Rex A. Palmer

The structure of ribonuclease at 2.5 Ångström resolution

The tertiary structure of ribonuclease-A crystallized from 40% aqueous ethanol has been determined by X-ray crystallography, using the method of isomorphous replacement with four heavy-metal derivatives. The unit cell of the crystal, with a = 30.31 A, b = 38.26 A, c = 52.91 A and β = 105 °55′, contains two molecules and is similar to the form studied in detail by Harker, Kartha & Bello at Buffalo, N.Y. The tertiary structures of the ribonucleases studied at the different centres are similar with regard to the α-carbon atom positions but the comparison cannot be extended further as no details of the Buffalo structure have been published. A preliminary comparison is made between the tertiary structures of RNase-A and that of RNase-S, whose atomic co-ordinates have been published. In both cases, the three amino acid residues, His-12, His-119 and Lys-41, responsible for activity have approximately the same relative positions with respect to one another, but there are some interesting differences between the two, which are pointed out in the text.

Preliminary crystallographic characterization of ricin agglutinin

The quaternary structure of ricin agglutinin (RCA) has been determined by x‐ray crystallography. The refined structure of ricin proved to be a successful search model using the molecular replacement method of phase determination. RCA forms an elongated molecule of dimensions 120 Å × 60 Å × 40 Å with two A chains at the center and a B chain at each end. The A chains are covalently associated via a disulfide bridge between Cys 156 of both chains. Additional contacts at residues 114–115 stabilize the dimer interface. The covalent association of RCA A chains was confirmed by gel filtration under reducing and nonreducing conditions. Proteins 28:586–589, 1997.

Structure, absolute configuration, and conformation of the antimalarial compound, Artemisinin

The crystal and molecular structure of the antimalarial compound Artemisinin (formerly known as Qinghaosu), C15H22O5 has been determined by direct methods. Crystals are orthorhombic colorless needles, space group P212121, Z = 4. Dc = 1.299 g cm −3, with unit cell parameters a = 6.3543(9), b = 9.439(3), c= 24.066(4) Å. The molecule incorporates a fused ring system containing a six-membered ring C which includes an oxygen bridge and a peroxy-bridge. The ring C has a distorted boat conformation and the C - O - O - C torsion angle is 47.8(2)°. Rings A and D have symmetrical chair and distorted chair conformations, repectively. Ring junctions A/B, A/D, and C/D are cis, junction B/D is trans. All inter-molecular contacts are van der Waals. The absolute configuration of Artemisinin was determined from the refined value of the Flack x parameter. [The atomic coordinates given in a previous structure analysis, “Crystal Structure and Absolute Configuration of Qinghaosu,” Qinghaosu Research Group, Institute of Biophysics, Academica Sinica, Scientia Sinica, Vol. XXIII No. 3, 380 (1980), do not display the molecule in its absolute configuration.]

Mistletoe lectin I forms a double trefoil structure

The quaternary structure of mistletoe lectin I (MLI), a type II ribosome inactivating protein, has been determined by X‐ray crystallography. A definitive molecular replacement solution was determined for MLI using the co‐ordinates of the homologue ricin as a search model. MLI exists as an [AB]2 dimer with internal crystallographic two‐fold symmetry. Domain I of the B chains is non‐covalently associated through interactions involving three looped chains (α, β, γ) in each molecule of the dimer, forming a double trefoil structure. The ricin molecule which shares 52% sequence homology with MLI has a disulphide bridge between Cys20 and Cys39 in the α loop. An evolutionary mutation has replaced Cys39 with serine in MLI. This mutation appears to allow the α loop the flexibility required to take up its place at the dimer interface, and also suggests a rationale for why ricin does not form dimers. Measurement of retention times using FPLC gel filtration confirms that dimerisation also occurs in solution between MLI B chains with an association constant K a=106 M.

Newly observed binding mode in pancreatic ribonuclease.

Dinucleotides containing guanine, when soaked into crystals of bovine pancreatic ribonuclease, have been found to bind in an unexpected manner, quite unlike interpretations of earlier X-ray diffraction studies. This finding has prompted a reexamination of three mononucleotide-RNase complexes from this laboratory resulting in a re-interpretation of the complex that involved a guanine mononucleotide.

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N‐acetylgalactosamine (GalNAc)‐specific lectin (SNA‐II) corresponding to slightly truncated B‐chains of a genuine Type‐II ribosome‐inactivating protein (Type‐II RIPs, SNA‐V), found in the same species. The three‐dimensional X‐ray structure of SNA‐II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 Å and 1.35 Å, respectively. In both crystal forms, the SNA‐II molecule folds into two linked ββ‐trefoil domains, with an overall conformation similar to that of the B‐chains of ricin and other Type‐II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high‐resolution structures of SNA‐II in complex with Gal and five Gal‐related saccharides (GalNAc, lactose, αα1‐methylgalactose, fucose, and the carcinoma‐specific Tn antigen) were determined at 1.55 Å resolution or better. Binding is observed in two saccharide‐binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X‐ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution. Proteins 2009.

