Trapping intermediates in the crystal: ligand binding to myoglobin

Crystal structures of the reactive short-lived species that occur in chemical or binding reactions can be determined using X-ray crystallography via time-resolved or kinetic trapping approaches. Recently, various kinetic trapping methods have been used to determine the structure of intermediates in ligand binding to myoglobin.     

On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy

Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen-hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.     

Crystallization and preliminary X-ray investigation of the recombinant Trypanosoma brucei rhodesiense calmodulin

Bipyramidal crystals of the recombinant calmodulin from Trypanosoma brucei rhodesiense were obtained by vapor diffusion against 55% (v/v) 2-methyl-2,4-pentanediol in 0.05 M cacodylate buffer, pH 5.6. When few nucleation events occurred, crystals grew to 0.25 × 0.25 × 1.20 mm. The space group of the crystal is I4₁22, with unit cell dimensions a = b = 56.88 Å, c = 230.11 Å, α = β = γ = 90°, z = 16. The molecular mass and volume of the unit cell suggest that there is one molecule in the asymmetric unit. The I/σ(I) ratio for data at 3.0 Å resolution was 3.67, indicating that the final structure can be refined at higher resolution. Molecular replacement methods and the PC-refinement technique have not yet yielded the structure under a variety of search conditions. We are currently investigating the multiple isomorphous replacement approach to determine this crystal structure. © 1995 Wiley-Liss, Inc.     

Crystallographic evidence for dioxygen interactions with iron proteins

The interaction of dioxygen with iron plays a key role in many important biological processes, such as dioxygen transport in the bloodstream and the reduction of dioxygen by iron in respiration. However, the catalytic mechanisms employed, for example in ligand oxidation, are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. This review outlines the structural evidence obtained by X-ray crystallographic methods for the nature of the interactions between dioxygen and the metal in iron-containing proteins. Proteins involved in iron transport or electron transfer are not included.     

Synthesis of 1H-1,2,3-triazole derivatives as new α-glucosidase inhibitors and their molecular docking studies

Inhibition of α-glucosidase is an effective strategy for controlling the post-prandial hyperglycemia in diabetic patients. For the identification of new inhibitors of this enzyme, a series of new (R)-1-(2-(4-bromo-2-methoxyphenoxy) propyl)-4-(4-(trifluoromethyl) phenyl)-1H-1,2,3-triazole derivatives were synthesized (8a–d and 10a–e). The structures were confirmed by NMR, mass spectrometry and, in case of compound 8a, by single crystal X-ray crystallography. The α-glucosidase inhibitory activities were investigated in vitro. Most derivatives exhibited significant inhibitory activity against α-glucosidase enzyme. Their structure-activity relationship and molecular docking studies were performed to elucidate the active pharmacophore against this enzyme. Compound 10b was the most active analogue with IC₅₀ value of 14.2 µM, while compound 6 was found to be the least active having 218.1 µM. A preliminary structure-activity relationship suggested that the presence of 1H-1,2,3-triazole ring in 1H-1,2,3-triazole derivatives is responsible for this activity and can be used as anti-diabetic drugs. The molecular docking studies of all active compounds were performed, in order to understand the mode of binding interaction and the energy of this class of compounds.     

Identification of canavalin as a proteolytically modified form of Jack bean α-d-mannosidase

The structure to 3.0 Å resolution of the protein canavalin from Jack beans has been determined by conventional X-ray crystallographic techniques. We present evidence that canavalin is a proteolytically modified form of the enzyme α-d-mannosidase. This is based on the facts that the purified precursor protein possesses substantial α-d-mannosidase activity; it comigrates on SDS-PAGE with Jack bean α-d-mannosidase prepared by other means; and its oligomer molecular weight and Zn²⁺ content are the same as those of Jack bean α-d-mannosidase.     

Structural differences in the inner part of Photosystem II between higher plants and cyanobacteria

A detailed comparison of key components in the Photosystem II complexes of higher plants and cyanobacteria was carried out. While the two complexes are overall very similar, significant differences exist in the relative orientation of individual components relative to one another. We compared a three-dimensional map of the inner part of plant PS II at 8 Å resolution, and a 5.5 Å projection map of the same complex determined by electron crystallography, to the recent 3.5–3.8 Å X-ray structures of cyanobacterial complexes. The largest differences were found in the rotational alignment of the cyt b_^559 subcomplex, and of the CP47 core antenna with respect to the D1/D2 reaction centre. Within the D1/D2 proteins, there are clear differences between plants and cyanobacteria at the stromal ends of membrane-spanning helices, even though these proteins are highly homologous. Notwithstanding these differences in the protein scaffold, the distances between the critical photosynthetic pigment cofactors seem to be precisely conserved. The different protein arrangements in the two complexes may reflect an adaptation to the two very different antenna systems, membrane-extrinsic phycobilisomes for cyanobacteria, and membrane-embedded chlorophyll a/b proteins in plants.     

