Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

High-resolution crystal structure of copper amine oxidase from Arthrobacter globiformis: assignment of bound diatomic molecules as O2.

The crystal structure of a copper amine oxidase from Arthrobacter globiformis was determined at 1.08 Šresolution with the use of low-molecular-weight polyethylene glycol (LMW PEG; average molecular weight ∼200) as a cryoprotectant. The final crystallographic R factor and Rfree were 13.0 and 15.0%, respectively. Several molecules of LMW PEG were found to occupy cavities in the protein interior, including the active site, which resulted in a marked reduction in the overall B factor and consequently led to a subatomic resolution structure for a relatively large protein with a monomer molecular weight of ∼70,000. About 40% of the presumed H atoms were observed as clear electron densities in the Fo - Fc difference map. Multiple minor conformers were also identified for many residues. Anisotropic displacement fluctuations were evaluated in the active site, which contains a post-translationally derived quinone cofactor and a Cu atom. Furthermore, diatomic molecules, most likely to be molecular oxygen, are bound to the protein, one of which is located in a region that had previously been proposed as an entry route for the dioxygen substrate from the central cavity of the dimer interface to the active site.

Structural basis of human p70 ribosomal S6 kinase-1 regulation by activation loop phosphorylation.

p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3'-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3'-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.

Structural basis for designing an array of engrailed homeodomains.

Small DNA-binding proteins that target desired sequences have the potential to act as a scaffold for molecular tools such as genome editing. In this study, an engrailed homeodomain (EHD) was chosen and it was evaluated whether it could be used as a molecular module that can connect to itself to recognize a longer target sequence. It was previously shown that two EHDs connected by a linker (EHD2) recognize a target sequence twice as long as that recognized by a single EHD in cells only when Arg53 in each EHD in the tandem protein is mutated to alanine {(EHD[R53A])2}. To investigate the recognition mechanism of (EHD[R53A])2, the crystal structure of the (EHD[R53A])2-DNA complex was determined at 1.6 Šresolution. The individual EHDs were found to adopt the typical homeodomain fold. Most importantly, the base-specific interactions in the major groove necessary for the affinity/specificity of wild-type EHD were preserved in (EHD[R53A])2. Bacterial assays confirmed that the base-specific interactions are retained under cellular conditions. These observations indicate that the R53A mutation only causes a loss of the arginine-phosphate interaction at the protein-DNA interface, which reduces the DNA-binding affinity compared with the wild type. It is therefore concluded that (EHD[R53A])2 precisely recognizes tandem target sites within cells, enabling the individual EHDs to concurrently bind to the target sites with modest binding affinity. This suggests that modulation of the binding activity of each EHD is vital to construct a protein array that can precisely recognize a sequence with multiple target sites.

Balance between DNA-binding affinity and specificity enables selective recognition of longer target sequences in vivo.

Although genome-editing enzymes such as TALEN and CRISPR/Cas9 are being widely used, they have an essential limitation in that their relatively high-molecular weight makes them difficult to be delivered to cells. To develop a novel genome-editing enzyme with a smaller molecular weight, we focused on the engrailed homeodomain (EHD). We designed and constructed proteins composed of two EHDs connected by a linker to increase sequence specificity. In bacterial one-hybrid assays and electrophoresis mobility shift assay analyses, the created proteins exhibited good affinity for DNA sequences consisting of two tandemly aligned EHD target sequences. However, they also bound to individual EHD targets. To avoid binding to single target sites, we introduced amino acid mutations to reduce the protein-DNA affinity of each EHD monomer and successfully created a small protein with high specificity for tandem EHD target sequences.

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography.

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Šresolution) and X-ray (1.1 Šresolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%-75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.

DNA conformational transitions inferred from re-evaluation of m|Fo| - D|Fc| electron-density maps.

Conformational flexibility of DNA plays important roles in biological processes such as transcriptional regulation and DNA packaging etc. To understand the mechanisms of these processes, it is important to analyse when, where and how DNA shows conformational variations. Recent analyses have indicated that conventional refinement methods do not always provide accurate models of crystallographic heterogeneities and that some information on polymorphism has been overlooked in previous crystallographic studies. In the present study, the m|Fo| - D|Fc| electron-density maps of double-helical DNA crystal structures were calculated at a resolution equal to or better than 1.5 Šand potential conformational transitions were found in 27% of DNA phosphates. Detailed analyses of the m|Fo| - D|Fc| peaks indicated that some of these unassigned densities correspond to ZI ↔ ZII or A/B → BI conformational transitions. A relationship was also found between ZI/ZII transitions and metal coordination in Z-DNA from the detected peaks. The present study highlights that frequent transitions of phosphate backbones occur even in crystals and that some of these transitions are affected by the local molecular environment.

