Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.

Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein ß-barrel across the time window of our measurements.

Juglone, a plant-derived 1,4-naphthoquinone, binds to hydroxylamine oxidoreductase and inhibits the electron transfer to cytochrome c554.

IMPORTANCE: Nitrification, the microbial conversion of ammonia to nitrate via nitrite, plays a pivotal role in the global nitrogen cycle. However, the excessive use of ammonium-based fertilizers in agriculture has disrupted this cycle, leading to groundwater pollution and greenhouse gas emissions. In this study, we have demonstrated the inhibitory effects of plant-derived juglone and related 1,4-naphthoquinones on the nitrification process in Nitrosomonas europaea. Notably, the inhibition mechanism is elucidated in which 1,4-naphthoquinones interact with hydroxylamine oxidoreductase, disrupting the electron transfer to cytochrome c554, a physiological electron acceptor. These findings support the notion that phytochemicals can impede nitrification by interfering with the essential electron transfer process in ammonia oxidation. The findings presented in this article offer valuable insights for the development of strategies aimed at the management of nitrification, reduction of fertilizer utilization, and mitigation of greenhouse gas emissions.

Structural insights into the epimerization of ß-1,4-linked oligosaccharides catalyzed by cellobiose 2-epimerase, the sole enzyme epimerizing non-anomeric hydroxyl groups of unmodified sugars.

Cellobiose 2-epimerase (CE) reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified ß1,4-linked oligosaccharides, including ß-1,4-mannobiose, cellobiose, and lactose. CE is responsible for conversion of ß1,4-mannobiose to 4-O-ß-d-mannosyl-d-glucose in mannan metabolism. However, the detailed catalytic mechanism of CE is unclear due to the lack of structural data in complex with ligands. We determined the crystal structures of halothermophile Rhodothermus marinus CE (RmCE) in complex with substrates/products or intermediate analogs, and its apo form. The structures in complex with the substrates/products indicated that the residues in the ß5-ß6 loop as well as those in the inner six helices form the catalytic site. Trp-322 and Trp-385 interact with reducing and non-reducing end parts of these ligands, respectively, by stacking interactions. The architecture of the catalytic site also provided insights into the mechanism of reversible epimerization. His-259 abstracts the H2 proton of the d-mannose residue at the reducing end, and consistently forms the cis-enediol intermediate by facilitated depolarization of the 2-OH group mediated by hydrogen bonding interaction with His-200. His-390 subsequently donates the proton to the C2 atom of the intermediate to form a d-glucose residue. The reverse reaction is mediated by these three histidines with the inverse roles of acid/base catalysts. The conformation of cellobiitol demonstrated that the deprotonation/reprotonation step is coupled with rotation of the C2-C3 bond of the open form of the ligand. Moreover, it is postulated that His-390 is closely related to ring opening/closure by transferring a proton between the O5 and O1 atoms of the ligand.

Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.

Among the epimerases specific to alginate, some of them in Azotobacter genera convert ß-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.

The c-di-GMP recognition mechanism of the PilZ domain of bacterial cellulose synthase subunit A.

In some Proteobacteria and Firmicutes such as Pseudomonas aeruginosa, Vibrio cholerae, Xanthomonas campestris, and Clostridium difficile, cyclic dimeric guanosine monophosphate (c-di-GMP) is known to regulate cellular processes, including motility, biofilm formation, and virulence, as a second messenger. Cellulose production in Acetobacter xylinum, a model organism of cellulose biosynthesis, also depends on by cellular c-di-GMP level. In cellulose-synthesizing bacteria, a terminal complex localized in the cell membrane synthesizes cellulose and regulates the production of cellulose sensed by c-di-GMP. Although previous studies indicated that the PilZ domain conserved in cellulose synthase subunit A (CeSA) was part of a receptor for c-di-GMP, the recognition mechanism by PilZ domain of CeSA remains unclear. In the present study, we studied the interaction between c-di-GMP and the PilZ domain of CeSA from a structural viewpoint. First, we solved the crystal structure of the PilZ domain of CeSA from A. xylinum (AxCeSA-PilZ) at 2.1Å resolution. Then, comparison of the sequence and structure of AxCeSA-PilZ to those of known structures of PilZ, such as VCA0042, PP4397, and PA4608, indicated the involvement of Lys573 and Arg643 of AxCeSA-PilZ in the recognition of c-di-GMP besides the RxxxR motif. Finally, the binding characteristics of c-di-GMP to AxCeSA-PilZ and mutants were determined with isothermal titration calorimetry, indicating that the residues corresponding to Lys573 and Arg643 in AxCeSA-PilZ generally contribute to the binding of c-di-GMP to PilZ.

