The crystal structures of the far-red fluorescent proteins eqFP650 and eqFP670 have been solved at 1.8 and 1.6 Å resolution, respectively. This permitted identification of the structural elements responsible for the bathochromic shift in both considered far-red fluorescent proteins.
The crystal structures of the far-red fluorescent proteins (FPs) eqFP650 (λex
max 592/650 nm) and eqFP670 (λex
max 605/670 nm), the successors of the far-red FP Katushka (λex
max 588/635 nm), have been determined at 1.8 and 1.6 Å resolution, respectively. An examination of the structures demonstrated that there are two groups of changes responsible for the bathochromic shift of excitation/emission bands of these proteins relative to their predecessor. The first group of changes resulted in an increase of hydrophilicity at the acylimine site of the chromophore due to the presence of one and three water molecules in eqFP650 and eqFP670, respectively. These water molecules provide connection of the chromophore with the protein scaffold via hydrogen bonds causing an ∼15 nm bathochromic shift of the eqFP650 and eqFP670 emission bands. The second group of changes observed in eqFP670 arises from substitution of both Ser143 and Ser158 by asparagines. Asn143 and Asn158 of eqFP670 are hydrogen bonded with each other, as well as with the protein scaffold and with the p-hydroxyphenyl group of the chromophore, resulting in an additional ∼20 nm bathochromic shift of the eqFP670 emission band as compared to eqFP650. The role of the observed structural changes was verified by mutagenesis.
far-red fluorescent proteins; cell imaging; tissue visualization; Katushka
The bacterial heat shock protein Hsp33 is a redox-regulated chaperone activated by oxidative stress. In response to oxidation, four cysteines within a Zn2+ binding C-terminal domain form two disulfide bonds with concomitant release of the metal. This leads to the formation of the biologically active Hsp33 dimer. The crystal structure of the N-terminal domain of the E. coli protein has been reported, but neither the structure of the Zn2+ binding motif nor the nature of its regulatory interaction with the rest of the protein are known. Here we report the crystal structure of the full-length B. subtilis Hsp33 in the reduced form. The structure of the N-terminal, dimerization domain is similar to that of the E. coli protein, although there is no domain swapping. The Zn2+ binding domain is clearly resolved showing the details of the tetrahedral coordination of Zn2+ by four thiolates. We propose a structure-based activation pathway for Hsp33.
rsTagRFP the first monomeric red fluorescent protein with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Förster resonance energy transfer (pcFRET). Yellow fluorescent proteins YPet and mVenus have been demonstrated to be excellent pcFRET donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans- isomers corresponding to its ON and OFF states. Unlike in other photoswitchable fluorescent proteins, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas the residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay.
KFP; Dronpa; TagRFP; PAmCherry; FRET
Crystal structures of S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine, cordycepin and adenine are presented.
S-Adenosyl-l-homocysteine hydrolase (SAHase) catalyzes the reversible breakdown of S-adenosyl-l-homocysteine (SAH) to adenosine and homocysteine. SAH is formed in methylation reactions that utilize S-adenosyl-l-methionine (SAM) as a methyl donor. By removing the SAH byproduct, SAHase serves as a major regulator of SAM-dependent biological methylation reactions. Here, the first crystal structure of SAHase of plant origin, that from the legume yellow lupin (LlSAHase), is presented. Structures have been determined at high resolution for three complexes of the enzyme: those with a reaction byproduct/substrate (adenosine), with its nonoxidizable analog (cordycepin) and with a product of inhibitor cleavage (adenine). In all three cases the enzyme has a closed conformation. A sodium cation is found near the active site, coordinated by residues from a conserved loop that hinges domain movement upon reactant binding. An insertion segment that is present in all plant SAHases is located near a substrate-pocket access channel and participates in its formation. In contrast to mammalian and bacterial SAHases, the channel is open when adenosine or cordycepin is bound and is closed in the adenine complex. In contrast to SAHases from other organisms, which are active as tetramers, the plant enzyme functions as a homodimer in solution.
S-Adenosyl-l-homocysteine hydrolase; Lupinus luteus
Crystal structures of the bacterial α1,6-fucosyltransferase NodZ in complex with GDP and GDP-fucose are presented.