Comparing reduced-dose sodium phosphate tablets to 2 L of polyethylene glycol: A randomized study.

AIM: To compare the tolerability and quality of bowel cleansing between 2 L polyethylene glycol (PEG) and reduced-dose sodium phosphate (NaP) tablets as a preparation for colonoscopy. METHODS: Two hundred patients were randomly assigned to the PEG or NaP groups at the same ratio. The NaP group patients took 30 tablets with 2 L of clear liquid, while the PEG group patients took 2L of PEG. Tolerability was assessed by a questionnaire about taste, volume, and the overall impression. The bowel cleansing quality was evaluated by colonoscopists. RESULTS: Although NaP showed better tolerability in terms of taste, volume and overall impression (P < 0.01, P < 0.01 and P = 0.02, respectively), the overall cleansing quality was better in the PEG group (P < 0.01). A subgroup analysis, stratified by sex and age, indicated that NaP was associated with better tolerability and equivalent bowel cleansing quality in females of < 50 years of age. CONCLUSION: Despite the better tolerability, the use of 30 NaP tablets with 2 L of clear liquid should be limited due to its lower cleansing quality; however, in certain cases the regimen may deserve consideration, particularly in cases involving young women.

DNA octaplex formation with an I-motif of water-mediated A-quartets: reinterpretation of the crystal structure of d(GCGAAAGC).

The crystal structure of the tetragonal form of d(gcGAAAgc) has been revised and reasonably refined including the disordered residues. The two DNA strands form a base-intercalated duplex, and the four duplexes are assembled according to the crystallographic 222 symmetry to form an octaplex. In the central region, the eight strands are associated by I-motif of double A-quartets. Furthermore, eight hydrated-magnesium cations link the four duplexes to support the octaplex formation. Based on these structural features, a proposal that folding of d(GAAA)n, found in the non-coding region of genomes, into an octaplex can induce slippage during replication to facilitate length polymorphism is presented.

Crystal structures of a DNA octaplex with I-motif of G-quartets and its splitting into two quadruplexes suggest a folding mechanism of eight tandem repeats.

Recent genomic analyses revealed many kinds of tandem repeats of specific sequences. Some of them are related to genetic diseases, but their biological functions and structures are still unknown. Two X-ray structures of a short DNA fragment d(gcGA[G]1Agc) show that four base-intercalated duplexes are assembled to form an octaplex at a low K+ concentration, in which the eight G5 residues form a stacked double G-quartet in the central part. At a higher K+ concentration, however, the octaplex is split into just two halves. These structural features suggest a folding process of eight tandem repeats of d(ccGA[G]4Agg), according to a double Greek-key motif. Such a packaging of the repeats could facilitate slippage of a certain sequence during DNA replication, to induce increase or decrease of the repeats.

Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson-Crick base pairs.

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4(1)22 with the unit-cell dimensions a = b = 53.4 A and c = 54.0 A, and the crystal structure has been determined at 2.5 A resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C2:C2+ (singly-protonated between the Watson- Crick sites), G3:G3 (between the minor groove sites), A4:A4 (between the major groove sites) and A5:A5 (between the Watson-Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex.

Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17.

Crystal structures of chitosanase from Bacillus sp. K17 (ChoK) have been determined at 1.5 A resolution in the active form and at 2.0 A resolution in the inactive form. This enzyme belongs to the family GH-8, out of 93 glycoside hydrolase families, and exhibits the substrate specificity of subclass II chitosanase. The catalytic site is constructed on the scaffold of a double-alpha(6)/alpha(6)-barrel, which is formed by six repeating helix-loop-helix motifs. This structure is quite different from those of the GH-46 chitosanases and of GH-5. Structural comparison with CelA (a cellulase belonging to the same family GH-8) suggests that the proton donor Glu122 is conserved, but the proton acceptor is the inserted Glu309 residue, and that the corresponding Asp278 residue in CelA is inactivated in ChoK. The four acidic residues, Asp179, Glu309, Asp183 and Glu107, can be involved in substrate recognition through interactions with the amino groups of the glucosamine residues bound in the -3, -2, -1 and +1 sites, respectively. The hydrophobic Trp235, Trp166, Phe413 and Tyr318 residues are highly conserved for binding of the hexose rings at the -3, -2, +1 and +2 sites, respectively. These structural features indicate that enzymes in GH-8 can be further divided into three subfamilies. Different types of chitosanases are discussed in terms of convergent evolution from different structural ancestors.