Structure of bacterial cellulose synthase subunit D octamer with four inner passageways.

The cellulose synthesizing terminal complex consisting of subunits A, B, C, and D in Acetobacter xylinum spans the outer and inner cell membranes to synthesize and extrude glucan chains, which are assembled into subelementary fibrils and further into a ribbon. We determined the structures of subunit D (AxCeSD/AxBcsD) with both N- and C-terminal His(6) tags, and in complex with cellopentaose. The structure of AxCeSD shows an exquisite cylinder shape (height: ∼65 Å, outer diameter: ∼90 Å, and inner diameter: ∼25 Å) with a right-hand twisted dimer interface on the cylinder wall, formed by octamer as a functional unit. All N termini of the octamer are positioned inside the AxCeSD cylinder and create four passageways. The location of cellopentaoses in the complex structure suggests that four glucan chains are extruded individually through their own passageway along the dimer interface in a twisted manner. The complex structure also shows that the N-terminal loop, especially residue Lys6, seems to be important for cellulose production, as confirmed by in vivo assay using mutant cells with axcesD gene disruption and N-terminus truncation. Taking all results together, a model of the bacterial terminal complex is discussed.

Structural snapshot of a glycoside hydrolase family 8 endo-ß-1,4-glucanase capturing the state after cleavage of the scissile bond.

Bacterial cellulose (BC), which is produced by bacteria, is a biodegradable and biocompatible natural resource. Because of its remarkable physicochemical properties, BC has attracted attention for the development and manufacture of biomedical and industrial materials. In the BC production system, the enzyme endo-ß-1,4-glucanase, which belongs to glycoside hydrolase family 8 (GH8), acts as a cleaner by trimming disordered cellulose fibers to produce high-quality BC. Understanding the molecular mechanism of the endo-ß-1,4-glucanase would help in developing a reasonable biosynthesis of BC. Nevertheless, all of the steps in the reaction of this endo-ß-1,4-glucanase are not clear. This study confirms the BC hydrolytic activity of the endo-ß-1,4-glucanase from the BC-producing bacterium Enterobacter sp. CJF-002 (EbBcsZ) and reports crystal structures of EbBcsZ. Unlike in previously reported GH8 endo-ß-1,4-glucanase structures, here the base catalyst was mutated (D242A) and the structure of this mutant bound to cellooligosaccharide [EbBcsZ(D242A)CPT] was analyzed. The EbBcsZ(D242A)CPT structure showed two cellooligosaccharides individually bound to the plus and minus subsites of EbBcsZ. The glucosyl unit in subsite -1 presented a distorted 5S1 conformation, a novel snapshot of a state immediately after scissile-bond cleavage. In combination with previous studies, the reaction process of endo-ß-1,4-glucanase is described and the ß-1,4-glucan-trimming mechanism of EbBcsZ is proposed. The EbBcsZ(D242A)CPT structure also showed an additional ß-1,4-glucan binding site on the EbBcsZ surface, which may help to accept the substrate.

Economic Evaluation of First-Line Pertuzumab Therapy in Patients with HER2-Positive Metastatic Breast Cancer in Japan.

