Crystal structure of the ϵ subunit of the proton-translocating ATP synthase from Escherichia coli

AbstractBackground: Proton-translocating ATP synthases convert the energy generated from photosynthesis or respiration into ATP. These enzymes, termed F0F1-ATPases, are structurally highly conserved. In Escherichia coli, F0F1-ATPase consists of a membrane portion, F0, made up of three different polypeptides (a, b and c) and an F1 portion comprising five different polypeptides in the stoichiometry α3β3γδϵ. The minor subunits γ, δ and ϵ are required for the coupling of proton translocation with ATP synthesis; the ϵ subunit is in close contact with the α, β , γ and c subunits. The structure of the ϵ subunit provides clues to its essential role in this complex enzyme.Results: The structure of the E. coli F0F1-ATPase ϵ subunit has been solved at 2.3 Å resolution by multiple isomorphous replacement. The structure, comprising residues 2–136 of the polypeptide chain and 14 water molecules, refined to an R value of 0.214 (Rfree = 0.288). The molecule has a novel fold with two domains. The N-terminal domain is a β sandwich with two five-stranded sheets. The C-terminal domain is formed from two α helices arranged in an antiparallel coiled-coil. A series of alanine residues from each helix form the central contacting residues in the helical domain and can be described as an ‘alanine zipper’. There is an extensive hydrophobic contact region between the two domains providing a stable interface. The individual domains of the crystal structure closely resemble the structures determined in solution by NMR spectroscopy.Conclusions: Sequence alignments of a number of ϵ subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function. In the crystal the N-terminal domains of two ϵ subunits make a close hydrophobic interaction across a crystallographic twofold axis. This region has previously been proposed as the contact surface between the ϵ and γ subunits in the complete F1-ATPase complex. In the crystal structure, we observe what is apparently a stable interface between the two domains of the ϵ subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing. This suggests that a gross conformational change in the ϵ subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out

Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to 2′-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C[subscript 439]) in α2 by a stable diferric tyrosyl radical (Y[subscript 122]•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y[subscript 122]•→ W[subscript 48]→ Y[subscript 356] in β2 to Y[subscript 731]→ Y[subscript 730]→ C[subscript 439] in α2. To study the details of this process, 3-aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated in place of Y[subscript 356] of β. The resulting protein, Y[subscript 356]NH[subscript 2]Y-β2, and the previously generated proteins Y[subscript 731]NH[subscript 2]Y-α2 and Y[subscript 730]NH[subscript 2]Y-α2 (NH[subscript 2]Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2–0.7 s[superscript –1]. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH[subscript 2]Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH[subscript 2]Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH[subscript 2]Y• formation is biphasic (k[subscript fast] of 9–46 s[superscript –1] and k[subscript slow] of 1.5–5.0 s[subscript –1]) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH[subscript 2]Y• formation, while the k[subscript cat] of 0.5 s[superscript –1] is proposed to be associated with rate-limiting oxidation by NH[subscript 2]Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y[subscript 730]NH[subscript 2]Y-α2 and Y[subscript 731]NH[subscript 2]Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.National Institutes of Health (U.S.) (GM29595

Evaluation of an individual sleep intervention programme in people undergoing peritoneal dialysis treatment

Objective: This study aimed at evaluate effects of a individually designed nonpharmacological intervention on sleep, activity and fatigue in peritoneal dialysis (PD) by use of both actigraphy registration and self-assessed questionnaires. Design: A prospective multiple baseline single-case experimental design. Methods: Two women and seven men with sleep problems, 48-77 years, treated with PD participated in a 17-week study. Two interventions were separately implemented. First, a pressure relieving mattress and second, a four week individual sleep hygiene and sleep scheduling intervention. The two interventions were evaluated both objectively by actigraphy and subjectively by questionnaires. Results: Totally 315 sleep-wake cycles from nine individuals were evaluated. Of the nine measured outcome variables i.e. sleep onset latency, nocturnal sleep duration, numbers and duration of napping, movement and fragmentation index (MFI), number of steps, metabolic equivalent unit (METs), sleep efficiency and fatigue, three patients improved clinically significantly in five or more of the outcomes. The other six patients also showed improvements but to a lesser degree. Physical activity advice was the intervention that yielded most sleep improvements. Conclusions: This study shows that patients on peritoneal dialysis treatment have a wide variety of sleep problems and that an individual sleep hygiene and sleep scheduling program can be applied with clinically significant improvements even in this heterogeneous and frail patient group. The intervention should be easy to use in daily clinical routines

