Elucidating polymorphs of crystal structures by intensity-based hierarchical clustering analysis of multiple diffraction data sets.

In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of the diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single-structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference to define the threshold used to group the isomorphous data sets (the `isomorphic threshold') poses a challenge. Here, unit-cell-based and intensity-based HCAs have been applied to data sets for apo trypsin and inhibitor-bound trypsin that were mixed post data acquisition to investigate the efficacy of HCA in classifying polymorphous data sets. Single-step intensity-based HCA successfully classified polymorphs with a certain `isomorphic threshold'. In data sets for several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested `isomorphic threshold'. Polymorphs were also detected in single crystals using data collected using the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis.

Characterization of a novel cysteine-less Cu/Zn-superoxide dismutase in Paenibacillus lautus missing a conserved disulfide bond.

Cu/Zn-superoxide dismutase (CuZnSOD) is an enzyme that binds a copper and zinc ion and also forms an intramolecular disulfide bond. Together with the copper ion as the active site, the disulfide bond is completely conserved among these proteins; indeed, the disulfide bond plays critical roles in maintaining the catalytically competent conformation of CuZnSOD. Here, we found that a CuZnSOD protein in Paenibacillus lautus (PaSOD) has no Cys residue but exhibits a significant level of enzyme activity. The crystal structure of PaSOD revealed hydrophobic and hydrogen-bonding interactions in substitution for the disulfide bond of the other CuZnSOD proteins. Also notably, we determined that PaSOD forms a homodimer through an additional domain with a novel fold at the N terminus. While the advantages of lacking Cys residues and adopting a novel dimer configuration remain obscure, PaSOD does not require a disulfide-introducing/correcting system for maturation and could also avoid misfolding caused by aberrant thiol oxidations under an oxidative environment.

Intrinsic structural vulnerability in the hydrophobic core induces species-specific aggregation of canine SOD1 with degenerative myelopathy-linked E40K mutation.

Canine degenerative myelopathy (DM), a fatal neurodegenerative disease in dogs, shares clinical and genetic features with amyotrophic lateral sclerosis, a human motor neuron disease. Mutations in the SOD1 gene encoding Cu/Zn superoxide dismutase (SOD1) cause canine DM and a subset of inherited human amyotrophic lateral sclerosis. The most frequent DM causative mutation is homozygous E40K mutation, which induces the aggregation of canine SOD1 but not of human SOD1. However, the mechanism through which canine E40K mutation induces species-specific aggregation of SOD1 remains unknown. By screening human/canine chimeric SOD1s, we identified that the humanized mutation of the 117th residue (M117L), encoded by exon 4, significantly reduced aggregation propensity of canine SOD1E40K. Conversely, introducing a mutation of leucine 117 to methionine, a residue homologous to canine, promoted E40K-dependent aggregation in human SOD1. M117L mutation improved protein stability and reduced cytotoxicity of canine SOD1E40K. Furthermore, crystal structural analysis of canine SOD1 proteins revealed that M117L increased the packing within the hydrophobic core of the ß-barrel structure, contributing to the increased protein stability. Our findings indicate that the structural vulnerability derived intrinsically from Met 117 in the hydrophobic core of the ß-barrel structure induces E40K-dependent species-specific aggregation in canine SOD1.

Crystal structural analysis of aldoxime dehydratase from Bacillus sp. OxB-1: Importance of surface residues in optimization for crystallization.

Aldoxime dehydratase (Oxd) is a heme enzyme that catalyzes aldoxime dehydration to the corresponding nitriles. Unlike many other heme enzymes, Oxd has a unique feature that the substrate binds directly to the heme. Therefore, it is thought that structural differences around the bound heme directly relate to differences in substrate selection. However sufficient structural information to discuss the substrate specificity has not been obtained. Oxd from Bacillus sp. OxB-1 (OxdB) shows unique substrate specificity and enantioselectivity compared to the Oxds whose crystal structures have already been reported. Here, we report the crystal structure of OxdB, which has not been reported previously. Although the crystallization of OxdB has been difficult, by adding a site-specific mutation to Glu85 located on the surface of the protein, we succeeded in crystallizing OxdB without reducing the enzyme activity. The catalytic triad essential for Oxd activity were structurally conserved in OxdB. In addition, the crystal structure of the Michaelis complex of OxdB and the diastereomerically pure substrate Z-2-(3-bromophenyl)-propanal oxime implied the importance of several hydrophobic residues for substrate specificity. Mutational analysis implicated Ala12 and Ala14 in the E/Z selectivity of bulky compounds. The N-terminal region of OxdB was shown to be shorter than those of Oxds from Pseudomonas chlororaphis and Rhodococcus sp. N-771, and have high flexibility. These structural differences possibly result in distinct preferences for aldoxime substrates based on factors such as substrate size.

