Ultra-high resolution X-ray structure of orthorhombic bovine pancreatic Ribonuclease A at 100K.

The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16000 keV (λ = 0.77 Å). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five [Formula: see text] moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free) = 0.129 (4279 reflections) and R1 = 0.112 (85,346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al. (Acta Crystallogr A 45:851-861, 1989). Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for Cß. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851-861, 1989) includes a sulphate anion which occupies approximately the same location as the [Formula: see text] phosphate group in protein nucleotide complexes (Borkakoti et al. in J Mol Biol 169:743-755, 1983). The present structure contains 5 [Formula: see text] groups SO41151-SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201-EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851-861, 1989).

Semi-Synthetic Analogues of Cryptolepine as a Potential Source of Sustainable Drugs for the Treatment of Malaria, Human African Trypanosomiasis, and Cancer.

The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3-6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3-6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3-6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.

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 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-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, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A 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.

Specific refolding pathway of viscumin A chain in membrane-like medium reveals a possible mechanism of toxin entry into cell.

How is a water-soluble globular protein able to spontaneously cross a cellular membrane? It is commonly accepted that it undergoes significant structural rearrangements on the lipid-water interface, thus acquiring membrane binding and penetration ability. In this study molecular dynamics (MD) simulations have been used to explore large-scale conformational changes of the globular viscumin A chain in a complex environment - comprising urea and chloroform/methanol (CHCl3/MeOH) mixture. Being well-packed in aqueous solution, viscumin A undergoes global structural rearrangements in both organic media. In urea, the protein is "swelling" and gradually loses its long-distance contacts, thus resembling the "molten globule" state. In CHCl3/MeOH, viscumin A is in effect turned "inside out". This is accompanied with strengthening of the secondary structure and surface exposure of hydrophobic epitopes originally buried inside the globule. Resulting solvent-adapted models were further subjected to Monte Carlo simulations with an implicit hydrophobic slab membrane. In contrast to only a few point surface contacts in water and two short regions with weak protein-lipid interactions in urea, MD-derived structures in CHCl3/MeOH reveal multiple determinants of membrane interaction. Consequently it is now possible to propose a specific pathway for the structural adaptation of viscumin A with respect to the cell membrane - a probable first step of its translocation into cytoplasmic targets.

Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K.

The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and Rfree = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6-Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S-S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported. Graphical abstract Conformations of PheB25 and PheD25 in three insulin structures: implications for biological activity? Insulin residues PheB25 and PheD25 are considered to be important for insulin receptor binding and changes in biological activity occur when these residues are modified. In porcine insulin and Intergen (II) PheB25 adopts conformation B and PheD25 conformation D. However, unexpectedly PheB25 in Insugen (I) human recombinant insulin adopts two distinct conformations corresponding to B and D, Figure 1 and PheD25 adopts a single conformation corresponding to B not D, Figure 2. Conformations of this residue in the ultra-high resolution structure of Insugen (I) are therefore unique within this set. Figures were produced with Biovia, Discovery Studio 2016.

Antagonist binding in the rat muscarinic receptor A study by docking and X-ray crystallography.

A series of agonists to the rat muscarinic receptor have been docked computationally to the active site of a homology model of rat M1 muscarinic receptor. The agonists were modelled on the X-ray crystal structure of atropine, which is reported here and the docking studies are shown to reproduce correctly the order of experimental binding affinities for the agonists as well as indicate where there appear to be inconsistencies in the experimental data. The crystal and molecular structure of atropine (tropine tropate; alpha-[hydroxymethyl]benzeneacetic acid 8-methyl[3.2.1]oct-3-yl ester C17H23NO3) has been determined by X-ray crystallography using an automated Patterson search method, and refined by full-matrix least-squares to a final R of 0.0452 for 2701 independent observed reflections and 192 parameters using Mo Kalpha radiation, lambda=0.71073A at 150K. The compound crystallises in space group Fdd2 with Z=16 molecules per unit cell.

X-ray structures of two forms of the antibiotic oligomycin A: an inhibitor of ATP synthase.

BACKGROUND: Corrections to the chemical and x-ray structures of two forms of the antibiotic oligomycin A are presented: the original and best known, form (E), from Streptomyces diastatochromogenes, and a new form (C) from Streptomyces diastaticus. METHOD: The crystal structures are isomorphous, crystallizing in space group P212121, with Z = 4[C45H73O11.CH3OH] per unit cell. Oligomycin A(E) refined with R1 = 0.0734, using Cu Kα x-radiation; and for Oligomycin A(C) R1 = 0.0651 using Mo Kα x-radiation. CONCLUSION: Serious corrections to the previously published structure of oligomycin A(C) are discussed and implemented. As a supplementary study geometry optimization of side group R4 of oligomycin A(E) was undertaken and achieved by energy minimization. These additional results clearly confirm the delocalization in this region observed in both x-ray structures. This result is contrary to the generally accepted formulation. Knowledge of the correct structures is important to those involved in the study and applications of the pharmacological and biological properties of these materials.

A comparative study of the single crystal X-ray determination and molecular modelling of the binding of oligomycin to ATP synthase.

