Structural Investigation of Diclofenac Binding to Ovine, Caprine, and Leporine Serum Albumins.

Free drug concentration in the blood sera is crucial for its appropriate activity. Serum albumin, the universal blood carrier protein, is responsible for transporting drugs and releasing them into the bloodstream. Therefore, a drug's binding to SA is especially important for its bioavailability and it is a key problem in the drug design process. In this paper, we present crystal structures of three animal serum albumin complexes: ovine, caprine, and leporine, with diclofenac, a popular non-steroidal anti-inflammatory drug that is used in therapy of chronic and acute pain. Details of diclofenac binding mode by the presented serum albumins are compared with analogous complexes of human and equine serum albumins. The analysis of the occupied binding pockets in crystal structures of the investigated serum albumins from different mammals shows that they have two common and a number of unique diclofenac binding sites. The most intriguing is the fact that the albumins from the described species are able to bind different numbers of molecules of this popular anti-inflammatory drug, but none of the binding sites overlap with ones in the human serum albumin.

Structural investigations of stereoselective profen binding by equine and leporine serum albumins.

Serum albumin, the most abundant transport protein of mammalian blood, interacts with various nonsteroidal anti-inflammatory drugs (NSAIDs) affecting their disposition, metabolism, and excretion. A big group of chiral NSAIDs transported by albumin, profens, is created by derivatives of 2-arylpropionic acid. The chiral center in the structures of profens is adjacent to the carboxylate moiety and often determines different pharmacological properties of profen enantiomers. This study describes crystal structures of two albumins, isolated from equine and leporine serum, in complexes with three profens: ibuprofen, ketoprofen, and suprofen. Based on three-dimensional structures, the stereoselectivity of albumin is discussed and referred to the previously published albumin complexes with drugs. Drug Site 2 (DS2) of albumin, the bulky hydrophobic pocket of subdomain IIIA with a patch of polar residues, preferentially binds (S)-enantiomers of all investigated profens. Almost identical binding mode of all these drugs clearly indicates the stereoselectivity of DS2 towards (S)-profens in different albumin species. Also, the affinity studies show that DS2 is the major site that presents high affinity towards investigated drugs. Additionally, crystallographic data reveal the secondary binding sites of ketoprofen in leporine serum albumin and ibuprofen in equine serum albumin, both overlapping with previously identified naproxen binding sites: the cleft formed between subdomains IIIA and IIIB close to the fatty acid binding site 5 and the niche created between subdomains IIA and IIIA, called fatty acid site 6.

Mapping the Transglycosylation Relevant Sites of Cold-Adapted ß-d-Galactosidase from Arthrobacter sp. 32cB.

ß-Galactosidase from Arthrobacter sp. 32cB (ArthßDG) is a cold-adapted enzyme able to catalyze hydrolysis of ß-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthßDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme's surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme's active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthßDG or other analogous galactosidases belonging to GH2 family.

Active Site Architecture and Reaction Mechanism Determination of Cold Adapted ß-d-galactosidase from Arthrobacter sp. 32cB.

ArthßDG is a dimeric, cold-adapted ß-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthßDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthßDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthßDG_E441Q/LACs and ArthßDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthßDG_E441Q/LACd, ArthßDG/ONPG (ligands bound in deep mode), and galactose ArthßDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 ßDGs, in ArthßDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.

Crystal structure and enzymatic properties of a broad substrate-specificity psychrophilic aminotransferase from the Antarctic soil bacterium Psychrobacter sp. B6.

Aminotransferases (ATs) are enzymes that are commonly used in the chemical and pharmaceutical industries for the synthesis of natural and non-natural amino acids by transamination reactions. Currently, the easily accessible enzymes from mesophilic organisms are most commonly used; however, for economical and ecological reasons the utilization of aminotransferases from psychrophiles would be more advantageous, as their optimum reaction temperature is usually significantly lower than for the mesophilic ATs. Here, gene isolation, protein expression, purification, enzymatic properties and structural studies are reported for the cold-active aromatic amino-acid aminotransferase (PsyArAT) from Psychrobacter sp. B6, a psychrotrophic, Gram-negative strain from Antarctic soil. Preliminary computational analysis indicated dual functionality of the enzyme through the ability to utilize both aromatic amino acids and aspartate as substrates. This postulation was confirmed by enzymatic activity tests, which showed that it belonged to the class EC 2.6.1.57. The first crystal structures of a psychrophilic aromatic amino-acid aminotransferase have been determined at resolutions of 2.19 Šfor the native enzyme (PsyArAT) and 2.76 Šfor its complex with aspartic acid (PsyArAT/D). Both types of crystals grew in the monoclinic space group P21 under slightly different crystallization conditions. The PsyArAT crystals contained a dimer (90 kDa) in the asymmetric unit, which corresponds to the active form of this enzyme, whereas the crystals of the PsyArAT/D complex included four dimers showing different stages of the transamination reaction.