OBJECTIVE: The purpose of this analysis was to evaluate the cost effectiveness of the combination of pertuzumab, trastuzumab, and docetaxel (PTD) for the treatment of patients with human epidermal growth factor receptor-2 (HER2)-positive breast cancer in Japan. METHODS: A partitioned survival analysis model was developed to predict costs and quality-adjusted life-years (QALYs) in a PTD arm and a trastuzumab plus docetaxel (TD) arm. Direct medical costs were considered from the perspective of the Japanese healthcare system. The time horizon of the model was set to 20 years. Data on overall survival and progression-free survival were derived from the CLEOPATRA trial. Cost parameters were estimated using a real-world claims database. Utilities were derived from published sources outside Japan. The incremental cost-effectiveness ratio (ICER) of PTD therapy compared with TD therapy was estimated. Sensitivity analysis was conducted to assess the uncertainty in parameter settings. RESULTS: Compared with TD therapy, PTD therapy incurred an additional cost of $US174,479 and conferred an additional 0.949 QALYs. This resulted in an ICER of $US183,901 per QALY gained. Utility weights for progression-free survival and progressed disease had a relatively large impact on the base-case result, but the ICERs remained higher than $US75,000 per QALY over the full range of model parameters. Based on a probabilistic sensitivity analysis, the probability that PTD is cost effective was estimated to be 3.3%. CONCLUSIONS: Applying a willingness-to-pay threshold of $US75,000 per QALY, PTD therapy as first-line therapy would not be cost effective. Further research is required on utilities and clinical benefits for Japanese patients with breast cancer.

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Crystallization and preliminary X-ray studies of azoreductases from Bacillus sp. B29.

Azoreductases from Bacillus sp. B29 are NADH-dependent flavoenzymes which contain a flavin mononucleotide (FMN) as a prosthetic group and exist as homodimers composed of 23 kDa subunits. These enzymes catalyze the reductive degradation of various azo compounds by a ping-pong mechanism. In order to determine the structure-function relationship of the azo-dye reduction mechanism, an X-ray crystallographic study of azoreductases was performed. Selenomethionine-labelled AzrA (SeMet-AzrA) and AzrC were crystallized by the hanging-drop vapour-diffusion method. A crystal of SeMet-AzrA diffracted to 2.0 A resolution and was determined to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.9, b = 69.0, c = 105.4 A. The native crystals of AzrC belonged to space group C2, with unit-cell parameters a = 192.0, b = 56.6, c = 105.5 A, beta = 115.7 degrees , and diffracted to 2.21 A resolution.

Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone.

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.

High-viscosity sample-injection device for serial femtosecond crystallography at atmospheric pressure.

A sample-injection device has been developed at SPring-8 Angstrom Compact Free-Electron Laser (SACLA) for serial femtosecond crystallography (SFX) at atmospheric pressure. Microcrystals embedded in a highly viscous carrier are stably delivered from a capillary nozzle with the aid of a coaxial gas flow and a suction device. The cartridge-type sample reservoir is easily replaceable and facilitates sample reloading or exchange. The reservoir is positioned in a cooling jacket with a temperature-regulated water flow, which is useful to prevent drastic changes in the sample temperature during data collection. This work demonstrates that the injector successfully worked in SFX of the human A2A adenosine receptor complexed with an antagonist, ZM241385, in lipidic cubic phase and for hen egg-white lysozyme microcrystals in a grease carrier. The injection device has also been applied to many kinds of proteins, not only for static structural analyses but also for dynamics studies using pump-probe techniques.

Crystal Structures of Human Orexin 2 Receptor Bound to the Subtype-Selective Antagonist EMPA.

Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.

A three-dimensional movie of structural changes in bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

Inhibition of lens epithelial cell migration at the intraocular lens optic edge: role of capsule bending and contact pressure.

