydrogen bonding patterns for the three structures that crystallize in different space groups despite relatively similar unit cell dimensions, but only in the case of cephaibol C does the packing emulate the formation of a membrane channel believed to be important for their biological function.     

Crystal structures of two functionally different thioredoxins in spinach chloroplasts

Thioredoxins are small ubiquitous proteins which act as general protein disulfide reductases in living cells. Chloroplasts contain two distinct thioredoxins ( f and m) with different phylogenetic origin. Both act as enzyme regulatory proteins but have different specificities towards target enzymes. Thioredoxin f (Trx f), which shares only low sequence identity with thioredoxin m (Trx m) and with all other known thioredoxins, activates enzymes of the Calvin cycle and other photosynthetic processes. Trx m shows high sequence similarity with bacterial thioredoxins and activates other chloroplast enzymes. The here described structural studies of the two chloroplast thioredoxins were carried out in order to gain insight into the structure/function relationships of these proteins. Crystal structures were determined for oxidized, recombinant thioredoxin f (Trx f-L) and at the N terminus truncated form of it (Trx f-S), as well as for oxidized and reduced thioredoxin m (at 2.1 and 2.3 A resolution, respectively). Whereas thioredoxin f crystallized as a monomer, both truncated thioredoxin f and thioredoxin m crystallized as non-covalent dimers. The structures of thioredoxins f and m exhibit the typical thioredoxin fold consisting of a central twisted five-stranded beta-sheet surrounded by four alpha-helices. Thioredoxin f contains an additional alpha-helix at the N terminus and an exposed third cysteine close to the active site. The overall three-dimensional structures of the two chloroplast thioredoxins are quite similar. However, the two proteins have a significantly different surface topology and charge distribution around the active site. An interesting feature which might significantly contribute to the specificity of thioredoxin f is an inherent flexibility of its active site, which has expressed itself crystallographically in two different crystal forms.     

Crystal structures of two homologous pathogenesis-related proteins from yellow lupine

Pathogenesis-related class 10 (PR10) proteins are restricted to the plant kingdom where they are coded by multigene families and occur at high levels. In spite of their abundance, their physiological role is obscure although members of a distantly related subclass (cytokinin-specific binding proteins) are known to bind plant hormones. PR10 proteins are of special significance in legume plants where their expression patterns are related to infection by the symbiotic, nitrogen-fixing bacteria. Here we present the first crystal structures of classic PR10 proteins representing two homologues from one subclass in yellow lupine. The general fold is similar and, as in a birch pollen allergen, consists of a seven-stranded beta-sheet wrapped around a long C-terminal helix. The mouth of a large pocket formed between the beta-sheet and the helix seems a likely site for ligand binding. The shape of the pocket varies because, in variance with the rigid beta-sheet, the helix shows unusual conformational variability consisting in bending, disorder, and axial shifting. A surface loop, proximal to the entrance to the internal cavity, shows an unusual structural conservation and rigidity in contrast to the high glycine content in its sequence. The loop is different from the so-called glycine-rich P-loops that bind phosphate groups of nucleotides, but it is very likely that it does play a role in ligand binding in PR10 proteins.     

Crystal structures of two archaeal Pelotas reveal inter-domain structural plasticity

Dom34 from Saccharomyces cerevisiae is one of the key players in no-go mRNA decay, a surveillance pathway by which an abnormal mRNA stalled during translation is degraded by an endonucleolytic cleavage. Its homologs called Pelota are found in other species. We showed previously that S. cerevisiae Dom34 (domain 1) has an endoribonuclease activity, which suggests its direct catalytic role in no-go decay. Pelota from Thermoplasma acidophilum and Dom34 from S. cerevisiae have been structurally characterized, revealing a tripartite architecture with a significant difference in their overall conformations. To gain further insights into structural plasticity of the Pelota proteins, we have determined the crystal structures of two archaeal Pelotas from Archaeoglobus fulgidus and Sulfolobus solfataricus. Despite the structural similarity of their individual domains to those of T. acidophilum Pelota and S. cerevisiae Dom34, their overall conformations are distinct from those of T. acidophilum Pelota and S. cerevisiae Dom34. Different overall conformations are due to conformational flexibility of the two linker regions between domains 1 and 2 and between domains 2 and 3. The observed inter-domain structural plasticity of Pelota proteins suggests that large conformational changes are essential for their functions.     

Novel structures of plant lectins and their complexes with carbohydrates.

Several novel structures of legume lectins have led to a thorough understanding of monosaccharide and oligosaccharide specificity, to the determination of novel and surprising quaternary structures and, most importantly, to the structural identification of the binding site for adenine and plant hormones. This deepening of our understanding of the structure/function relationships among the legume lectins is paralleled by advances in two other plant lectin families - the monocot lectins and the jacalin family. As the number of available crystal structures increases, more parallels between plant and animal lectins become apparent.     