Crystal and molecular structure of 5-carbamyl-5H-dibenzo[b,f] azepine

The structure of the title compound has been determined by direct methods from diffractometer data, and refined by full-matrix least squares. Crystals are monoclinicP21/n,a=7.534(1),b=11.150(2),c=13.917(3) Å,β=92.94(4)°,Z=4,Dx=1.34 (3) g cm−3,R=0.084 for 1259 observed reflections. The azepine ring has a boat conformation. The fused benzene rings are planar. Molecules are packed as hydrogen-bonded dimers through the carboxamide groups. The atomic charge distribution over the fused ring system is approximately symmetrical.

The refined structure of ribonuclease-A at 1.45 Å resolution

Features of the refined X-ray crystal structure of bovine pancreatic ribonuclease-A at 1.45 Å resolution are described. The positions of the protein atoms have been determined within the range 0.004–0.05 Å, and of solvent atoms, assumed to be oxygens, within the range 0.08–0.13 Å. The present model contains 127 solvent molecules, taken to be water, and a sulfate anion located in the active site. Mean square atomic displacement parameters,Uiso, refined for each atom, give an indication of the mobility of different parts of the structure. Main-chainUiso values tend to be less than side-chain values, having an average value of 0.15 Å2 compared to 0.25 Å2. Both main-chain and side-chain averageUiso values tend to increase with distance from the center of gravity of the molecule. Side-chain averageUiso values also tend to increase with the number of atoms in the side-chain, with different distributions for ring and chain type residues. Side-chain conformations have been analyzed and found on the whole to follow commonly observed distributions. A notable exception to this is the active-site residue His-119 which occupies two distinct sites. Apart from two small clusters of eight and seven atoms respectively, the solvent molecules are distributed in quite small numbers on the protein surface. The solvent clusters occur in the active-site region and, together with the sulfate anion, appear to stabilize residues in this region. Sixty-three solvent atoms have only one identified hydrogen bond contact. Of the rest, 36 form two, 22 form three, and 6 form four hydrogen bonds. There is a marked tendency for the mean square displacement parameter,Uiso, for the solvent atoms to be lower for atoms with many hydrogen bond contacts than for those with fewer contacts.

The role of structural domains in RIP II toxin model membrane binding

The interaction of plant toxin ricin and MLI binding subunits to liposomes containing monosialoganglioside (GM1), bearing a terminal galactose residue, has been examined as a possible receptor model. For the first time we demonstrate that ricin B‐chain but not ricin provokes liposome aggregation at 10 M% GM1 concentration, whereas in the presence of either ricin A‐chain or galactose the aggregation is inhibited. The B‐subunit of plant toxin MLI from Viscum album has similar lectin specificity and activity but cannot aggregate GM1 liposomes. The ability of the B‐chain to aggregate liposomes adds a new crucial step in the toxin transmembrane penetration mechanism. We demonstrate here possible ricin B‐chain interactions with membranes proceeding via two sites, namely (a) a galactose‐binding domain and (b) a hydrophobic interchain domain. In close contact with two phospholipid bilayers, ricin B‐chain may determine the geometry of the fusion site. These events can provoke A‐chain translocation which follows membrane fusion.

An X-ray refinement study on the binding of ribonuclease-A to cytidine-N(3)-oxide 2′-phosphate

Abstract The X-ray structure of the inhibitor complex of bovine ribonuclease-A with cytidine-N(3)-oxide 2′-phosphate (O(3)-2′CMP) has been determined at 2.3 A resolution and refined by restrained least squares to R = 0.195 for 5240 reflections. Little disturbance of the protein main-chain atoms has taken place, but incorporation of the inhibitor molecule is associated with significant movement of side-groups, particularly within the active site. The PO42− group of the inhibitor molecule occupies the SO42− site of the native protein (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38, 2210–2217), while the modified pyrimidine rng is located in the B1 region and the ribose moiety is close to site R1 (Haar, W., Maurer, W. and Ruterjans, H. (1974) Eur. J. Biochem. 44, 201–211). Hydrogen bond interactions occur between the PO42− group and side-groups of His-12, Lys-41 and His-119. Lys-41 has moved from its native position to establish a direct contact with the PO42− group (a water molecule being expelled in the process) and His-119 now occupies exclusively the minor native site. Thr-45 plays an important role in stabilizing the modified pyrimidine base by forming hydrogen bond interactions both between O(γ) and the base N(4) atom and the NH amide group and O(3) of the base. Phe-120 does not form any close association with the planar base of the inhibitor.