Heme controls the structural rearrangement of its sensor protein mediating the hemolytic bacterial survival.

Hemes (iron-porphyrins) are critical for biological processes in all organisms. Hemolytic bacteria survive by acquiring b-type heme from hemoglobin in red blood cells from their animal hosts. These bacteria avoid the cytotoxicity of excess heme during hemolysis by expressing heme-responsive sensor proteins that act as transcriptional factors to regulate the heme efflux system in response to the cellular heme concentration. Here, the underlying regulatory mechanisms were investigated using crystallographic, spectroscopic, and biochemical studies to understand the structural basis of the heme-responsive sensor protein PefR from Streptococcus agalactiae, a causative agent of neonatal life-threatening infections. Structural comparison of heme-free PefR, its complex with a target DNA, and heme-bound PefR revealed that unique heme coordination controls a >20 Å structural rearrangement of the DNA binding domains to dissociate PefR from the target DNA. We also found heme-bound PefR stably binds exogenous ligands, including carbon monoxide, a by-product of the heme degradation reaction.

X-ray dose-dependent structural changes of the [2Fe-2S] ferredoxin from Chlamydomonas reinhardtii.

Plant-type ferredoxin (Fd) is an electron transfer protein in chloroplast. Redox-dependent structural change of Fd controls its association with and dissociation from Fd-dependent enzymes. Among many X-ray structures of oxidized Fd have been reported so far, very likely a given number of them was partially reduced by strong X-ray. To understand the precise structural change between reduced and oxidized Fd, it is important to know whether the crystals of oxidized Fd may or may not be reduced during the X-ray experiment. We prepared the thin plate-shaped Fd crystals from Chlamydomonas reinhardtii and monitored its absorption spectra during experiment. Absorption spectra of oxidized Fd crystals were clearly changed to that of reduced form in an X-ray dose-dependent manner. In another independent experiment, the X-ray diffraction images obtained from different parts of one single crystal were sorted and merged to form two datasets with low and high X-ray doses. An Fo-Fo map calculated from the two datasets showed that X-ray reduction causes a small displacement of the iron atoms in the [2Fe-2S] cluster. Both our spectroscopic and crystallographic studies confirm X-ray dose-dependent reduction of Fd, and suggest a structural basis for its initial reduction step especially in the core of the cluster.

Kinetic and structural insight into a role of the re-face Tyr328 residue of the homodimer type ferredoxin-NADP+ oxidoreductase from Rhodopseudomonas palustris in the reaction with NADP+/NADPH.

Among the thioredoxin reductase-type ferredoxin-NAD(P)+ oxidoreductase (FNR) family, FNR from photosynthetic purple non­sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP+/NADPH using steady state and pre-steady state kinetic approaches. The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of Kd for NADP+ and KM for NADPH in the diaphorase assay; however, the kcat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP+ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s-1). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP+/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP+/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP+/NADPH and/or reaction with electron transfer proteins.

Structural basis for the heme transfer reaction in heme uptake machinery from Corynebacteria.

The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.

Structural characterization of HypX responsible for CO biosynthesis in the maturation of NiFe-hydrogenase.

Several accessory proteins are required for the assembly of the metal centers in hydrogenases. In NiFe-hydrogenases, CO and CN- are coordinated to the Fe in the NiFe dinuclear cluster of the active center. Though these diatomic ligands are biosynthesized enzymatically, detail mechanisms of their biosynthesis remain unclear. Here, we report the structural characterization of HypX responsible for CO biosynthesis to assemble the active site of NiFe hydrogenase. CoA is constitutionally bound in HypX. Structural characterization of HypX suggests that the formyl-group transfer will take place from N10-formyl-THF to CoA to form formyl-CoA in the N-terminal domain of HypX, followed by decarbonylation of formyl-CoA to produce CO in the C-terminal domain though the direct experimental results are not available yet. The conformation of CoA accommodated in the continuous cavity connecting the N- and C-terminal domains will interconvert between the extended and the folded conformations for HypX catalysis.

