Publications by authors named "Grzegorz Bujacz"

60 Publications

Synergy of Solid-State NMR, Single-Crystal X-ray Diffraction, and Crystal Structure Prediction Methods: A Case Study of Teriflunomide (TFM).

Cryst Growth Des 2021 Jun 10;21(6):3328-3343. Epub 2021 May 10.

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.

In this work, for the first time, we present the X-ray diffraction crystal structure and spectral properties of a new, room-temperature polymorph of teriflunomide (TFM), CSD code 1969989. As revealed by DSC, the low-temperature TFM polymorph recently reported by Gunnam et al. undergoes a reversible thermal transition at -40 °C. This reversible process is related to a change in value, from 2 to 1, as observed by variable-temperature H-C cross-polarization (CP) magic-angle spinning (MAS) solid-state NMR, while the crystallographic system is preserved (triclinic). Two-dimensional C-H and H-H double-quantum MAS NMR spectra are consistent with the new room-temperature structure, including comparison with GIPAW (gauge-including projector augmented waves) calculated NMR chemical shifts. A crystal structure prediction procedure found both experimental teriflunomide polymorphs in the energetic global minimum region. Differences between the polymorphs are seen for the torsional angle describing the orientation of the phenyl ring relative to the planarity of the TFM molecule. In the low-temperature structure, there are two torsion angles of 4.5 and 31.9° for the two = 2 molecules, while in the room-temperature structure, there is disorder that is modeled with ∼50% occupancy between torsion angles of -7.8 and 28.6°. These observations are consistent with a broad energy minimum as revealed by DFT calculations. PISEMA solid-state NMR experiments show a reduction in the C-H dipolar coupling in comparison to the static limit for the aromatic CH moieties of 75% and 51% at 20 and 40 °C, respectively, that is indicative of ring flips at the higher temperature. Our study shows the power of combining experiments, namely DSC, X-ray diffraction, and MAS NMR, with DFT calculations and CSP to probe and understand the solid-state landscape, and in particular the role of dynamics, for pharmaceutical molecules.
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http://dx.doi.org/10.1021/acs.cgd.1c00123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8273857PMC
June 2021

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

Materials (Basel) 2021 Jun 17;14(12). Epub 2021 Jun 17.

Institute of Molecular and Industrial Biotechnology, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland.

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 (ArAT) from 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 ArAT 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 ArAT, 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.
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http://dx.doi.org/10.3390/ma14123351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8235216PMC
June 2021

Structural variety of heterosynthons in linezolid cocrystals with modified thermal properties.

Acta Crystallogr B Struct Sci Cryst Eng Mater 2020 Oct 17;76(Pt 5):892-912. Epub 2020 Sep 17.

Centre of Molecular and Macromolecular Studies of Polish Academy of Sciences, Sienkiewicza 112, Lodz, 90-363, Poland.

In a search for new crystalline forms of linezolid with modified thermal properties five cocrystals of this wide range antibiotic with aromatic acids were obtained via mechanochemical grinding and analyzed with single crystal X-ray diffraction, solid-state NMR spectroscopy, powder X-ray diffraction and DSC measurements. The coformers used in this study were benzoic acid, p-hydroxybenzoic acid, protocatechuic acid, γ-resorcylic acid and gallic acid. In each of the cocrystals distinct structural features have been found, including a variable amount of water and different heterosynthons, indicating that there is more than one type of intermolecular interaction preferred by the linezolid molecule. Basing on the frequency of the observed supramolecular synthons, the proposed hierarchy of the hydrogen-bond acceptor sites of linezolid (LIN) is C=O > C=O > C-O-C > C-N-C > C-O-C. In addition, aromatic-aromatic interactions were found to be important in the stabilization of the analyzed structures. The obtained cocrystals show modified thermal properties, with four of them having melting points lower than the temperature of the phase transition from linezolid form II to linezolid form III. Such a change in this physicochemical property allows for the future application of melting-based techniques of introducing linezolid into drug delivery systems. In addition a change in water solubility of linezolid upon cocrystalization was evaluated, but only in the case of the cocrystal with protocatechuic acid was there a significant (43%) improvement in solubility in comparison with linezolid.
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http://dx.doi.org/10.1107/S2052520620010896DOI Listing
October 2020

Understanding the formation of apremilast cocrystals.