Structural studies of bovine, equine, and leporine serum albumin complexes with naproxen.

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein-ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA-NPS), equine (ESA-NPS), and leporine (LSA-NPS) determined to 2.58 Å (C2), 2.42 Å (P61), and 2.73 Å (P212121) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA-NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA-NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein.

Crystallographic identification of an unexpected protein complex in silkworm haemolymph.

The first crystal structure of a complex formed by two storage proteins, SP2 and SP3, isolated from their natural source, mulberry silkworm (Bombyx mori L.) haemolymph, has been determined. The structure was solved by molecular replacement using arylphorin, a protein rich in aromatic amino-acid residues, from oak silkworm as the initial model. The quality of the electron-density maps obtained from the X-ray diffraction experiment allowed the authors to detect that the investigated crystal structure was composed of two different arylphorins: SP2 and SP3. This discovery was confirmed by N-terminal sequencing. SP2 has been extensively studied previously, whereas only a few reports on SP3 are available. However, to date no structural studies have been reported for these proteins. These studies revealed that SP2 and SP3 exist in the silkworm body as a heterohexamer formed by one SP2 trimer and one SP3 trimer. The overall fold, consisting of three haemocyanin-like subdomains, of SP2 and SP3 is similar. Both proteins contain a conserved N-glycosylation motif in their structures.

Structures of bovine, equine and leporine serum albumin.

Serum albumin first appeared in early vertebrates and is present in the plasma of all mammals. Its canonical structure supported by a conserved set of disulfide bridges is maintained in all mammalian serum albumins and any changes in sequence are highly correlated with evolution of the species. Previous structural investigations of mammalian serum albumins have only concentrated on human serum albumin (HSA), most likely as a consequence of crystallization and diffraction difficulties. Here, the crystal structures of serum albumins isolated from bovine, equine and leporine blood plasma are reported. The structure of bovine serum albumin (BSA) was determined at 2.47 Å resolution, two crystal structures of equine serum albumin (ESA) were determined at resolutions of 2.32 and 2.04 Å, and that of leporine serum albumin (LSA) was determined at 2.27 Å resolution. These structures were compared in detail with the structure of HSA. The ligand-binding pockets in BSA, ESA and LSA revealed different amino-acid compositions and conformations in comparison to HSA in some cases; however, much more significant differences were observed on the surface of the molecules. BSA, which is one of the most extensively utilized proteins in laboratory practice and is used as an HSA substitute in many experiments, exhibits only 75.8% identity compared with HSA. The higher resolution crystal structure of ESA highlights the binding properties of this protein because it includes several bound compounds from the crystallization solution that provide additional structural information about potential ligand-binding pockets.

High-resolution structure of Bombyx mori lipoprotein 7: crystallographic determination of the identity of the protein and its potential role in detoxification.

Three crystal structures of a lipoprotein (Bmlp7) of unknown function, a member of the 30 kDa lipoprotein family from mulberry silkworm (Bombyx mori L.) haemolymph, have been determined. The 1.33 Å resolution structure is an excellent example of how a precise crystallographic study can contribute to protein identification. The correct sequence of this haemolymph-isolated protein was assigned thanks to superb-quality electron-density maps. Two unexpected cadmium cations were found in this crystal structure [Bmlp7-I(Cd)] and their presence may be connected to a detoxification mechanism in this insect. For a comparison of the metal-binding sites, the crystal structure of a platinum complex (Bmlp7-Pt) was also solved at 1.94 Å resolution. The third (2.50 Å resolution) structure, of the native protein harvested in a different season (Bmlp7-II), corresponds to a different polymorph with an altered pattern of intermolecular interactions and with a total absence of cadmium ions and highlights the possible involvement of Bmlp7 in the response to environmental pollution. The N-terminal domain of Bmlp7 has a fold resembling a clockwise spiral created by six helices and can be classified as a VHS domain. The C-terminal domain is folded as a ß-trefoil. The biological function of Bmlp7 is unknown, but its structural homology to sugar-binding proteins suggests that, in analogy to other 30 kDa haemolymph lipoproteins, it could play a role as an anti-apoptotic factor or function in the immune response of the insect to fungal infections.