Crystal structure of the starch-binding domain of glucoamylase from Aspergillus niger.

Glucoamylases are widely used commercially to produce glucose syrup from starch. The starch-binding domain (SBD) of glucoamylase from Aspergillus niger is a small globular protein containing a disulfide bond. The structure of A. niger SBD has been determined by NMR, but the conformation surrounding the disulfide bond was unclear. Therefore, X-ray crystal structural analysis was used to attempt to clarify the conformation of this region. The SBD was purified from an Escherichia coli-based expression system and crystallized at 293 K. The initial phase was determined by the molecular-replacement method, and the asymmetric unit of the crystal contained four protomers, two of which were related by a noncrystallographic twofold axis. Finally, the structure was solved at 2.0 Šresolution. The SBD consisted of seven ß-strands and eight loops, and the conformation surrounding the disulfide bond was determined from a clear electron-density map. Comparison of X-ray- and NMR-determined structures of the free SBD showed no significant difference in the conformation of each ß-strand, but the conformations of the loops containing the disulfide bond and the L5 loop were different. In particular, the difference in the position of the Cα atom of Cys509 between the X-ray- and NMR-determined structures was 13.3 Å. In addition, the B factors of the amino-acid residues surrounding the disulfide bond are higher than those of other residues. Therefore, the conformation surrounding the disulfide bond is suggested to be highly flexible.

Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters.

Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin.

In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the [2Fe-2S] cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Šresolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.

Crystal structures of copper-depleted and copper-bound fungal pro-tyrosinase: insights into endogenous cysteine-dependent copper incorporation.

Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 Å resolution, respectively, revealing that Phe(513) on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193-201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19-26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys(92), which is covalently bound to His(94) via an unusual thioether linkage in the holo-form, and Cys(522) and Cys(525) of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information.

Concentration-dependent oligomerization of cross-linked complexes between ferredoxin and ferredoxin-NADP+ reductase.

Ferredoxin-NADP(+) reductase (FNR) forms a 1:1 complex with ferredoxin (Fd), and catalyzes the electron transfer between Fd and NADP(+). In our previous study, we prepared a series of site-specifically cross-linked complexes of Fd and FNR, which showed diverse electron transfer properties. Here, we show that X-ray crystal structures of the two different Fd-FNR cross-linked complexes form oligomers by swapping Fd and FNR moieties across the molecules; one complex is a dimer from, and the other is a successive multimeric form. In order to verify whether these oligomeric structures are formed only in crystal, we investigated the possibility of the oligomerization of these complexes in solution. The mean values of the particle size of these cross-linked complexes were shown to increase with the rise of protein concentration at sub-milimolar order, whereas the size of dissociable wild-type Fd:FNR complex was unchanged as analyzed by dynamic light scattering measurement. The oligomerization products were detected by SDS-PAGE after chemical cross-linking of these complexes at the sub-milimolar concentrations. The extent and concentration-dependent profile of the oligomerizaion were differentiated between the two cross-linked complexes. These results show that these Fd-FNR cross-linked complexes exhibit concentration-dependent oligomerization, possibly through swapping of Fd and FNR moieties also in solution. These findings lead to the possibility that some native multi-domain proteins may present similar phenomenon in vivo.

Structures of cyanobacteriochromes from phototaxis regulators AnPixJ and TePixJ reveal general and specific photoconversion mechanism.