Acta Crystallogr B Struct Sci Cryst Eng Mater 2019 Oct 7;75(Pt 5):803-814. Epub 2019 Sep 7.

Centre of Molecular and Macromolecular Studies PAS, Sienkiewicza 112, Lodz, 90363, Poland.

Apremilast (APR), an anti-psoriatic agent, easily forms isostructural cocrystals and solvates with aromatic entities, often disobeying at the same time Kitaigorodsky's rule as to the saturation of possible hydrogen-bonding sites. In this paper the reasons for this peculiar behavior are investigated, employing a joint experimental and theoretical approach. This includes the design of cocrystals with coformers having a high propensity towards the formation of both aromatic-aromatic and hydrogen-bonding interactions, determination of their structure, using solid-state NMR spectroscopy and X-ray crystallography, as well as calculations of stabilization energies of formation of the obtained cocrystals, followed by crystal structure prediction calculations and solubility measurements. The findings indicate that the stabilization energies of cocrystal formation are positive in all cases, which results from strain in the APR conformation in these crystal forms. On the other hand, solubility measurements show that the Gibbs free energy of formation of the apremilast:picolinamide cocrystal is negative, suggesting that the formation of the studied cocrystals is entropy driven. This entropic stabilization is associated with the disorder observed in almost all known cocrystals and solvates of APR.
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http://dx.doi.org/10.1107/S205252061900917XDOI Listing
October 2019

Application of 1-Hydroxy-4,5-Dimethyl-Imidazole 3-Oxide as Coformer in Formation of Pharmaceutical Cocrystals.

Pharmaceutics 2020 Apr 15;12(4). Epub 2020 Apr 15.

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.

Two, well defined binary crystals with 1-Hydroxy-4,5-Dimethyl-Imidazole 3-Oxide (HIMO) as coformer and thiobarbituric acid (TBA) as well barbituric acid (BA) as Active Pharmaceutical Ingredients (APIs) were obtained by cocrystallization (from methanol) or mechanochemically by grinding. The progress of cocrystal formation in a ball mill was monitored by means of high-resolution, solid state NMR spectroscopy. The C CP/MAS, N CP/MAS and H Very Fast (VF) MAS NMR procedures were employed to inspect the tautomeric forms of the APIs, structure elucidation of the coformer and the obtained cocrystals. Single crystal X-ray studies allowed us to define the molecular structure and crystal packing for the coformer as well as the TBA/HIMO and BA/HIMO cocrystals. The intermolecular hydrogen bonding, π-π interactions and CH-π contacts responsible for higher order organization of supramolecular structures were determined. Biological studies of HIMO and the obtained cocrystals suggest that these complexes are not cytotoxic and can potentially be considered as therapeutic materials.
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http://dx.doi.org/10.3390/pharmaceutics12040359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238160PMC
April 2020

C5-Substituted 2-Selenouridines Ensure Efficient Base Pairing with Guanosine; Consequences for Reading the NNG-3' Synonymous mRNA Codons.

Int J Mol Sci 2020 Apr 20;21(8). Epub 2020 Apr 20.

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz; Poland.

5-Substituted 2-selenouridines (R5Se2U) are post-transcriptional modifications present in the first anticodon position of transfer RNA. Their functional role in the regulation of gene expression is elusive. Here, we present efficient syntheses of 5-methylaminomethyl-2-selenouridine (, mnm5Se2U), 5-carboxymethylaminomethyl-2-selenouridine (, cmnm5Se2U), and Se2U () alongside the crystal structure of the latter nucleoside. By using pH-dependent potentiometric titration, pa values for the N3H groups of - were assessed to be significantly lower compared to their 2-thio- and 2-oxo-congeners. At physiological conditions (pH 7.4), Se2-uridines and preferentially adopted the zwitterionic form (, ca. 90%), with the positive charge located at the amino alkyl side chain and the negative charge at the Se2-N3-O4 edge. As shown by density functional theory (DFT) calculations, this form efficiently bound to guanine, forming the so-called "new wobble base pair", which was accepted by the ribosome architecture. These data suggest that the tRNA anticodons with wobble R5Se2Us may preferentially read the 5'-NNG-3' synonymous codons, unlike their 2-thio- and 2-oxo-precursors, which preferentially read the 5'-NNA-3' codons. Thus, the interplay between the levels of U-, S2U- and Se2U-tRNA may have a dominant role in the epitranscriptomic regulation of gene expression via reading of the synonymous 3'-A- and 3'-G-ending codons.
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http://dx.doi.org/10.3390/ijms21082882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216251PMC
April 2020