Isolation, purification, crystallization and preliminary X-ray studies of two 30†kDa proteins from silkworm haemolymph.

Juvenile hormone-binding protein (JHBP) and the low-molecular-mass lipoprotein PBMHP-12 belong to a group of 30 kDa proteins that comprise the major protein component of the haemolymph specific to the fifth-instar larvae stage of the mulberry silkworm Bombyx mori L. Proteins from this group are often essential for the development of the insect. In a project aimed at crystallographic characterization of B. mori JHBP (BmJHBP), it was copurified together with PBMHP-12. Eventually, the two proteins were isolated and crystallized separately. The BmJHBP crystals were orthorhombic (space group C222(1)) and the PBMHP-12 crystals were triclinic. The crystals diffracted X-rays to 2.9 Š(BmJHBP) and 1.3 Š(PBMHP-12) resolution.

Study of host-guest interactions in benzodiazacoronands by means of solid state NMR spectroscopy, X-ray diffraction and quantum mechanical computations.

In this work we present solid state data for five host-guest complexes formed by N-(4,19-dioxo-2,8,15,21-tetraoxa-5,18-diazatricyclohexacosa-1(25),9(14),10,12,22(26),23-hexaen-26-yl)-benzamide (1) belonging to the group of benzodiazacoronands, achiral compounds for which chiral crystals were found (J. Kalisiak and J. Jurczak, Cryst. Growth Des., 2006, 6, 20). The X-ray structure was resolved for four of them. It was found that 1 crystallizes in P2(1)/c, P1 and P2(1)/n achiral space groups. Differentiation of molecular packing and the presence of guest molecules within the crystal lattice were analyzed with solid state NMR. An attempt was made to correlate changes in (13)C δ(ii) and (15)N δ(ii) chemical shift tensor values, obtained from analysis of spinning sidebands of 1D and 2D (2D PASS) NMR spectra, with changes in the strength of hydrogen bonding. Quantum mechanical DFT GIAO calculations of NMR shielding parameters carried out on structures with coordinates taken from XRD were employed for signals assignment and verification of structural constraints.

Structural Evidence of Active Site Adaptability towards Different Sized Substrates of Aromatic Amino Acid Aminotransferase from Psychrobacter Sp. B6.

Aromatic amino acid aminotransferases present a special potential in the production of drugs and synthons, thanks to their ability to accommodate a wider range of substrates in their active site, in contrast to aliphatic amino acid aminotransferases. The mechanism of active site adjustment toward substrates of psychrophilic aromatic amino acid aminotransferase (PsyArAT) from Psychrobacter sp. B6 is discussed based on crystal structures of complexes with four hydroxy-analogs of substrates: phenylalanine, tyrosine, tryptophan and aspartic acid. These competitive inhibitors are bound in the active center of PsyArAT but do not undergo transamination reaction, which makes them an outstanding tool for examination of the enzyme catalytic center. The use of hydroxy-acids enabled insight into substrate binding by native PsyArAT, without mutating the catalytic lysine and modifying cofactor interactions. Thus, the binding mode of substrates and the resulting analysis of the volume of the catalytic site is close to a native condition. Observation of these inhibitors' binding allows for explanation of the enzyme's adaptability to process various sizes of substrates and to gain knowledge about its potential biotechnological application. Depending on the character and size of the used inhibitors, the enzyme crystallized in different space groups and showed conformational changes of the active site upon ligand binding.

Cryoprotection properties of salts of organic acids: a case study for a tetragonal crystal of HEW lysozyme.

