Molecular structure and function of bacterial nitric oxide reductase

AbstractThe crystal structure of the membrane-integrated nitric oxide reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and a hydrophilic domain, where the heme c is located, while the larger NorB subunit consists of 12 trans-membrane helices, which contain heme b and the catalytically active binuclear center (heme b3 and non-heme FeB). The roles of the 5 well-conserved glutamates in NOR are discussed, based on the recently solved structure. Glu211 and Glu280 appear to play an important role in the catalytic reduction of NO at the binuclear center by functioning as a terminal proton donor, while Glu215 probably contributes to the electro-negative environment of the catalytic center. Glu135, a ligand for Ca2+ sandwiched between two heme propionates from heme b and b3, and the nearby Glu138 appears to function as a structural factor in maintaining a protein conformation that is suitable for electron-coupled proton transfer from the periplasmic region to the active site. On the basis of these observations, the possible molecular mechanism for the reduction of NO by cNOR is discussed. This article is part of a Special Issue entitled: Respiratory Oxidases

Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp A11-2, reveal potential for expanding its substrate selectivity

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate–FMN–enzyme complex remains unknown. Herein, we report the crystal structures of the DBT–FMN–TdsC and DBTO–FMN–TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications

Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex

熱、炎症などに関与するプロスタグランジン受容体EP3シグナリング複合体の可視化 --緑内障、高眼圧症治療薬の合理的設計に貢献--. 京都大学プレスリリース. 2022-09-15.Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E₂. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5

Liposome collapse resulting from an allosteric interaction between 2,6‐dimethyl‐β‐cyclodextrins and lipids

Although heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin (DMe‐β‐CDx) has been reported to exhibit higher cytotoxicity than many other cyclodextrins because of the way in which it abstracts cholesterols from liposomes, we have identified another reason for its cytotoxicity based on its interaction with lipids. These interactions exhibited nonlinear sigmoidal responses with Hill coefficient values (n) in the range of 3.0‒3.6, which indicated that this phenomenon involves positive allosterism. Furthermore, analysis by mass spectroscopy revealed that the lipid•DMe‐β‐CDx complexes had stoichiometric ratios in the range of 1:1‒1:4.Electronic supplementary information (ESI) available: Average hydrodynamic diameter values and UV-vis absorption, 1H NMR, NOESY and CSI-MS spectra. See DOI: 10.1039/c5ra14970cThis work was supported by a JSPS KAKENHI a Grant‐in‐Aid for Scientific Research (B) (Grant No. 25288037)

Crystal structure of the anion exchanger domain of human erythrocyte band 3

Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1CTD) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1CTD, and to propose a possible transport mechanism that could explain why selected mutations lead to disease

Porphyrin‐uptake in liposomes and living cells using an exchange method with cyclodextrin

The water‐solubilisation of porphyrin derivatives is very important for biological applications. Although liposomal drug carriers for porphyrin derivatives have shown significant promise in the field of medicinal chemistry (e.g., as sensitisers for photodynamic therapy), it is currently not possible to prepare lipid‐membrane‐incorporated tetraphenylporphyrin (TPP) with a high concentration of TPP using conventional methods. In this study, we have succeeded in preparing lipid-membrane‐incorporated TPP and zinc(II) tetraphenylporphyrin (ZnTPP) from the corresponding TPP or ZnTPP•cyclodextrin complex using the exchange method in lipid‐membranes composed of liposomes. Furthermore, the exchange method allowed for the incorporation of TPP or ZnTPP into the plasma membranes of HeLa cells. However, it was not possible to prepare lipid‐membrane‐incorporated porphyrin derivatives with polar and hydrophilic groups in the meso positions using this exchange reaction.Electronic supplementary information (ESI) available: Experimental procedures, 1H NMR spectra, DLS measurements, cryo-TEM images, phase contrast and fluorescence images. See DOI: 10.1039/c5ra24985This work was supported by JSPS KAKENHI a Grant‐in‐Aid for Scientific Research (B) (Grant No. 25288037) and a Grant‐in‐Aid for Young Scientists (A) (Grant No. 24681028)