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

Biochim Biophys Acta Proteins Proteom 2019 09 10;1867(9):776-786. Epub 2019 Jun 10.

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland.

Crystal structures of cold-adapted β-d-galactosidase (EC 3.2.1.23) from the Antarctic bacterium Arthrobacter sp. 32cB (ArthβDG) 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 ArthβDG 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.
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http://dx.doi.org/10.1016/j.bbapap.2019.06.001DOI Listing
September 2019

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

Int J Biol Macromol 2018 Feb 18;107(Pt B):1629-1640. Epub 2017 Oct 18.

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 90-924 Lodz, Stefanowskiego 4/10, Poland; Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ijbiomac.2017.10.024DOI Listing
February 2018

Approach toward the Understanding of Coupling Mechanism for EDC Reagent in Solvent-Free Mechanosynthesis.

Org Lett 2017 10 22;19(19):5360-5363. Epub 2017 Sep 22.

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences , Sienkiewicza 112, 90-363 Lodz, Poland.

A unique approach in mechanosynthesis, joining solid-state NMR spectroscopy, X-ray crystallography, and theoretical calculations, is employed for the first time to study the mechanism of the formation of the C-N amide bond using EDC·HCl as a coupling reagent. It has been proved that EDC·HCl, which in the crystal lattice exists exclusively in the cyclic form (X-ray data), easily undergoes transformation to a pseudocyclic stable intermediate in reaction with carboxylic acid forming a low-melt phase (differential scanning calorimetry, solid-state NMR). The obtained intermediate is reactive and can be further used for synthesis of amides in reaction with appropriate amines.
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http://dx.doi.org/10.1021/acs.orglett.7b02637DOI Listing
October 2017

Ferrocene-Biotin Conjugates: Synthesis, Structure, Cytotoxic Activity and Interaction with Avidin.

Chempluschem 2016 Nov 19;81(11):1191-1201. Epub 2016 Aug 19.

Department of Organic Chemistry, Faculty of Chemistry, University of Łódź, Tamka 12, 41-403, Łódź, Poland.

Friedel-Crafts acylation of ferrocene with d-biotin, d-homobiotin and d-desthiobiotin gave ferrocenyl ketones. These compounds were diastereoselectively reduced to the corresponding alcohols using (R)- and (S)-Me-CBS-oxazaborolidine-borane complexes as reducing agents. The alcohols were further transformed into azido and finally to amino derivatives with retention of configuration, as confirmed by X-ray crystallography. Ferrocenylbiotin alcohols smoothly underwent dehydration to (E)-alkenes as the major isomers by heating in diluted acetic acid. The synthesized compounds retained high affinity for avidin. They also exhibited high cytotoxicity toward cancer cells expressing various levels of sodium-dependent multivitamin transporter (SMVT) in the absence of biotin in the medium, whereas the presence of free biotin decreased their antiproliferative activity. This revealed that these biotin-ferrocene conjugates might be used as biologically active agents against cancer cells, although there was no clear relationship between their cytotoxicity and cellular SMVT level.
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http://dx.doi.org/10.1002/cplu.201600320DOI Listing
November 2016

Structural Characterization of the Avidin Interactions with Fluorescent Pyrene-Conjugates: 1-Biotinylpyrene and 1-Desthiobiotinylpyrene.

Molecules 2016 Sep 27;21(10). Epub 2016 Sep 27.

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 90-924 Łódź, Stefanowskiego 4/10, Poland.