PURPOSE: To evaluate the inhibitory effect of a sharp intraocular lens (IOL) optic edge, a sharp capsule bend, and contact pressure between the optic edge and posterior capsule on lens epithelial cell (LEC) migration. SETTING: Department of Ophthalmology, Kyorin University, Tokyo, Japan. METHODS: This in vitro laboratory study evaluated a tumble-polished convex-plano IOL (CP group), an AcrySof IOL (Alcon) with a sharp edge (AS group), a new IOL with a round ridge (RR group), and a new IOL with a sharp ridge (SR group). The 2 new IOLs have high ridges and high angled loops that create firm contact between the ridge and posterior capsule. After sham cataract surgery, an IOL and a capsular tension ring (CTR) were implanted in the capsular bag of rabbit eyes. The extracted capsular bags containing the CTR and IOL were cultured. The inhibitory effect of each IOL on cell migration was analyzed. Furthermore, LEC migration on the posterior capsule was compared in culture between capsules having a sharp right angle and those with gradually curving bends. RESULTS: The inhibitory effect on cell migration was statistically greatest in the SR group followed by the RR, AS, and CP groups. A sharp capsule bend did not inhibit cell migration. CONCLUSIONS: The results suggest that inhibition of cell migration at the optic edge is regulated by the degree of contact pressure between the optic edge and posterior capsule. A sharp capsule bend might indicate strong contact but does not in itself inhibit cell migration.

[Results of surgery on white cataract using trypan blue capsule staining to visualize capsulorrhexis].

PURPOSE: To evaluate surgery on white cataracts using trypan blue capsule staining. METHODS: A retrospective study comprised 64 eyes of 60 patients with white cataract that had surgery with trypan blue capsule staining. The average age was 62.4 years and progress observation periods were 5.6 months. The rate of successful continuous curvilinear capsulorrhexis(CCC), complications, visual acuity, intraocular pressure(IOP), flare value, and corneal endothelial cell loss were studied. RESULTS: The CCC was completed uneventfully in 93.8% eyes. Posterior capsule rupture occurred in 2 eyes, and early perforation in 1 eye. Accidental vitreous staining and endothelial staining with trypan blue were observed in 1 eye each. There were no postoperative complications associated with trypan blue. Forty-five eyes had a best corrected visual acuity of 0.8 or better at the last visit. Twelve eyes had some ocular pathology resulting in visual loss, and a reliable visual acuity test could not be performed in 6 eyes. The mean postoperative IOP was within the normal range. The mean postoperative flare returned to within normal range 1 month after surgery. The mean corneal endothelial loss was 13.7%, and that of eyes with nucleus of grade 2 or softer was only 2.9%. CONCLUSIONS: Cataract surgery using trypan blue was safe and effective in managing white cataracts.

[Vitrectomy for endophthalmitis after cataract surgery].

PURPOSE: To identify risk factors of poor visual outcome with vitrectomy for early-onset endophthalmitis after cataract surgery. PATIENTS AND METHODS: Clinical records of 29 consecutive eyes with endophthalmitis developing within 6 weeks after cataract surgery and that underwent therapeutic vitrectomy between June 1996 and April 2001 were retrospectively reviewed. Twenty-two of the eyes received intravitreal injections of vancomycin and ceftazidime at the time of vitrectomy, and all patients received intravenous antibiotics. Eyes were divided into two groups; group A consisted of 22 eyes with a final visual acuity of 0.2 or greater, and group B consisted of 7 eyes with a final visual acuity of less than 0.2. RESULTS: Fifteen eyes (52%) in group A achieved a visual acuity of 0.5 or better and 8(28%) achieved a visual acuity of 1.0, while 4 eyes in group B developed phthisis bulbi. For eyes with a preoperative visual acuity of hand motions or worse, there was no correlation between final visual acuity and preoperative visual acuity. The overall culture-positive rate was 57%. In group A, methicillin-resistant Staphylococcus epidermidis was identified in 6 eyes, methicillin-resistant Staphylococcus aureus (MRSA) in 3 eyes and enterococcus in 2 eyes. In group B, alpha-hemolytic streptococcus (AHS) was identified in 4 eyes, aspergillus in 1 eye, and MRSA in 1 eye. All isolates were sensitive to vancomycin with the exception of the aspergillus. AHS infection appeared to be associated with wound failure from the initial cataract surgery and a poor visual outcome. Among 3 of the eyes that developed phthisis bulbi, intravitreal injection of antibiotics was not performed. CONCLUSION: Early vitrectomy and intravitreal injection of vancomycin may improve visual outcomes, but infection with AHS may be associated with cataract surgery wound failure and poor visual outcomes.