Active site geometry of glucose-1-phosphate uridylyltransferase

Glucose-1-phosphate uridylyltransferase, or UGPase, catalyzes the production of UDP-glucose from glucose-1-phosphate and UTP. Because of the biological role of UDP-glucose in glycogen synthesis and in the formation of glycolipids, glycoproteins, and proteoglycans, the enzyme is widespread in nature. Recently this laboratory reported the three-dimensional structure of UGPase from Escherichia coli. While the initial X-ray analysis revealed the overall fold of the enzyme, details concerning its active site geometry were limited because crystals of the protein complexed with either substrates or products could never be obtained. In an effort to more fully investigate the active site geometry of the enzyme, UGPase from Corynebacterium glutamicum was subsequently cloned and purified. Here we report the X-ray structure of UGPase crystallized in the presence of both magnesium and UDP-glucose. Residues involved in anchoring the ligand to the active site include the polypeptide chain backbone atoms of Ala 20, Gly 21, Gly 117, Gly 180, and Ala 214, and the side chains of Glu 36, Gln 112, Asp 143, Glu 201, and Lys 202. Two magnesium ions are observed coordinated to the UDP-glucose. An alpha- and a beta-phosphoryl oxygen, three waters, and the side chain of Asp 142 ligate the first magnesium, whereas the second ion is coordinated by an alpha-phosphoryl oxygen and five waters. The position of the first magnesium is conserved in both the glucose-1-phosphate thymidylyltransferases and the cytidylyltransferases. The structure presented here provides further support for the role of the conserved magnesium ion in the catalytic mechanisms of the sugar-1-phosphate nucleotidylyltransferases.     

Progress towards structural elucidation of Photosystem II

In recent years Photosystem II, and in particular the oxygen evolving component of the enzyme, have been the subject of intense biochemical and biophysical analysis. To date no high resolution structural model of the complex has been produced. As a consequence unambiguous interpretation of much experimental data has proven difficult, leading to a lack of consensus over many basic questions regarding the mechanisms involved, the oligomerization state of the enzyme in vivo and even the exact biochemical composition.This review is a summary of the progress towards the production of a structural model of PS II-derived from either X-ray crystallography or electron microscopy based techniques-and the current opinions, which have arisen from these structural analyses, on the structural topology and assemblage of the various subunits that constitute the complex.Abbreviations C₁₂-M dodecyl maltoside - CP chlorophyll protein - cyt b-559 cytochrome b-559 - DMPC dimyristoyl phosphatidyl choline - EC electron crystallography - EM electron microscopy - LHC II light harvesting complex II - OEC oxygen evolving complex - OG octyl--glucopyranoside - PS I Photosystem I - PS II Photosystem II - Tris N-tris (hydroxymethyl) amino ethane     

The role and structure of mitochondrial carriers

We have identified a novel CARD-containing protein from EST database. BinCARD (Bcl10-interacting protein with CARD). BinCARD was ubiquitously expressed. Co-immunoprecipitation, In vitro binding, mammalian two-hybrid, and immunostaining assays revealed that BinCARD interacted with Bcl10 through CARD. BinCARD potently suppressed NF-kappa B activation induced by Bcl10 and decreased the amounts of phosphorylated Bcl10. Mutations at the residue Leu17 or Leu65, which is highly conserved in CARD, abolished the inhibitory effects of BinCARD on both Bcl10-induced activation of NF-kappa B and phosphorylation of Bcl10. Further, expression of BinCARD inhibited Bcl10 phosphorylation induced by T cell activation signal. These results suggest that BinCARD interacts with Bcl10 to inhibit Bcl10-mediated activation of NF-kappa B and to suppress Bcl10 phosphorylation.     

Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.

Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAIV, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.     

Two conformational states of Candida rugosa lipase.

The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole do not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap.     

Characterization of two crystal forms of Clostridium thermocellum endoglucanase CelC

Endoglucanase CelC from Clostridium thermocellum expressed in Escherichia coli has been crystallized in two different crystal forms by the hanging drop method. Crystals of form I were grown with polyethylene glycol as a precipitant. They are orthorhombic, space group P2₁2₁2₁, with cell dimensions a =51.4 Å, b =84.3 Å, and c =87.5 Å. Crystals of form II, obtained in ammonium sulfate solutions, belong to the tetragonal space group P4₁2₁2 (or P4₃2₁2) with cell dimensions of a = b = 130.7 Å and c = 69.6 Å. Diffraction data to 2.8 Å resolution were observed for both crystal forms with a rotating anode generator. Preliminary oscillation images of the orthorhombic form I crystals using a synchrotron radiation source show diffraction to 2.2 Å resolution, indicating that these crystals are suitable for high resolution crystallographic analysis. © 1994 Wiley-Liss, Inc.     