Avidin is a tetrameric protein that belongs to the calycin superfamily. It has been studied mainly because of its extraordinary affinity to biotin, which led to a wide range of applications based on the avidin-biotin system. In the present study, we report the first crystal structures of avidin in a complex with two novel fluorescent pyrene derivatives: 1-biotinylpyrene (B9P) and 1-desthiobiotinylpyrene (D9P). The crystal structures were solved by molecular replacement using the coordinates of avidin molecule as a starting model and the final models of avidin/B9P and avidin/D9P were refined to resolutions of 2.0 Å and 2.1 Å, respectively. Our data reveal changes in loop conformation as well as in overall fold and quaternary arrangement of the avidin upon the binding of these fluorescent probes. Moreover, the crystal structures allowed analysis of the details of the interactions between the protein and the pyrene derivatives. Structural description of the complexes will contribute to the design of conjugates for expanding the capabilities of avidin-biotin technology.
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http://dx.doi.org/10.3390/molecules21101270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274289PMC
September 2016

Crystal and molecular structure of hexagonal form of lipase B from Candida antarctica.

Acta Biochim Pol 2016 30;63(1):103-109. Epub 2015 Dec 30.

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Heteroorganic Chemistry, Łódź, Poland.

During crystallization screenings of commercially available hydrolytic enzymes, the new, hexagonal crystal form of CAL-B, has been discovered and hereby reported. The NAG molecules, which were closing the glycosylation site in the orthorhombic form, in hexagonal structure make the glycosylation site open. It is unknown whether the opening and closing of the glycosylation site by the 'lid' NAG molecules, could be related to the opening and closing of the active center of the enzyme upon substrate binding and product release.
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http://dx.doi.org/10.18388/abp.2015_1065DOI Listing
December 2016

Crystallographic and CD probing of ligand-induced conformational changes in a plant PR-10 protein.

J Struct Biol 2016 Jan 28;193(1):55-66. Epub 2015 Nov 28.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland. Electronic address:

Plant pathogenesis-related class 10 (PR-10) proteins are a family of abundant proteins initially identified as elements of the plant defense system. The key structural feature suggesting PR-10 functionality is a huge hydrophobic cavity created in the protein interior by a scaffold composed of an extended β-sheet wrapped around a long and flexible C-terminal α-helix. Several crystallographic and NMR studies have shown that the cavity can accommodate a variety of small molecule ligands, including phytohormones. The article describes ∼1.3 Å resolution crystal structures of a Lupinus luteus PR-10 isoform LlPR-10.1A, in its free form and in complex with trans-zeatin, a naturally occurring plant hormone belonging to the cytokinin group. Moreover we present the structure of the same protein where the saturation with zeatin is not complete. This set of three crystal structures allows us to track the structural adaptation of the protein upon trans-zeatin docking, as well as the sequence of the ligand-binding events, step-by-step. In addition, titration of LlPR-10.1A with trans-zeatin monitored in solution by CD spectra, confirmed the pattern of structural adaptations deduced from the crystallographic studies. The ligand-biding mode shows no similarity to other zeatin complexes of PR-10 proteins. The present work, which describes the first atomic models of the same PR-10 protein with and without a physiological ligand, reveals that the conformation of LlPR-10.1A undergoes a significant structural rearrangement upon trans-zeatin binding.
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http://dx.doi.org/10.1016/j.jsb.2015.11.008DOI Listing
January 2016

Thermal stability and conformation of antiparallel duplexes formed by P-stereodefined phosphorothioate DNA/LNA chimeric oligomers with DNA and RNA matrices.