Rhizobial NodZ α1,6-fucosyltransferase (α1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5′-diphosphate-β-l-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two α1,6-FucT structures have been determined, both without any donor or acceptor molecule that could highlight the structural background of the catalytic mechanism. Here, the first crystal structures of α1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which is a byproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35 Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme–product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-l-glucosamine (penta-NAG). The structure has been determined at 1.98 Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among α1,2-, α1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand βC2 and helix αC3. In addition, there is a shift of the αC3 helix itself upon GDP-Fuc binding.
glycosyltransferases; fucosyltransferases; family GT-23 glycosyltransferases; chitooligosaccharide fucosylation; Nod-factor biosynthesis; nodulation; Nod factors; legume–rhizobium symbiosis; nitrogen fixation
In certain AMPPNP-containing protein structures, the nitrogen bridging the two terminal phosphate groups can be deprotonated.
Many different proteins utilize the chemical energy provided by the cofactor adenosine triphosphate (ATP) for their proper function. A number of structures in the Protein Data Bank (PDB) contain adenosine 5′-(β,γ-imido)triphosphate (AMPPNP), a nonhydrolysable analog of ATP in which the bridging O atom between the two terminal phosphate groups is substituted by the imido function. Under mild conditions imides do not have acidic properties and thus the imide nitrogen should be protonated. However, an analysis of protein structures containing AMPPNP reveals that the imide group is deprotonated in certain complexes if the negative charges of the phosphate moieties in AMPPNP are in part neutralized by coordinating divalent metals or a guanidinium group of an arginine.
imidodiphosphate; adenosine 5′-(β,γ-methylene)triphosphate; AMPPNP
Crystal structures of the human mitochondrial helicase hSuv3 in complex with AMPPNP and with a short strand of RNA are presented.
Suv3 is a helicase that is involved in efficient turnover and surveillance of RNA in eukaryotes. In vitro studies show that human Suv3 (hSuv3) in complex with human polynucleotide phosphorylase has RNA degradosome activity. The enzyme is mainly localized in mitochondria, but small fractions are found in cell nuclei. Here, two X-ray crystallographic structures of human Suv3 in complex with AMPPNP, a nonhydrolysable analog of ATP, and with a short five-nucleotide strand of RNA are presented at resolutions of 2.08 and 2.9 Å, respectively. The structure of the enzyme is very similar in the two complexes and consists of four domains. Two RecA-like domains form the tandem typical of all helicases from the SF2 superfamily which together with the C-terminal all-helical domain makes a ring structure through which the nucleotide strand threads. The mostly helical N-terminal domain is positioned externally with respect to the core of the enzyme. Most of the typical helicase motifs are present in hSuv3, but the protein shows certain unique characteristics, suggesting that Suv3 enzymes may constitute a separate subfamily of helicases.
mitochondrial helicases; human Suv3; SF2 helicases
A repetitive measurement of the same diffraction image allows to judge the performance of a data collection facility.
The accuracy of X-ray diffraction data depends on the properties of the crystalline sample and on the performance of the data-collection facility (synchrotron beamline elements, goniostat, detector etc.). However, it is difficult to evaluate the level of performance of the experimental setup from the quality of data sets collected in rotation mode, as various crystal properties such as mosaicity, non-uniformity and radiation damage affect the measured intensities. A multiple-image experiment, in which several analogous diffraction frames are recorded consecutively at the same crystal orientation, allows minimization of the influence of the sample properties. A series of 100 diffraction images of a thaumatin crystal were measured on the SBC beamline 19BM at the APS (Argonne National Laboratory). The obtained data were analyzed in the context of the performance of the data-collection facility. An objective way to estimate the uncertainties of individual reflections was achieved by analyzing the behavior of reflection intensities in the series of analogous diffraction images. The multiple-image experiment is found to be a simple and adequate method to decompose the random errors from the systematic errors in the data, which helps in judging the performance of a data-collection facility. In particular, displaying the intensity as a function of the frame number allows evaluation of the stability of the beam, the beamline elements and the detector with minimal influence of the crystal properties. Such an experiment permits evaluation of the highest possible data quality potentially achievable at the particular beamline.
diffraction data precision; signal-to-noise ratio; measurement uncertainty; beamline performance
Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviral activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.