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme

Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography

温度による酵素の構造変化を分子動画撮影 様々な生体高分子のダイナミクスを決定する新たな方法論. 京都大学プレスリリース. 2023-09-19.Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics

通常型膵癌進展におけるFibroblast activation protein α 発現に関する臨床病理学的検討

通常型膵癌(以下膵癌)は極めて予後不良な癌であり,病理学的には癌周囲に豊富な間質を伴う。固形癌の間質は癌の増殖,浸潤,転移や抗癌剤に対する抵抗性獲得に重要な役割を果たし,なかでも癌関連線維芽細胞(Carcinoma associated fibroblast:CAF)と呼ばれる癌間質の活性化線維芽細胞は癌進展に深く関与している.今回我々は膵癌組織のCAFに発現するFibroblast activation protein α(以下FAP)の臨床病理学的な意義について検討した.対象は2006年4月から2010年4月の間に当院で切除した膵癌症例30例である.抗FAP抗体を用いて免疫組織学的検討を行った.FAPは主として癌周囲の間質に検出され,一部には膵癌細胞にも認められたが,今回の検討ではFAP陽性の線維芽細胞をCAFとみなし,線維芽細胞のFAP発現強度と臨床病理学的因子を比較検討した.FAP陽性線維芽細胞は28/30例(93%)に認められた.FAP発現強度の内訳は,1+:8例,2+:16 例,3+:4例であった.陰性/弱陽性群(陰性・1+)と強陽性群(2+・3+)での生存率を比較したところ,それぞれの全生存日数の中央値は862日と352日であり強陽性群の生存率は有意に低かった(P<0.05).またFAPの発現強度と膵癌細胞の分化度は有意な関連性を示し,FAPの発現が強いほど有意に低分化であった.さらに多変量解析ではFAPの強陽性は膵癌患者の予後を規定する独立因子の一つであった.以上の成績から膵癌組織の線維芽細胞におけるFAPの発現は膵癌患者の予後を規定する重要なバイオマーカーであることが明らかとなり,膵癌細胞の分化度と相関することを初めて明らかにした.Pancreatic cancer is associated with an extremely poor prognosis, and pathologically shows abundant stroma surrounding cancer cells. The stroma of solid cancer plays an important role in cancer growth, invasion, metastasis, and the acquisition of resistance to anticancer drugs. In particular, activated fibroblasts in the cancer stroma called carcinomaassociated fibroblasts (CAFs) are closely involved in cancer progression. In this study, we evaluated the clinicopathological importance of fibroblast activation proteinα (FAP) that is expressed in CAFs in pancreatic cancer tissue. The subjects consisted of 30 patients with pancreatic cancer who underwent resection in our hospital between April 2006 and April 2010.Immunohistological evaluation was performed using the anti-FAP antibody.FAP was detected mainly in the stroma surrounding pancreatic cancer cells and partly in cancer cells. In this study, FAP-positive fibroblasts were regarded as CAFs, and the associations between the FAP expression level in fibroblasts and clinicopathological factors were evaluated. FAP-positive fibroblasts were observed in 28(93%) of the 30 patients. The FAP expression level was 1+ in 8 patients, 2+ in 16, and 3+ in 4. The survival rate was compared between the negative/weakly positive group (negative, 1+) and strongly positive group (2+, 3+). The medians of all survival periods were 862 and 352 days in the negative/weakly positive and strongly positive groups, respectively, and the survival rate was significantly lower in the latter (p<0.05). In addition, the FAP expression level was significantly correlated with the differentiation level of pancreatic cancer cells; when the FAP expression level was higher, differentiation was significantly poorer. Multivariate analysis revealed strongly FAP-positive fibroblasts to be an independent prognostic factor in patients with pancreatic cancer. These results show that FAP expression in fibroblasts in pancreatic cancer tissue is an important prognostic biomarker in patients with pancreatic cancer and correlated with the differentiation level of pancreatic cancer cells