Org Biomol Chem 2015 Oct;13(39):10032-40

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363 Łódź, Poland.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of LNA-type nucleosides (LNA-OTPs, 2a-d; B' = Thy, Ade(Bz), Cyt(Bz), Gua(dmf), respectively) were synthesized and separated into pure P-diastereomers. X-ray analysis allowed for assignment of the absolute configuration of the phosphorus atom in the detritylated, fast-eluting diastereomer 2a. The diastereomerically pure LNA-OTP monomers were used in solid phase synthesis of P-stereodefined chimeric PS-(DNA/LNA) 11-mers containing 2-3 LNA units. Formally, among the phosphorothioate oligomers the biggest enhancement in thermal stability of Watson-Crick paired duplexes was found for [SP-PS]-(DNA/LNA)/RNA duplexes (on average 8.2 °C per LNA nucleotide), followed by [RP-PS]-(DNA/LNA)/RNA (6.3 °C), [RP-PS]-(DNA/LNA)/DNA (3.8 °C) and [SP-PS]-(DNA/LNA)/DNA (2.4 °C per LNA nucleotide). However, detailed analysis of the thermal dissociation data showed that the thermal stability of (PS-LNA)-containing duplexes does not depend on the spatial orientation of the sulfur atoms. This conclusion received support from CD measurements.
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http://dx.doi.org/10.1039/c5ob01474cDOI Listing
October 2015

Synthesis and the absolute configuration of both enantiomers of 4,5-dihydroxy-3-(formyl)cyclopent-2-enone acetonide as a new chiral building block for prostanoid synthesis.

Org Biomol Chem 2015 Jan;13(3):807-16

Department of Heteroorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza Str. 112, 90-363 Łódź, Poland.

The synthesis of both enantiomers of 4,5-dihydroxy-3-(formyl)cyclopent-2-enone acetonide (5) was accomplished in five steps starting from meso-tartaric acid (6). The key steps involved are preparation of the isopropylidene protected 3-[(dimethoxyphosphoryl)methyl]-4,5-dihydroxycyclopent-2-enone (9), resolution of the diastereoisomeric products 10 of the Horner reaction of racemic 9 with (R)-glyceraldehyde acetonide and the final regioselective ozonolysis of the exocyclic carbon–carbon double bond of the separated dienones 10 leading to both enantiomeric title compounds 5. The absolute configuration of both enantiomers was initially assigned based on the comparison of the chiroptical properties obtained from the DFT calculations with the experimental data and finally confirmed by X-ray analysis.
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http://dx.doi.org/10.1039/c4ob01535eDOI Listing
January 2015

Crystal structure of Bombyx mori lipoprotein 6: comparative structural analysis of the 30-kDa lipoprotein family.

PLoS One 2014 7;9(11):e108761. Epub 2014 Nov 7.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Lodz, Poland.

The 30-kDa lipoprotein (LP) family of mulberry silkworm comprises major hemolymph proteins specific to the fifth instar larvae. The family consists of 46 members, 24 of which are referred to as typical 30-kDa LPs. To date, two crystal structures of 30-kDa LPs from Bombyx mori have been described (Bmlp3 and Bmlp7). Here, we present the crystal structure of Bmlp6, another 30-kDa LP member. Bmlp6 is comprised of two domains characteristic of this family, the VHS-type N-terminal domain and β-trefoil C-terminal domain. The structures of the three 30-kDa LPs have been compared and a number of differences are noted, including loop conformation, the surface electrostatic potential, and the potential binding cavities. We discuss the observed structural differences in the light of the potential different roles of the particular 30-kDa LP members in silkworm physiology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108761PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224370PMC
July 2015

Crystallographic identification of an unexpected protein complex in silkworm haemolymph.

Acta Crystallogr D Biol Crystallogr 2013 Dec 19;69(Pt 12):2353-64. Epub 2013 Nov 19.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.

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.
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http://dx.doi.org/10.1107/S0907444913021823DOI Listing
December 2013

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of human histidine triad nucleotide-binding protein 2 (hHINT2).

Acta Crystallogr Sect F Struct Biol Cryst Commun 2013 Jul 28;69(Pt 7):783-7. Epub 2013 Jun 28.

Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Sienkiewicza 112, 90-363 Łódź, Poland.