Fragmented sleep: An unrevealed problem in peritoneal dialysis patients

Objective. The aim of this study was to describe the sleep--wake cycle, sleep quality, fatigue and Health Related Quality of Life (HRQoL) measured with questionnaires, actigraphy and a sleep diary during a one-week period in patients undergoing peritoneal dialysis (PD) treatment at home. A further aim was to explore differences compared with patients with coronary artery disease (CAD) and individuals from the general population. Material and methods. In this study one-week actigraphy registration, four questionnaires (Uppsala Sleep Inventory, SF-36, FACIT-fatigue, International Restless Legs Study Groups form) and a sleep diary were used. Results. Data from 68 participants and 470 nights were collected. PD patients (n == 28) had more fragmented sleep (p andlt; 0.001) and worse sleep efficiency (SE%) (p andlt; 0.0001) than the CAD (n == 22) and the population (n == 18) groups. Pruritus (57%), restless legs (46%) and fatigue (89%) were prevalent in PD patients. Pruritus correlated with fragmented sleep (r == --0.45, p == 0.01) and SE (r == --0.49, p == 0.01). In HRQoL, the physical component score was decreased in the PD and CAD groups (p andlt; 0.01) compared to the population group. Conclusions. To the authors knowledge this study is the first to demonstrate that PD patients have deteriorated sleep, with serious fragmentation measured by a one-week actigraphy registration. Further, PD patients exhibit worse sleep quality than CAD patients and individuals in the population. Evaluation of sleep in clinical practice is highly recommended since PD patients are vulnerable individuals with extended self-care responsibilities and at risk for comorbidity secondary to insufficient sleep. Future research on whether PD patients sleep problems and fatigue can be improved by an individual non-pharmacological intervention programme is required.Original Publication:Pia Yngman Uhlin, Anna Johansson, Anders Fernström, Sussanne Börjeson and Ulla Edéll-Gustafsson, Fragmented sleep: An unrevealed problem in peritoneal dialysis patients, 2011, SCANDINAVIAN JOURNAL OF UROLOGY AND NEPHROLOGY, (45), 3, 206-215.http://dx.doi.org/10.3109/00365599.2011.557025Copyright: Informa Healthcarehttp://informahealthcare.com

Higher response rates in patients with severe chronic skin graft-versus-host disease treated with extracorporeal photopheresis

Introduction: Different forms of graft-versus-host disease (GVHD) remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The prognosis for steroid-refractory chronic GVHD (cGVHD) remains poor. Our aim was to evaluate extracorporeal photopheresis (ECP) treatment in cGVHD patients with different organ involvement to detect subgroups of patients with the best response. Material and methods: Thirty-four patients who underwent HSCT and developed moderate (n = 7) or severe (n = 27) steroid-refractory or steroid-dependent cGVHD treated with ECP were included in the analysis. A matched cGVHD control patient group untreated with ECP was collected for comparison. Results: Compared to the control group and the stable/progressive disease (SD/PD) patients, individuals with complete/partial remission have higher overall survival and lower transplant-related mortality. Furthermore, patients with complete and partial remission (CR/PR) had significantly higher levels of albumin and platelets after ECP treatment compared to patients with stable or progressive cGVHD (SD/PD). Corticosteroid treatment and other immunosuppressive agents could successfully be tapered in the CR/PR group compared to the SD/PD patients. In this study patients with skin cGVHD are those with the highest rate of CR/PR after ECP treatment. Conclusions: Our results suggest that ECP treatment is safe and effective for patients with predominantly skin, oral and liver cGVHD

Kinetics of Radical Intermediate Formation and Deoxynucleotide Production in 3-Aminotyrosine-Substituted Escherichia coli

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to 2′-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C[subscript 439]) in α2 by a stable diferric tyrosyl radical (Y[subscript 122]•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y[subscript 122]•→ W[subscript 48]→ Y[subscript 356] in β2 to Y[subscript 731]→ Y[subscript 730]→ C[subscript 439] in α2. To study the details of this process, 3-aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated in place of Y[subscript 356] of β. The resulting protein, Y[subscript 356]NH[subscript 2]Y-β2, and the previously generated proteins Y[subscript 731]NH[subscript 2]Y-α2 and Y[subscript 730]NH[subscript 2]Y-α2 (NH[subscript 2]Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2–0.7 s[superscript –1]. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH[subscript 2]Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH[subscript 2]Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH[subscript 2]Y• formation is biphasic (k[subscript fast] of 9–46 s[superscript –1] and k[subscript slow] of 1.5–5.0 s[subscript –1]) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH[subscript 2]Y• formation, while the k[subscript cat] of 0.5 s[superscript –1] is proposed to be associated with rate-limiting oxidation by NH[subscript 2]Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y[subscript 730]NH[subscript 2]Y-α2 and Y[subscript 731]NH[subscript 2]Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.National Institutes of Health (U.S.) (GM29595

A hot oxidant, 3-NO[subscript 2]Y[subscript 122] radical, unmasks conformational gating in reductase

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y[superscript •]) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y[subscript 122][superscript •]→W[subscript 48]→Y[subscript 356] within β to Y[subscript 731]→Y[subscript 730]→C[subscript 439] within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO[subscript 2]Y) has recently been incorporated site-specifically in place of Y[subscript 122] in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO[subscript 2]Y[superscript •]) and was inactive. In the present paper we show that incubation of apo-Y[subscript 122]NO[subscript 2]Y-β2 with Fe[superscript 2+] and O[subscript 2] generates a diferric-NO[subscript 2]Y[superscript •] that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO[subscript 2]Y[superscript •]/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO[subscript 2]Y[superscript •] is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s[superscript −1]), that dCDP is produced at 107 s[superscript −1], and that a new Y[superscript •] is produced at 97 s[superscript −1]. Studies with pathway mutants suggest that the new Y[superscript •] is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y[subscript 122]NO[subscript 2]Y-β2, a mechanism for PCET uncoupling in NO[subscript 2]Y[superscript •]-RNR is proposed.National Institutes of Health (U.S.) (Grant number GM29595