Recently published X-ray structures of three common forms, A, B and C, of oligomycin, including absolute configurations, are investigated to examine their binding to ATP Synthase. The X-ray studies reveal regions with differences in three-dimensional structure and hydrogen bonding propensity between the oligomycins, which may be associated with their potential to bind to sites on ATP Synthase. Computational docking studies carried out using MOE with the X-ray structures and an homology model of the F(O) domain of ATP Synthase from Escherichia coli, are used to derive an induced fit pocket. Docking of all oligomycins to this pocket indicate that the B and C forms bind more tightly than the A form. Consideration of the single crystal X-ray data alone indicate the B form may be the best inhibitor and that O(24) is the most important ligating group for binding, this is supported by the docking data. The latter reveals Asn214 and other key proton translocating residues to be the main residues contacted by the inhibitor. These data allow the binding modes of different forms of oligomycin to be deduced from X-ray single crystal data supported by molecular modelling and computational docking studies.

Two new cyclosporin folds observed in the structures of the immunosuppressant cyclosporin G and the formyl peptide receptor antagonist cyclosporin H at ultra-high resolution.

Cyclosporins are cyclic undecapeptides of fungal origin the best known of which, CsA, is a lead clinical immunosuppressant; CsG is a potential clinical immunosuppressant differing from CsA in residue 2 (L-alpha-amino-butyric acid in CsA, L-norvaline in CsG); and CsH is an inverse formyl peptide receptor agonist, differing from CsA in the chiral inversion of MeVal-11 from L to D. Crystal structure determinations of CsG and CsH were undertaken to identify structural and surface features important for biological activity and the future design of new cyclosporin derivatives. Ultra-high resolution X-ray structures (0.80 to 0.87 A resolution) determined for two crystal forms of both CsH and CsG in the presence and absence of Mg2+ are described. A major outcome of this study is the observation that the local change in chirality between CsA and CsH is associated with a major structural transformation from open beta-sheet in CsA to a highly convoluted conformation in CsH. CsG also possesses a completely novel cloverleaf motif with no H-bonded secondary structure features in spite of the minimal chemical difference with CsA. Unlike CsA, the structures of both CsH and CsG are heavily solvated. This study therefore shows that the chemical differences between the three cyclosporins, CsA, CsG and CsH can invoke unpredictably major differences in their 3D structures. The 9-11 cis-peptide bond in CsA moves to 11-1 in CsG, influencing the overall molecular conformation, while the peptide bonds in the highly convoluted loop conformation of CsH are all trans.

Crystal structure at 3 A of mistletoe lectin I, a dimeric type-II ribosome-inactivating protein, complexed with galactose.

The X-ray structure of mistletoe lectin I (MLI), a type-II ribosome-inactivating protein (RIP), cocrystallized with galactose is described. The model was refined at 3.0 A resolution to an R-factor of 19.9% using 21 899 reflections, with Rfree 24.0%. MLI forms a homodimer (A-B)2 in the crystal, as it does in solution at high concentration. The dimer is formed through contacts between the N-terminal domains of two B-chains involving weak polar and non-polar interactions. Consequently, the overall arrangement of sugar-binding sites in MLI differs from those in monomeric type-II RIPs: two N-terminal sugar-binding sites are 15 A apart on one side of the dimer, and two C-terminal sugar-binding sites are 87 A apart on the other side. Galactose binding is achieved by common hydrogen bonds for the two binding sites via hydroxy groups 3-OH and 4-OH and hydrophobic contact by an aromatic ring. In addition, at the N-terminal site 2-OH forms hydrogen bonds with Asp27 and Lys41, and at the C-terminal site 3-OH and 6-OH undergo water-mediated interactions and C5 has a hydrophobic contact. MLI is a galactose-specific lectin and shows little affinity for N-acetylgalactosamine. The reason for this is discussed. Structural differences among the RIPs investigated in this study (their quaternary structures, location of sugar-binding sites, and fine sugar specificities of their B-chains, which could have diverged through evolution from a two-domain protein) may affect the binding sites, and consequently the cellular transport processes and biological responses of these toxins.

A new gene encoding the ribosome-inactivating protein from mistletoe extracts.

Extracts from mistletoe (Viscum album L.) contain three main toxic proteins--the lectins MLI (also known as viscumin), MLII and MLIII. A catalytic subunit of the mistletoe plant toxic lectin MLIII has been cloned and expressed in Escherichia coli cells. The structure and immunochemical properties of recombinant MLIII A-subunit were investigated using a panel of monoclonal antibodies against ML-toxins. Ribosome-inactivating activity of the recombinant MLIII A-subunit was determined in a cell-free system exhibiting inhibition of endogenous protein synthesis. The comparative analysis of nucleotide and deduced amino acid sequences of the cloned MLIII A and the native MLI A-subunits was performed, revealing the main differences in the primary structure of these proteins. Antigenicity analysis of the MLIII A-subunit has revealed a new epitope D179-E184 that is not present in viscumin. The role of toxic lectins with respect to the immunological properties of mistletoe extracts is discussed.