Histidine triad nucleotide-binding protein 2 (HINT2) is a mitochondrial adenosine phosphoramidase mainly expressed in the pancreas, liver and adrenal gland. HINT2 possibly plays a role in apoptosis, as well as being involved in steroid biosynthesis, hepatic lipid metabolism and regulation of hepatic mitochondria function. The expression level of HINT2 is significantly down-regulated in hepatocellular carcinoma patients. To date, endogenous substrates for this enzyme, as well as the three-dimensional structure of human HINT2, are unknown. In this study, human HINT2 was cloned, overexpressed in Escherichia coli and purified. Crystallization was performed at 278 K using PEG 4000 as the main precipitant; the crystals, which belonged to the tetragonal space group P41212 with unit-cell parameters a = b = 76.38, c = 133.25 Å, diffracted to 2.83 Å resolution. Assuming two molecules in the asymmetric unit, the Matthews coefficient and the solvent content were calculated to be 2.63 Å(3) Da(-1) and 53.27%, respectively.
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http://dx.doi.org/10.1107/S1744309113015200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3702325PMC
July 2013

Two crystal structures of Bombyx mori lipoprotein 3 - structural characterization of a new 30-kDa lipoprotein family member.

PLoS One 2013 16;8(4):e61303. Epub 2013 Apr 16.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

The 30-kDa family of lipoproteins from insect hemolymph has been the focus of a number of studies over the last few years. Recently, four crystal structures of Bombyx mori lipoprotein 7 have been determined. Here we report two crystal structures of another member of the 30-kDa lipoprotein family, Bombyx mori lipoprotein 3 (Bmlp3). The protein was isolated from its natural source, mulberry silkworm hemolymph. It crystallized in two different crystal forms, Bmlp3-p21 (space group P21) and Bmlp3-c2 (space group C2). The crystal structures were solved by molecular replacement using the coordinates of Bmlp7 as a starting model. The crystals of Bmlp3-p21 diffracted X-rays to 2.4 Å resolution and of Bmlp3-c2 to 2.1 Å resolution. Bmlp3 has an overall fold characteristic of 30-kDa lipoproteins, with a VHS-type N-terminal domain and β-trefoil C-terminal domain. Structural comparison of Bmlp3 and Bmlp7 shows that the loops present in the C-terminal domain are flexible and participate in dimer formation. Additionally, new putative binding sites of Bmlp3 have been analyzed in detail and the electrostatic potential of the protein surface at physiological pH 7.4 conditions has been calculated. The results of these calculations are the starting point for an explanation of the recently reported cell-penetrating properties of the 30-kDa lipoproteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061303PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628942PMC
November 2013

Structural investigation of the interactions of biotinylruthenocene with avidin.

Chem Biol Interact 2013 Jun 18;204(1):6-12. Epub 2013 Apr 18.

Institute of Technical Biochemistry, Lodz University of Technology, 90-924 Lodz, Stefanowskiego 4/10, Poland.

The crystal structure of avidin, a protein from hen egg white, was determined in the form of a complex with biotinylruthenocene. This biotin-derived organometallic ligand is a potential anticancer agent for targeted therapy based upon avidin-biotin technology. Isothermal titration calorimetry experiments, involving avidin complexes with biotin (vitamin H or B7) derivatives, show differences in their affinity to the protein in comparison to its avidin-biotin complex, the strongest known biochemical interaction in Nature. The crystal structure of the first complex of avidin with biotinylruthenocene, determined at 2.5Å resolution (PDB: 4I60), shows unique interactions of the ruthenocene moiety with avidin. Biotin derivatives exhibit weaker affinity to avidin then biotin, which allows their wider use in biotechnology. The specific properties of biotinylruthenocene and the knowledge of its interactions with avidin may be useful in biochemical, medical, and nanotechnological applications.
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http://dx.doi.org/10.1016/j.cbi.2013.04.005DOI Listing
June 2013

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

Acta Crystallogr D Biol Crystallogr 2012 Sep 18;68(Pt 9):1140-51. Epub 2012 Aug 18.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

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 β-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.
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http://dx.doi.org/10.1107/S0907444912021555DOI Listing
September 2012

New active HIV-1 protease inhibitors derived from 3-hexanol: conformation study of the free inhibitors in crystalline state and in complex with the enzyme.

Chem Biol Drug Des 2012 May 7;79(5):798-809. Epub 2012 Mar 7.

Institute of Technical Biochemistry, Technical University of Łódź, Stefanowskiego 4/10, Łódź, Poland.