Currently, the great majority of the data that are used for solving macromolecular structures by X-ray crystallography are collected at cryogenic temperatures. Selection of a suitable cryoprotectant, which ensures crystal stability at low temperatures, is critical for the success of a particular diffraction experiment. The effectiveness of salts of organic acids as potential cryoprotective agents is presented in the following work. Sodium formate, acetate, malonate and citrate were tested, as were sodium potassium tartrate and acetate in the form of potassium and ammonium salts. For each salt investigated, the minimal concentration that was required for successful cryoprotection was determined over the pH range 4.5-9.5. The cryoprotective ability of these organic salts depends upon the number of carboxylic groups; the lowest concentration required for cryoprotection was observed at neutral pH. Case-study experiments conducted using the tetragonal form of hen egg-white lysozyme (HEWL) confirmed that salts of organic acids can successfully act as cryoprotective agents of protein crystals grown from high concentrations of inorganic salts. When crystals are grown from solutions containing a sufficient concentration of organic acid salts no additional cryoprotection is needed as the crystals can safely be frozen directly from the crystallizing buffers.

H-type lectins - Structural characteristics and their applications in diagnostics, analytics and drug delivery.

Lectins are ubiquitous carbohydrate-binding proteins that interact with sugar moieties in a highly specific manner. H-type lectins represent a new group of lectins that were identified in invertebrates. These lectins share structural homology and bind mainly to N-acetylgalactosamine (GalNAc). Recent structural studies on the H-type lectins provided a detailed description of the GalNAc-lectin interaction that is already exploited in a number of biomedical applications. Two members of the H-type lectin family, Helix pomatia agglutinin (HPA) and Helix aspersa agglutinin (HAA), have already been extensively used in many diagnostic tests due their ability to specifically recognize GalNAc. This ability is especially important because aberrant glycosylation patterns of proteins expressed by cancer cells contain GalNAc. In addition, H-type lectins were utilized in diagnostics of other non-cancer diseases and represent great potential as components of drug delivery systems. Here, we present an overview of the H-type lectins and their applications in diagnostics, analytics and drug delivery.

Modeling of isotope effects on binding oxamate to lactic dehydrogenase.

A new crystal structure of the rabbit muscle L-lactic dehydrogenase (PDB code 3H3F) has been determined. The independent unit of this structure contains two tetramers, each of them with a unique constitution of two active sites with the open loop conformation and two with the loops closed over the actives sites. On the basis of this structure, interactions of an inhibitor, oxamate anion, with the protein have been modeled using different hybrid schemes that involved B3LYP/6-31++G(d,p) DFT theory level in the QM layer. In ONIOM calculations, either Amber (QM:MM) or one of the three semiempirical parametrizations, AM1, PM3, and RM1 (QM:QM) was used, while in the traditional QM/MM scheme, the OPLS-AA force field was used for the outer layer. Normal modes of vibrations of oxamate in aqueous solution and in the active site of the enzyme were used to calculate binding isotope effects. On the basis of the comparison of the values obtained theoretically with those experimentally determined for the oxygen atoms of the carboxylic group of oxamate it was concluded that the DFT/OPLS-AA scheme, applied to the dimer consisting of two chains, one with the open loop and the other with the closed loop conformation, provides the best description of the active site. Calculations of the binding isotope effects of the other atoms of oxamate suggest that nitrogen isotope effect may be useful for the experimental differentiation between open and closed loop conformations.

Structural features of cold-adapted dimeric GH2 ß-D-galactosidase from Arthrobacter sp. 32cB.

Crystal structures of cold-adapted ß-d-galactosidase (EC 3.2.1.23) from the Antarctic bacterium Arthrobacter sp. 32cB (ArthßDG) have been determined in an unliganded form resulting from diffraction experiments conducted at 100 K (at resolution 1.8 Å) and at room temperature (at resolution 3.0 Å). A detailed comparison of those two structures of the same enzyme was performed in order to estimate differences in their molecular flexibility and rigidity and to study structural rationalization for the cold-adaptation of the investigated enzyme. Furthermore, a comparative analysis with structures of homologous enzymes from psychrophilic, mesophilic, and thermophilic sources has been discussed to elucidate the relationship between structure and cold-adaptation in a wider context. The performed studies confirm that the structure of cold-adapted ArthßDG maintains balance between molecular stability and structural flexibility, which can be observed independently on the temperature of conducted X-ray diffraction experiments. Obtained information about proper protein function under given conditions provide a guideline for rational engineering of proteins in terms of their temperature optimum and thermal stability.

Structural studies of two thermostable laccases from the white-rot fungus Pycnoporus sanguineus.