Lectin; Entry Inhibitor; HIV; Man9; High Mannose Oligosaccharide; Branched Carbohydrates; Griffithsin
In the crystal structure of the racemic title compound, C22H25NO2, solved and refined against sychrotron diffraction data, the hydroxy group and the carbonyl O atom participate in the formation of O—H⋯O hydrogen bonds between pairs of enantiomers related by a crystallographic centre of symmetry.
The crystal of the title compound, C21H23NO2, was chosen from a conglomerate formed by a racemic mixture. An intramolecular hydrogen bond is formed between hydroxy group and heterocyclic N atom of the azabicyclo[3.2.1]octan-3-one system. The crystal structure is stabilized by C—H⋯O interactions between aliphatic C—H groups and the carbonyl O atom. For the title chiral crystal, the highly redundant and accurate diffraction data set collected with low energy copper radiation gave a Flack parameter of 0.12 (18) for anomalous scattering effects originating from O atoms.
The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix.
The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.
anomalous diffraction; ATP-dependent proteases; protein domains; structure quality; Lon protease
Using energy and density guided Rosetta refinement to improve molecular replacement, we have determined the crystal structure of the protease (PR) encoded by xenotropic murine leukemia virus-related virus (XMRV). Despite overall similarity of XMRV PR to other retropepsins, the topology of its dimer interface more closely resembles the monomeric, pepsin-like enzymes. Thus, XMRV PR may represent a distinct evolutionary branch of the family of aspartic proteases.
The crystal structure of the title compound, C14H17NO2, solved and refined against synchrotron diffraction data, contains one formula unit in an asymmetric unit. In the crystal, molecules form right-handed helices located at the 21 screw axis parallel to the a-axis direction, generated by O—H⋯N hydrogen bonding between the hydroxy group and carbonitrile group of an adjacent molecule.
This article, largely based on personal experiences of the authors, reviews the early history of the application of synchrotron radiation to structural biology, and particularly protein crystallography, to show the tremendous impact that this experimental innovation has had on these disciplines.
The introduction of synchrotron radiation sources almost four decades ago has led to a revolutionary change in the way that diffraction data from macromolecular crystals are being collected. Here a brief history of the development of methodologies that took advantage of the availability of synchrotron sources are presented, and some personal experiences with the utilization of synchrotrons in the early days are recalled.
macromolecular crystallography; structural biology; science history
The crystal structure of a Z-DNA hexamer duplex d(CGCGCG)2 determined at ultra high resolution of 0.55 Å and refined without restraints, displays a high degree of regularity and rigidity in its stereochemistry, in contrast to the more flexible B-DNA duplexes. The estimations of standard uncertainties of all individually refined parameters, obtained by full-matrix least-squares optimization, are comparable with values that are typical for small-molecule crystallography. The Z-DNA model generated with ultra high-resolution diffraction data can be used to revise the stereochemical restraints applied in lower resolution refinements. Detailed comparisons of the stereochemical library values with the present accurate Z-DNA parameters, shows in general a good agreement, but also reveals significant discrepancies in the description of guanine-sugar valence angles and in the geometry of the phosphate groups.
The crystal structure of the title compound, C28H38O11, solved and refined against synchrotron diffraction data, contains two formula units in the asymmetric unit. In both molecules, the dihydropyran ring along with its methyl substituents is disordered and adopts two alternative half-chair conformations. The occupancy of the major conformers of the two molecules refined to 0.858 (5) and 0.523 (5).