A prospective, open-label, flexible-dose study of quetiapine in the treatment of delirium.

BACKGROUND: Delirium is an organic psychiatric syndrome characterized by fluctuating consciousness and impaired cognitive functioning. High-potency typical neuroleptics have traditionally been used as first-line drugs in the treatment of delirium. However, these drugs are frequently associated with undesirable adverse events including extrapyramidal symptoms (EPS). The purpose of the present open-label, flexible-dose study was to provide preliminary data on the usefulness and safety of quetiapine for patients with delirium. METHOD: Twelve patients with DSM-IV delirium were treated with flexible doses of open-label quetiapine (mean +/- SD dosage = 44.9 +/- 31.0 mg/day). To evaluate the usefulness and safety of quetiapine, scores from the Delirium Rating Scale, Japanese version, were assessed every day (for 1 outpatient, at least twice per week), and scores from the Mini-Mental State Examination, Japanese version, and the Drug-Induced Extrapyramidal Symptom Scale were assessed at baseline and after remission of delirium. Data were gathered from April to October 2001. RESULTS: All patients achieved remission of delirium several days after starting quetiapine (mean +/- SD duration until remission = 4.8 +/- 3.5 days). Quetiapine treatment was well tolerated, and no clinically relevant change in EPS was detected. CONCLUSION: Quetiapine may be a useful alternative to conventional neuroleptics in the treatment of delirium due to its rapid onset and relative lack of adverse events. Further double-blind, placebo-controlled studies are warranted.

Direct interactions between Z-DNA and alkaline earth cations, discovered in the presence of high concentrations of MgCl2 and CaCl2.

In this study, crystals of Z-DNA hexamer d(CGCGCG) complexed with MgCl2 and CaCl2 were obtained in the presence of high concentrations of alkaline earth salts (500mM) using a temperature control technique, and their crystal structures were determined at 1.3Å resolution. Mg(2+) and Ca(2+) cations in these structures tend to interact directly with phosphate groups of Z-DNA duplexes; however, they tend to form water-mediated interactions with Z-DNA in the presence of lower concentrations of alkaline earth salts. In these crystals, a DNA duplex was laid along its c-axis and interacted with its 6 neighboring DNA duplexes through coordination bonds of PO…(Mg(2+) or Ca(2+))…OP. A symmetrical hexagonal Z-DNA duplex assembly model may explain DNA condensation caused by alkaline earth salts. These structures offer insights into the functions of alkaline earth cations essential to the structures and assembly of Z-DNA duplexes.

Local conformational changes in the DNA interfaces of proteins.

When a protein binds to DNA, a conformational change is often induced so that the protein will fit into the DNA structure. Therefore, quantitative analyses were conducted to understand the conformational changes in proteins. The results showed that conformational changes in DNA interfaces are more frequent than in non-interfaces, and DNA interfaces have more conformational variations in the DNA-free form. As expected, the former indicates that interaction with DNA has some influence on protein structure. The latter suggests that the intrinsic conformational flexibility of DNA interfaces is important for adjusting their conformation for DNA. The amino acid propensities of the conformationally changed regions in DNA interfaces indicate that hydrophilic residues are preferred over the amino acids that appear in the conformationally unchanged regions. This trend is true for disordered regions, suggesting again that intrinsic flexibility is of importance not only for DNA binding but also for interactions with other molecules. These results demonstrate that fragments destined to be DNA interfaces have an intrinsic flexibility and are composed of amino acids with the capability of binding to DNA. This information suggests that the prediction of DNA binding sites may be improved by the integration of amino acid preference for DNA and one for disordered regions.

Insights into the structures of DNA damaged by hydroxyl radical: crystal structures of DNA duplexes containing 5-formyluracil.