Laccases are enzymes that have the ability to catalyze the oxidation of a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water. The active site of those proteins contains four copper ions, classified into three types. Laccases are interesting enzymes for study from the point of view of their structure, function and application because of their role in lignin degradation. Structural studies of two thermostable laccases produced by the strain Pycnoporus sanguineus CS43 (PsLacI and PsLacII) were performed. Both isoforms of PsLac show high thermal stability, at 60°C and 50°C, respectively, and they remained active at a high concentration of organic solvents. However, PsLacI has a higher thermal and pH stability and tolerance against inhibitors, and is a more efficient catalyst for ABTS and DMP (laccases substrate) than PsLacII. Based on the determined crystal structures we achieved insights into the structural factors relevant for the enzymatic properties of PsLacI and PsLacII. N-glycosylation site Asn354, which is very often present in structures of fungal laccases from other species, was not present in PsLac. This observation may be of particular significance due to the close distance between Asn354 and the substrate-binding pocket. This results in better access to the hydrophobic cavity for a particular substrate. Furthermore, we identified significant differences in the region of substrate-binding pocket, which confer PsLacI a markedly better performance than PsLacII.

Crystal structures of serum albumins from domesticated ruminants and their complexes with 3,5-diiodosalicylic acid.

Serum albumin (SA) is the most abundant protein in plasma and is the main transporter of molecules in the circulatory system of all vertebrates, with applications in medicine, the pharmaceutical industry and molecular biology. It is known that albumins from different organisms vary in sequence; thus, it is important to know the impact of the amino-acid sequence on the three-dimensional structure and ligand-binding properties. Here, crystal structures of ovine (OSA) and caprine (CSA) serum albumins, isolated from sheep and goat blood, are described, as well those of their complexes with 3,5-diiodosalicylic acid (DIS): OSA-DIS (2.20 Šresolution) and CSA-DIS (1.78 Šresolution). The ligand-free OSA structure was determined in the trigonal space group P3221 at 2.30 Šresolution, while that of CSA in the orthorhombic space group P212121 was determined at 1.94 Šresolution. Both albumins are also capable of crystallizing in the triclinic space group P1, giving isostructural crystals that diffract to around 2.5 Šresolution. A comparison of OSA and CSA with the closely related bovine serum albumin (BSA) shows both similarities and differences in the distribution of DIS binding sites. The investigated serum albumins from domesticated ruminants in their complexes with DIS are also compared with the analogous structures of equine and human serum albumins (ESA-DIS and HSA-DIS). Surprisingly, despite 98% sequence similarity, OSA binds only two molecules of DIS, whereas CSA binds six molecules of this ligand. Moreover, the binding of DIS to OSA and CSA introduced changes in the overall architecture of the proteins, causing not only different conformations of the amino-acid side chains in the binding pockets, but also a significant shift of the whole helices, changing the volume of the binding cavities.

Structure and dynamics of L-selenomethionine in the solid state.

L-selenomethionine 1 crystallizes in P2(1) space group with two molecules in the asymmetric unit. Solid-state NMR spectroscopy is used for searching of structure and dynamics of 1 in the crystal lattice. The distinct molecular motion of side chains for A and B molecules of 1 is apparent from measurements of relaxation parameters (1H 1rho, 13C T1) and analysis of CSA data (2D-PASS experiment). The 13C delta(ii) and 77Se delta(ii) parameters are correlated with theoretical shielding parameters obtained by means DFT GIAO calculations. Attempt to explain the mechanism of phase transition of crystals of 1 at 313K is presented.

Study of molecular dynamics and the solid state phase transition mechanism for unsymmetrical thiopyrophosphate using X-ray diffraction, DFT calculations and NMR spectroscopy.

Differential scanning calorimetry (DSC) and low-temperature X-ray diffraction studies showed that 2-thio-(5,5-dimethyl-1,3,2-dioxaphosphorinanyl)2'-oxo-dineopentyl-thiophosphate (compound 1) undergoes reversible phase transition at 203 K related to the change of symmetry of the crystallographic unit. Solid state NMR spectroscopy was used to establish the dynamic processes of aliphatic groups and the phosphorus skeleton. 13C and 31P variable temperature NMR studies as well as T1 and T1rho measurements of relaxation times revealed the different mode of molecular motion for each neopentyl residue directly bonded to phosphorus. It is concluded that molecular dynamics of aliphatic groups causes different van der Waals interactions in the crystal lattice and is the driving force of phase transition for compound 1. Finally, we showed that very sharp phase transition temperature makes compound 1 an excellent candidate as a low-temperature NMR thermometer in the solid phase.