Four novel linear non-peptidic HIV-1 protease inhibitors derived from 2,5-diamino-1,6-diphenyl-3-hexanol were synthesized and characterized. All of them exhibit tight binding to HIV-1 protease, with inhibition constants K(i) in the range 20 pm-5 nm. The investigated inhibitors were crystallized, and their crystal structures were determined by X-ray diffraction. In all cases, the conformations found in the crystalline state differ significantly from the conformations obtained by computational docking of the inhibitor in the binding cleft of native HIV-1 protease. Owing to the prevalence of hydrophobic substituents in all these inhibitors, the conformational mobility in water solution is restricted to their compact forms. The spectrum of low-energy conformations in solution dramatically changes during the formation of inhibitor crystals (phenyl ring stacking as a leading motif) or during the formation of a complex with HIV-1 protease (elongated conformation suitable to fit the enzyme pockets as a factor responsible for tight binding). High conformational flexibility and low conformational stress in the molecules of these inhibitors most likely increase their biological activity in comparison with more rigid compounds.
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http://dx.doi.org/10.1111/j.1747-0285.2012.01328.xDOI Listing
May 2012

The influence of the stereochemistry of alanine residue on the solid state conformation and crystal packing of opioid peptides containing D-Ala or L-Ala in message domain--XRD and NMR study.

J Phys Chem B 2011 Aug 25;115(32):9910-9. Epub 2011 Jul 25.

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.

In this work, an X-ray diffraction (XRD) and solid state NMR study of two tetrapeptides with different stereochemistry of alanine residue is presented using Tyr-(D-Ala)-Phe-Gly (1), an N-terminal sequence of opioid peptide dermorphin, and its biologically inactive analog Tyr-(L-Ala)-Phe-Gly (2). Single-crystal XRD proved that 1 crystallized under different conditions from exclusively one structure: a monoclinic crystal with P2(1) space group. In contrast, 2 very easily formed at least three crystallographic modifications, 2a (monoclinic P2(1)), 2b (orthorhombic P2(1)2(1)2) and 2c (tetragonal P4(1)2(1)2). Solid-state NMR spectroscopy was employed to investigate the structure and molecular dynamics of 1, 2a, and 2b. By employing different NMR experiments (dipolar dephasing and PILGRIM) and an analysis of the (13)C principal elements of the chemical shift tensor (CST), it was proven that the main skeleton of tetrapeptides is rigid, whereas significant differences in the molecular motion of the aromatic residues were observed. Comparing current data with those of previous studies (J. Phys. Chem. B2004, 108, 4535-4545 and Cryst. Growth Des. 2009, 9, 4050-4059), it can be assumed that an important preorganization mechanism anticipating the formation of peptide crystals containing D-Ala in sequence is the intramolecular CH-π interaction, which occurs for the amino acid with D stereochemistry. This effect may be responsible for the formation of only one crystallographic form of D-Ala peptides.
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http://dx.doi.org/10.1021/jp205570yDOI Listing
August 2011

Crystal structures of the apo form of β-fructofuranosidase from Bifidobacterium longum and its complex with fructose.

FEBS J 2011 May 18;278(10):1728-44. Epub 2011 Apr 18.

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Lodz, Poland.

We solved the 1.8 Å crystal structure of β-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of β-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed β-propeller and a C-terminal β-sandwich module. The active site is located in the centre of the β-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 β-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn α-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 Å crystal structure of the β-fructofuranosidase complex with β-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.
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http://dx.doi.org/10.1111/j.1742-4658.2011.08098.xDOI Listing
May 2011

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

Acta Crystallogr Sect F Struct Biol Cryst Commun 2011 Mar 25;67(Pt 3):372-6. Epub 2011 Feb 25.

Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

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 Å (BmJHBP) and 1.3 Å (PBMHP-12) resolution.
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http://dx.doi.org/10.1107/S1744309110054564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3053166PMC
March 2011

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

Phys Chem Chem Phys 2011 Apr 8;13(14):6423-33. Epub 2011 Mar 8.

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.

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.
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http://dx.doi.org/10.1039/c0cp02401eDOI Listing
April 2011

Crystal structures of NodS N-methyltransferase from Bradyrhizobium japonicum in ligand-free form and as SAH complex.