Positions of His-64 and a bound water in human carbonic anhydrase II upon binding three structurally related inhibitors.

The 3-dimensional structure of human carbonic anhydrase II (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, 1a, 1b, and 1c, has been determined by X-ray crystallographic methods. The 3 inhibitors (1a = C8H12N2O4S3) vary only in the length of the substituent on the 4-amino group: 1a, proton; 1b, methyl; and 1c, ethyl. The binding constants (Ki's) for 1a, 1b, and 1c to HCAII are 1.52, 1.88, and 0.37 nM, respectively. These structures were solved to learn if any structural cause could be found for the difference in binding. In the complex with inhibitors 1a and 1b, electron density can be observed for His-64 and a bound water molecule in the native positions. When inhibitor 1c is bound, the side chain attached to the 4-amino group is positioned so that His-64 can only occupy the alternate position and the bound water is absent. While a variety of factors contribute to the observed binding constants, the major reason 1c binds tighter to HCAII than does 1a or 1b appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in binding and overcomes the small penalty for putting the His-64 side chain in a higher energy state.     

RIKEN structural genomics beamlines at the SPring-8; high throughput protein crystallography with automated beamline operation

RIKEN Structural Genomics Beamlines, BL26B1 & BL26B2 at the SPring-8, have been constructed for the structural genomics research. The main feature of the beamline is full automation of the successive data collections to maximize the beam-time efficiency. The beamline optics adopted a standard design commonly used for the SPring-8 bending magnet beamlines. Beamline instruments are operated by centralized control system through the computer network to achieve the automatic operation. The core part of the beamline development is a sample management system composed of sample changer robots for laboratory and beamline, and a networked sample database. BL26B1 has started user operation, and the automatic operation with the sample management system has been implemented at BL26B2.     

Crystal packing modifies ligand binding affinity: the case of aldose reductase

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.     

Damage detection by the UvrABC pathway: crystal structure of UvrB bound to fluorescein-adducted DNA

UvrB is the damage recognition element of the highly conserved UvrABC pathway that functions in the removal of bulky DNA adducts. Pivotal to this is the formation of a damage detection complex that relies on the ability of UvrB to locate and sequester diverse lesions. Whilst structures of UvrB bound to DNA have recently been reported, none address the issue of lesion recognition. Here, we describe the crystal structure of UvrB bound to a pentanucleotide containing a single fluorescein-adducted thymine that reveals a unique mechanism for damage detection entirely dependent on the exclusion of lesions larger than an undamaged nucleotide.     

Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus

Neuraminidases from different subtypes of influenza virus are characterized by the absence of serological cross-reactivity and an amino acid sequence homology of approximately 50%. The three-dimensional structure of the neuraminidase antigen of subtype N9 from an avian influenza virus (A/tern/Australia/G70c/75) has been determined by X-ray crystallography and shown to be folded similarly to neuraminidase of subtype N2 isolated from a human influenza virus. This result demonstrates that absence of immunological cross-reactivity is no measure of dissimilarity of polypeptide chain folding. Small differences in the way in which the subunits are organized around the molecular fourfold axis are observed. Insertions and deletions with respect to subtype N2 neuraminidase occur in four regions, only one of which is located within the major antigenic determinants around the enzyme active site.     

Use of novel selenomethionine-resistant yeast to produce selenomethionyl protein suitable for structural analysis

Yeast is widely used to determine the tertiary structure of eukaryotic proteins, because of its ability to undergo post-translational modifications such as glycosylation. A mutant lacking S -adenosylmethionine synthesis has been reported as a suitable host for producing selenomethionine derivatives, which can help solve phase problems in protein crystallography. However, the mutant required external addition of S -adenosylmethionine for cell proliferation. Here, a selenomethionine-resistant Pichia pastoris mutant that showed S -adenosylmethionine autotrophy was isolated. Human lysozyme expressed by the mutant under the control of constitutive promoter contained selenomethionine at 65% occupancy, sufficient for use as a selenomethionine derivative for single-wavelength anomalous dispersion phasing.     

Crystallographic study of inhibitors of tRNA-guanine transglycosylase suggests a new structure-based pharmacophore for virtual screening

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the pathogenicity of Shigellae. As the crystal structure of this protein is known, it is a putative target for the structure-based design of inhibitors. Here we report a crystallographic study of several new ligands exhibiting a 2,6-diamino-3H-quinazolin-4-one scaffold, which has been shown recently to be a promising template for TGT-inhibitors. Crystal structure analysis of these complexes has revealed an unexpected movement of the side-chain of Asp102. A detailed analysis of the water network disrupted by this rotation has lead to the derivation of a new composite pharmacophore. A virtual screening has been performed based on this pharmacophore hypothesis and several new inhibitors of micromolar binding affinity with new skeletons have been discovered.