Conformational properties of Pro-Pro sequences. I. Crystal structures of two dipeptides with L-Pro-L-Pro and L-Pro-D-Pro sequences

The crystal structures of Bu^tCO-L -Pro-L -Pro-NHMe, H₂O (1: monoclinic; P2₁; a = 6.662, b = 11.067, c = 12.205 Å; β = 96.28°) and Bu^tCO-L -Pro-D -Pro-NHMe (2: monoclinic; P2₁; a = 10.770, b = 15.039, c = 11.325 Å; β = 110.00°) have been solved by x-ray diffraction. Structure 1 accommodates an open disposition with intermolecular interactions involving the water molecule, while 2 is βII-folded by an intramolecular i + 3 → i hydrogen bond. In both derivatives, small thermal parameters are indicative of fairly fixed conformations for the proline rings. Comparison between conformations of either isolated or adjacent L -Pro residues in the crystal structures of unstrained oligopeptides shows that the conformational properties of L -Pro-L -Pro sequences are probably a simple combination of those found for isolated L -Pro residues.     

Crystal structures and amidolytic activities of two glycosylated snake venom serine proteinases

We deduced that Agkistrodon actus venom serine proteinases I and II, previously isolated from the venom of A. acutus (Zhu, Z., Gong, P., Teng, M., and Niu, L. (2003) Acta Crystallogr. Sect. D Biol. Crystallogr. 59, 547-550), are encoded by two almost identical genes, with only the single substitution Asp for Asn at residue 62. Amidolytic assays indicated that they possess slightly different enzymatic properties. Crystal structures of A. actus venom serine proteinases I and II were determined at resolution of 2.0 and 2.1 A with the identification of trisaccharide (NAG(301)-FUC(302)-NAG(303)) and monosaccharide (NAG(301)) residues in them, respectively. The substrate binding sites S3 of the two proteinases appear much shallower than that of Trimeresurus stejnegeri venom plasminogen activator despite the overall structural similarity. Based on structural analysis, we showed that these Asn(35)-linked oligosaccharides collide spatially with some inhibitors, such as soybean trypsin inhibitor, and would therefore hinder their inhibitory binding. Difference of the carbohydrates in both the proteinases might also lead to their altered catalytic activities.     

Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides

Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.     

Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface.

The site on influenza virus N9 neuraminidase recognized by NC41 monoclonal antibody comprises 19 amino acid residues that are in direct contact with 17 residues on the antibody. Single sequence changes in some of the neuraminidase residues in the site markedly reduce antibody binding. However, two mutants have been found within the site, Ile368 to Arg and Asn329 to Asp selected by antibodies other than NC41, and these mutants bind NC41 antibody with only slightly reduced affinity. The three-dimensional structures of the two mutant N9-NC41 antibody complexes as derived from the wild-type complex are presented. Both structures show that some amino acid substitutions can be accommodated within an antigen-antibody interface by local structural rearrangements around the mutation site. In the Ile368 to Arg mutant complex, the side-chain of Arg368 is shifted by 2.9 A from its position in the uncomplexed mutant and a shift of 1.3 A in the position of the light chain residue HisL55 with respect to the wild-type complex is also observed. In the other mutant, the side-chain of Asp329 appears rotated by 150 degrees around C alpha-C beta with respect to the uncomplexed mutant, so that the carboxylate group is moved to the periphery of the antigen-antibody interface. The results provide a basis for understanding some of the potential structural effects of somatic hypermutation on antigen-antibody binding in those cases where the mutation in the antibody occurs at antigen-contacting residues, and demonstrate again the importance of structural context in evaluating the effect of amino acid substitutions on protein structure and function.     

Crystal structures of two Bacillus carboxylesterases with different enantioselectivities

Naproxen esterase (NP) from Bacillus subtilis Thai I-8 is a carboxylesterase that catalyzes the enantioselective hydrolysis of naproxenmethylester to produce S-naproxen (E > 200). It is a homolog of CesA (98% sequence identity) and CesB (64% identity), both produced by B. subtilis strain 168. CesB can be used for the enantioselective hydrolysis of 1,2-O-isopropylideneglycerol (solketal) esters (E > 200 for IPG-caprylate). Crystal structures of NP and CesB, determined to a resolution of 1.75 Å and 2.04 Å, respectively, showed that both proteins have a canonical α/β hydrolase fold with an extra N-terminal helix stabilizing the cap subdomain. The active site in both enzymes is located in a deep hydrophobic groove and includes the catalytic triad residues Ser130, His274, and Glu245. A product analog, presumably 2-(2-hydroxyethoxy)acetic acid, was bound in the NP active site. The enzymes have different enantioselectivities, which previously were shown to result from only a few amino acid substitutions in the cap domain. Modeling of a substrate in the active site of NP allowed explaining the different enantioselectivities. In addition, Ala156 may be a determinant of enantioselectivity as well, since its side chain appears to interfere with the binding of certain R-enantiomers in the active site of NP. However, the exchange route for substrate and product between the active site and the solvent is not obvious from the structures. Flexibility of the cap domain might facilitate such exchange. Interestingly, both carboxylesterases show higher structural similarity to meta-cleavage compound (MCP) hydrolases than to other α/β hydrolase fold esterases.     

Crystal structures of two potent nonamidine inhibitors bound to factor Xa

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.     

Thermodynamic analysis of amyloid fibril structures reveals a common framework for stability in amyloid polymorphs

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