J Mol Biol 2010 Dec 21;404(5):874-89. Epub 2010 Oct 21.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

NodS is an S-adenosyl-L-methionine (SAM)-dependent N-methyltransferase that is involved in the biosynthesis of Nod factor (NF) in rhizobia, which are bacterial symbionts of legume plants. NF is a modified chitooligosaccharide (COS) signal molecule that is recognized by the legume host, where it initiates symbiotic processes leading to atmospheric nitrogen fixation. We report the crystal structure of recombinant NodS protein from Bradyrhizobium japonicum, which infects lupine and serradella legumes. Two crystal forms--ligand-free NodS and NodS in complex with S-adenosyl-L-homocysteine, which is a by-product of the methylation reaction--were obtained, and their structures were refined to resolutions of 2.43 Å and 1.85 Å, respectively. Although the overall fold (consisting of a seven-stranded β-sheet flanked by layers of helices) is similar to those of other SAM-dependent methyltransferases, NodS has specific features reflecting the unique character of its oligosaccharide substrate. In particular, the N-terminal helix and its connecting loop get ordered upon SAM binding, thereby closing the methyl donor cavity and shaping a long surface canyon that is clearly the binding site for the acceptor molecule. Comparison of the two structural forms of NodS suggests that there are also other conformational changes taking place upon the binding of the donor substrate. As an enzyme that methylates a COS substrate, NodS is the first example among all SAM-dependent methyltransferases to have its three-dimensional structure elucidated. Gaining insight about how NodS binds its donor and acceptor substrates helps to better understand the mechanism of NodS activity and the basis of its functional difference in various rhizobia.
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http://dx.doi.org/10.1016/j.jmb.2010.10.016DOI Listing
December 2010

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

Acta Crystallogr D Biol Crystallogr 2010 Jul 19;66(Pt 7):789-96. Epub 2010 Jun 19.

Institute of Technical Biochemistry, Technical University of Lodz, Poland.

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.
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http://dx.doi.org/10.1107/S0907444910015416DOI Listing
July 2010

'Hot' macromolecular crystals.

Cryst Growth Des 2009 Dec;10(2):580

Department of Molecular Physiology and Biological Physics, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA.

Transcriptional regulator protein TM1030 from the hyperthermophile Thermotoga maritima, as well as its complex with DNA, was crystallized at a wide range of temperatures. Crystallization plates were incubated at 4, 20, 37 and 50° C over 3 weeks. The best crystals of TM1030 in complex with DNA were obtained at 4, 20 and 37° C, while TM1030 alone crystallized almost equally well in all temperatures. The crystals grown at different temperatures were used for X-ray diffraction experiments and their structures were compared. Surprisingly, the models of TM1030 obtained from crystals grown at different temperatures are similar in quality. While there are some examples of structures of proteins grown at elevated temperatures in the PDB, these temperatures appear to be underrepresented. Our studies show that crystals of some proteins may be grown and are stable at broad range of temperatures. We suggest that crystallization experiments at elevated temperatures could be used as a standard part of the crystallization protocol.
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http://dx.doi.org/10.1021/cg900971hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2809425PMC
December 2009

Structural investigation of biologically active phenolic compounds isolated from European tree species.

Molecules 2009 Oct 16;14(10):4147-58. Epub 2009 Oct 16.

Institute of Technical Biochemistry, Technical University of Łódź, ul. Stefanowskiego 4/10, 90-924 Łódź, Poland.

X-ray structures of two compounds isolated from wood knots of coniferous trees, namely dihydrokaempferol (3,5,8,13-tetrahydroxyflavanon) and lariciresinol (3,14-dimetoxy-7,10-epoxylignan-4,15,19-triol), are presented here. Diffraction data for the Dihydrokaempferol crystals were collected on a CAD4 diffractometer and on a synchrotron for the lariciresinol crystal. The investigated compounds inhibit lipid peroxidation and lariciresinol is additionally a good scavenger of superoxide radicals. The structural data presented in this work provide a useful basis for designing more active compounds with potential use as antioxidants.
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http://dx.doi.org/10.3390/molecules14104147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255330PMC
October 2009
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