Fast-FT is a fluorescent timer (FT) engineered from DsRed-like fluorescent protein mCherry. Crystal structures of Fast-FT (chromophore Met66-Tyr67-Gly68) and its precursor with blocked blue-to-red conversion Blue102 (chromophore Leu66-Tyr67-Gly68) have been determined at the resolution of 1.15 Å and 1.81 Å, respectively. Structural data suggest that blue-to-red conversion, taking place in Fast-FT and in related FTs, is associated with the oxidation of Cα2-Cβ2 bond of Tyr67. Site directed mutagenesis revealed a crucial role of Arg70 and Tyr83 in the delayed oxidation of Cα2-Cβ2 bond, introducing the timing factor in maturation of the timer. Substitutions Ser217Ala and Ser217Cys in Fast-FT substantially slow down formation of an intermediate blue chromophore but do not affect much blue-to-red conversion, whereas mutations Arg70Lys or Trp83Leu, having little effect on the blue chromophore formation rate, markedly accelerates formation of the red chromophore. The chromophore of FTs adopts a cis-conformation stabilized by a hydrogen bond between its phenolate oxygen and the side chain hydroxyl of Ser146. In Blue102, a bulky side chain of Ile146 precludes the chromophore from adopting a “cis-like” conformation, blocking its blue-to-red conversion. Both Fast-FT and Blue102 structures revealed hydrolytic degradation of the chromophores. In Fast-FT, chromophore-forming Met66 residue is eliminated from the polypeptide chain, whereas Leu66 in Blue102 is cleaved out from the chromophore, decarboxylated and remains attached to the preceding Phe65. Hydrolysis of the chromophore competes with chromophore maturation starting from either the keto or enolate intermediates and is driven by the same residues that participate in chromophore maturation.
The crystal structure of the title water-soluble analogue of vitamin E, trolox amide, C14H19NO3, solved and refined against synchrotron diffraction data, contains two molecules in the asymmetric unit. In both molecules, the heterocyclic ring is in a half-chair conformation. The crystal packing features a herring-bone pattern generated by N—H⋯O hydrogen bonds between the hydroxy and amide groups. O—H⋯O hydrogen bonds also occur.
The crystal structure of the title compound, C90H100N6O24S2·C4H8O2·0.25C6H14, solved and refined against synchrotron diffraction data, contains two formula units in the asymmetric unit with the all-trans n-hexane molecule having half-occupancy and one of the ethyl acetate molecules disordered over two positions. The two symmetry-independent disulfide molecules are assembled by approximate face-to-face and face-to-edge interactions between their 4-nitrobenzoyloxy groups into an intertwined dimer having a double-helix-type structure. The centrally placed disulfide bridges in the two symmetry-independent molecules exhibit different helicity as shown by the C—S—S—C torsion angles of 71.0 (1) and −92.5 (1)°.
The crystal structure of the Taz2 zinc-finger domain of the human p300 transcriptional coactivator was determined using the anomalous diffraction signal of the bound Zn ions. Crystal contacts suggested a possible novel mode of Taz2–peptide ligand interactions.
CBP and its paralog p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. The structure of a segment of human p300 protein (residues 1723–1836) corresponding to the extended zinc-binding Taz2 domain has been investigated. The crystal structure was solved by the SAD approach utilizing the anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. Residues 1813–1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal-contact interactions with the peptide-binding site of Taz2, providing additional insights into the mechanism of the recognition of diverse transactivation domains (TADs) by Taz2. On the basis of these results and molecular modeling, a hypothetical model of the binding of phosphorylated p53 TAD1 to Taz2 has been proposed.
zinc-finger proteins; anomalous diffraction; protein recognition; transcription regulation
The problem of undercounting of citations that are published only in supplementary material is studied for the journals Nature, Science, Cell and the Proceedings of the National Academy of Sciences (USA).
citations; supplementary material; editorial
The title compound, C23H26F2N2O4, is a dipeptidic inhibitor of γ-secretase, one of the enzymes involved in Alzheimer’s disease. The molecule adopts a compact conformation, without intramolecular hydrogen bonds. In the crystal structure, one of the amide N atoms forms the only intermolecular N—H⋯O hydrogen bond; the second amide N atom does not form hydrogen bonds. High-resolution synchrotron diffraction data permitted the unequivocal location and refinement without restraints of all H atoms, and the identification of the characteristic shift of the amide H atom engaged in the hydrogen bond from its ideal position, resulting in a more linear hydrogen bond. Significant residual densities for bonding electrons were revealed after the usual SHELXL refinement, and modeling of these features as additional interatomic scatterers (IAS) using the program PHENIX led to a significant decrease in the R factor from 0.0411 to 0.0325 and diminished the r.m.s. deviation level of noise in the final difference Fourier map from 0.063 to 0.037 e Å−3.