Hydroxyl radicals are potent mutagens that attack DNA to form various base and ribose derivatives. One of the major damaged thymine derivatives is 5-formyluracil (fU), which induces pyrimidine transition during replication. In order to establish the structural basis for such mutagenesis, the crystal structures of two kinds of DNA d(CGCGRATfUCGCG) with R = A/G have been determined by X-ray crystallography. The fU residues form a Watson-Crick-type pair with A and two types of pairs (wobble and reversed wobble) with G, the latter being a new type of base pair between ionized thymine base and guanine base. In silico structural modeling suggests that the DNA polymerase can accept the reversed wobble pair with G, as well as the Watson-Crick pair with A.

The structure of a d(gcGAACgc) duplex containing two consecutive bulged A residues in both strands suggests a molecular switch.

In previous studies, it was reported that DNA fragments with the sequence d(gcGXYAgc) (where X = A or G and Y = A, T or G) form a stable base-intercalated duplex (Bi-duplex) in which the central X and Y residues are not involved in any base-pair interactions but are alternately stacked on each other between the two strands. To investigate the structural stability of the Bi-duplex, the crystal structure of d(gcGAACgc) with a point mutation at the sixth residue of the sequence, d(gcGAAAgc), has been determined. The two strands are associated in an antiparallel fashion to form two types of bulge-containing duplexes (Bc-duplexes), I and II, both of which are quite different from the Bi-duplex of the parent sequence. In both Bc-duplexes, three Watson-Crick G.C base pairs constitute the stem regions at the two ends. The A(4) residues are bulged in to form a pair with the corresponding A(4) residue of the opposite strand in either duplex. The A(4).A(4)* pair formation is correlated to the orientations of the adjacent A(5) residues. A remarkable difference between the two Bc-duplexes is seen at the A(5) residue. In Bc-duplex I, it is flipped out and comes back to interact with the G(3) residue. In Bc-duplex II, the A(5) residue extends outwards to interact with the G(7) residue of the neighbouring Bc-duplex I. These results indicate that trans sugar-edge/Hoogsteen (sheared-type) G(3).A(6)* base pairs are essential in the formation of a Bi-duplex of d(gcGXYAgc). On the other hand, the alternative conformations of the internal loops containing two consecutive bulged A residues suggest molecular switching.

Octaplex formation of d(GCGAAAGC) with the double A-quartet.

We reported that the sequence gcGA[X]1Agc adopts a specific structure to form several multiplexes. In the case of X=G, an octaplex formation occurs, in which the stacked double G-quartet stabilizes the architecture through potassium cation mediations. In the case of a mutant X=A, too, it has been found that the oligomers form an octaplex. The eight central A residues form a stacked double A-quartet, which is a first example of adenine associations. Several water molecules occupy the centre to stabilize the quartets, so that the octaplex seems to be swollen as compared with that of X=G. In any multiplex formations, the building block is a base-intercalated duplexes.

The octaplex structure of d(GCGAGAGC) with I-motif of guanine quartet, controlled by potassium concentration.

Crystal structures of DNA octamer with the sequence d(GCGAGAGC) have been determined by X-ray analyses to investigate the specific DNA structural motifs that are useful for designing various functional DNA molecules. The octamers are assembled to form an octaplex with G-quartets and water-mediated A-quartets. At relatively high potassium concentration, however, the octaplex is split into two quadruplexes, each of which contains two G-duets. The two crystal forms suggest a dynamic formation of an octaplex from two quadruplexes in solution.

X-ray structure of d(CGAA), parallel-stranded DNA duplex with homo base pairs.

To clarify whether the parallel right-handed duplex found in the d(GCGAAAGCT) crystal is possible to occur without the anti-parallel part, the crystal structures of d(CGAA) at pH6.0 and pH7.0 have been determined by X-ray analysis. There are two independent duplexes in the crystallographic asymmetric unit. In each duplex, two chains are aligned in parallel fashion to form a right-handed double helix through the homo base pair formations, the sugar puckers being essentially C2'-endo. The C1:C1 pair is achieved by hemi-protonation. The G2:G2, A3:A3 and A4:A4 pairs occur between the minor groove sites, between the major groove sites and between the Watson-Crick sites, respectively. In every pairing, the two bases are located at the trans positions to each other. These structural features are the same as those at pH7.0, suggesting that the C1:C1 pair is hemi-protonated even at the neutral condition.