Crystal structure of Ruminococcus albus cellobiose 2-epimerase: structural insights into epimerization of unmodified sugar.

Enzymatic epimerization is an important modification for carbohydrates to acquire diverse functions attributable to their stereoisomers. Cellobiose 2-epimerase (CE) catalyzes interconversion between d-glucose and d-mannose residues at the reducing end of ß-1,4-linked oligosaccharides. Here, we solved the structure of Ruminococcus albus CE (RaCE). The structure of RaCE showed strong similarity to those of N-acetyl-D-glucosamine 2-epimerase and aldose-ketose isomerase YihS with a high degree of conservation of residues around the catalytic center, although sequence identity between them is low. Based on structural comparison, we found that His184 is required for RaCE activity as the third histidine added to two essential histidines in other sugar epimerases/isomerases. This finding was confirmed by mutagenesis, suggesting a new catalytic mechanism for CE involving three histidines.

Cellulose complementing factor (Ccp) is a new member of the cellulose synthase complex (terminal complex) in Acetobacter xylinum.

The cellulose complementing factor (Ccp) is known to be involved in cellulose production in the Acetobacter species. However, its precise functions remain unclear. In the current study, we identified the coding region of the ccpAx gene (ccp gene from Acetobacter xylinum) and the localization of the CcpAx in cells by generating fusion proteins tagged to an enhanced green fluorescent protein (EGFP). From the results of N-terminal sequencing of CcpAx-EGFP-fusion protein, which recovered 65% of cellulose-producing abilities of the wild-type to the ccpAx gene-knockout mutant, the ccpAx gene was determined to encode a protein with the molecular weight of 8 kDa. The amino acid sequence deduced had high similarities with the C-terminal regions of Ccp proteins from other Acetobacter species. Fluorescence microscopy analysis showed that CcpAx was longitudinally localized along with one side of the cell membrane. Additionally, the localization of AxCeSD, which is thought to be a member of the cellulose synthase complex [terminal complex (TC)] in A. xylinum, was determined in the same manner as CcpAx. Fluorescence microscopy analysis showed that AxCeSD had a localization pattern similar to that of CcpAx. Pulldown assays and isothermal titration calorimetry analysis clearly showed a significant interaction between CcpAx and AxCeSD. Taken together, these data strongly suggest that CcpAx functions as a member of the TC in A. xylinum.

Inhibition of posterior capsule opacification by a capsular adhesion-preventing ring.

OBJECTIVE: To assess the inhibitory effect of a capsular adhesion-preventing ring (CAPR) that facilitates aqueous humor circulation into the capsular bag on posterior capsule opacification (PCO) formation after cataract surgery. METHODS: After phacoemulsification, a polymethyl methacrylate intraocular lens with (n=5) or without (n=5) a CAPR was implanted in rabbit eyes. The inhibitory effect of the CAPR on PCO formation was assessed by stereoscopic microscopy and histologic examination 8 weeks after surgery. RESULTS: All eyes in which a CAPR was implanted demonstrated remarkably less PCO than the control eyes. Neither anteroposterior capsular adhesion nor regeneration of lens fiber occurred in 2 eyes in the CAPR group. The remaining 3 eyes with a CAPR showed partial capsular adhesion and limited lens fiber regeneration in the resultant closed capsular space. CONCLUSIONS: The CAPR appears to prevent PCO formation by separating the anterior and posterior capsules and allowing circulation of aqueous humor, including growth inhibitory factors, into the equatorial space of the capsule through the holes and grooves in the ring. CLINICAL RELEVANCE: A CAPR may be useful for preventing PCO in the clinical setting.