Cyanobacteriochromes are cyanobacterial tetrapyrrole-binding photoreceptors that share a bilin-binding GAF domain with photoreceptors of the phytochrome family. Cyanobacteriochromes are divided into many subclasses with distinct spectral properties. Among them, putative phototaxis regulators PixJs of Anabaena sp. PCC 7120 and Thermosynechococcus elongatus BP-1 (denoted as AnPixJ and TePixJ, respectively) are representative of subclasses showing red-green-type and blue/green-type reversible photoconversion, respectively. Here, we determined crystal structures for the AnPixJ GAF domain in its red-absorbing 15Z state (Pr) and the TePixJ GAF domain in its green-absorbing 15E state (Pg). The overall structure of these proteins is similar to each other and also similar to known phytochromes. Critical differences found are as follows: (i) the chromophore of AnPixJ Pr is phycocyanobilin in a C5-Z,syn/C10-Z,syn/C15-Z,anti configuration and that of TePixJ Pg is phycoviolobilin in a C10-Z,syn/C15-E,anti configuration, (ii) a side chain of the key aspartic acid is hydrogen bonded to the tetrapyrrole rings A, B and C in AnPixJ Pr and to the pyrrole ring D in TePixJ Pg, (iii) additional protein-chromophore interactions are provided by subclass-specific residues including tryptophan in AnPixJ and cysteine in TePixJ. Possible structural changes following the photoisomerization of the chromophore between C15-Z and C15-E are discussed based on the X-ray structures at 1.8 and 2.0-Å resolution, respectively, in two distinct configurations.

Cloning, expression, crystallization and preliminary X-ray studies of the ferredoxin-NAD(P)+ reductase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

Ferredoxin-NADP(+) reductase (FNR) is a flavoenzyme that catalyses the reduction of NADP(+) in the final step of the photosynthetic electron-transport chain. FNR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TeFNR) contains an additional 9 kDa domain at its N-terminus relative to chloroplastic FNRs and is more thermostable than those from mesophilic cyanobacteria. With the aim of understanding the structural basis of the thermostability of TeFNR and assigning a structural role to the small additional domain, the gene encoding TeFNR with and without an additional domain was engineered for heterologous expression and the recombinant proteins were purified and crystallized. Crystals of TeFNR without the additional domain belonged to space group P2(1), with unit-cell parameters a = 55.05, b = 71.66, c = 89.73 Å, α = 90, ß = 98.21, γ = 90°.

N-terminal structure of maize ferredoxin:NADP+ reductase determines recruitment into different thylakoid membrane complexes.

To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.

Crystallization and preliminary X-ray studies of an electron-transfer complex of ferredoxin and ferredoxin-dependent glutamate synthase from the cyanobacterium Leptolyngbya boryana.

Ferredoxin (Fd) dependent glutamate synthase (Fd-GOGAT) is a key enzyme involved in nitrogen assimilation that catalyzes the two-electron reductive conversion of Gln and 2-oxoglutarate to two molecules of Glu. Fd serves as an electron donor for Fd-GOGAT and the two proteins form a transient electron-transfer complex. In this study, these two proteins were cocrystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed at 2.65 Å resolution. The crystals belonged to space group P4(3), with unit-cell parameters a = b = 84.95, c = 476.31 Å.

Chlorophyll biosynthesis: spotlight on protochlorophyllide reduction.

Photosynthetic organisms require chlorophyll or bacteriochlorophyll for their light trapping and energy transduction activities. The biosynthetic pathways of chlorophyll and bacteriochlorophyll are similar in most of their early steps, except for the reduction of protochlorophyllide (Pchlide) to chlorophyllide. Whereas angiosperms make use of a light-dependent enzyme, cyanobacteria, algae, bryophytes, pteridophytes and gymnosperms contain an additional, light-independent enzyme dubbed dark-operative Pchlide oxidoreductase (DPOR). Anoxygenic photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides rely solely on DPOR. Recent atomic resolution of reductase and catalytic components of DPOR from R. sphaeroides and R. capsulatus, respectively, have revealed their similarity to nitrogenase components. In this review, we discuss the two fundamentally different mechanisms of Pchlide reduction in photosynthetic organisms.

Asymmetric dimeric structure of ferredoxin-NAD(P)+ oxidoreductase from the green sulfur bacterium Chlorobaculum tepidum: implications for binding ferredoxin and NADP+.

Ferredoxin-NAD(P)(+) oxidoreductase (FNR) catalyzes the reduction of NAD(P)(+) to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 A resolution, which reveals a unique structure-function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by pi-pi stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.