Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.

Twisting and Protonation of Retinal Chromophore Regulate Channel Gating of Channelrhodopsin C1C2.

Channelrhodopsins (ChRs) are light-gated ion channels and central optogenetic tools that can control neuronal activity with high temporal resolution at the single-cell level. Although their application in optogenetics has rapidly progressed, it is unsolved how their channels open and close. ChRs transport ions through a series of interlocking elementary processes that occur over a broad time scale of subpicoseconds to seconds. During these processes, the retinal chromophore functions as a channel regulatory domain and transfers the optical input as local structural changes to the channel operating domain, the helices, leading to channel gating. Thus, the core question on channel gating dynamics is how the retinal chromophore structure changes throughout the photocycle and what rate-limits the kinetics. Here, we investigated the structural changes in the retinal chromophore of canonical ChR, C1C2, in all photointermediates using time-resolved resonance Raman spectroscopy. Moreover, to reveal the rate-limiting factors of the photocycle and channel gating, we measured the kinetic isotope effect of all photoreaction processes using laser flash photolysis and laser patch clamp, respectively. Spectroscopic and electrophysiological results provided the following understanding of the channel gating: the retinal chromophore highly twists upon the retinal Schiff base (RSB) deprotonation, causing the surrounding helices to move and open the channel. The ion-conducting pathway includes the RSB, where inflowing water mediates the proton to the deprotonated RSB. The twisting of the retinal chromophore relaxes upon the RSB reprotonation, which closes the channel. The RSB reprotonation rate-limits the channel closing.

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.

Consensus mutagenesis approach improves the thermal stability of system xc - transporter, xCT, and enables cryo-EM analyses.

System xc - is an amino acid antiporter that imports L-cystine into cells and exports intracellular L-glutamate, at a 1:1 ratio. As L-cystine is an essential precursor for glutathione synthesis, system xc - supports tumor cell growth through glutathione-based oxidative stress resistance and is considered as a potential therapeutic target for cancer treatment. System xc - consists of two subunits, the light chain subunit SLC7A11 (xCT) and the heavy chain subunit SLC3A2 (also known as CD98hc or 4F2hc), which are linked by a conserved disulfide bridge. Although the recent structures of another SLC7 member, L-type amino acid transporter 1 (LAT1) in complex with CD98hc, have provided the structural basis toward understanding the amino acid transport mechanism, the detailed molecular mechanism of xCT remains unknown. To revealthe molecular mechanism, we performed single-particle analyses of the xCT-CD98hc complex. As wild-type xCT-CD98hc displayed poor stability and could not be purified to homogeneity, we applied a consensus mutagenesis approach to xCT. The consensus mutated construct exhibited increased stability as compared to the wild-type, and enabled the cryoelectron microscopy (cryo-EM) map to be obtained at 6.2 Å resolution by single-particle analysis. The cryo-EM map revealed sufficient electron density to assign secondary structures. In the xCT structure, the hash and arm domains are well resolved, whereas the bundle domain shows some flexibility. CD98hc is positioned next to the xCT transmembrane domain. This study provides the structural basis of xCT, and our consensus-based strategy could represent a good choice toward solving unstable protein structures.

Neutrophil Elastase Inhibition Ameliorates Endotoxin-induced Myocardial Injury Accompanying Degradation of Cardiac Capillary Glycocalyx.

Myocardial injury in sepsis may be caused by a burst of several inflammatory mediators, leading to vascular endothelial injuries. However, the contribution of neutrophil elastase (NE) to myocardial injury in sepsis is still unknown. We aimed to evaluate whether endotoxemia-induced myocardial injury is associated with NE. Lipopolysaccharide (LPS) was injected intraperitoneally at a dose of 20 mg/kg into granulocyte-colony-stimulating-factor knockout mice (G-CSF-KO), which have few neutrophils, and littermate control mice. The survival rate of G-CSF-KO mice 48 hours after LPS injection was significantly greater than that of control mice. The serum level of troponin I in G-CSF-KO mice was significantly lower than that in control mice. In addition, the concentration of inflammatory cytokine interleukin-6 (IL-6) was significantly decreased 6 and 12 hours after LPS administration compared with that in control mice. Ultrastructural analysis revealed that vascular endothelial structures and the endothelial glycocalyx in G-CSF-KO mice were clearly preserved. Next, mice were injected with 0.2 mg/kg sivelestat (an NE inhibitor) after LPS administration. The survival rate was significantly higher and the serum level of troponin I was lower in sivelestat-injected mice than in control mice, respectively. Furthermore, IL-6 levels were significantly decreased 6 and 12 hours after LPS administration compared with those in control mice. Vascular endothelial structures and the endothelial glycocalyx in sivelestat-treated mice were clearly preserved at the ultrastructural level. In conclusion, NE is significantly associated with myocardial injury in endotoxemia. Inhibition of NE may be a useful tool for the management of endotoxemia.

Factors Enhancing Serum Syndecan-1 Concentrations: A Large-Scale Comprehensive Medical Examination.

Endothelial disorders are related to various diseases. An initial endothelial injury is characterized by endothelial glycocalyx injury. We aimed to evaluate endothelial glycocalyx injury by measuring serum syndecan-1 concentrations in patients during comprehensive medical examinations. A single-center, prospective, observational study was conducted at Asahi University Hospital. The participants enrolled in this study were 1313 patients who underwent comprehensive medical examinations at Asahi University Hospital from January 2018 to June 2018. One patient undergoing hemodialysis was excluded from the study. At enrollment, blood samples were obtained, and study personnel collected demographic and clinical data. No treatments or exposures were conducted except for standard medical examinations and blood sample collection. Laboratory data were obtained by the collection of blood samples at the time of study enrolment. According to nonlinear regression, the concentrations of serum syndecan-1 were significantly related to age (p = 0.016), aspartic aminotransferase concentration (AST, p = 0.020), blood urea nitrogen concentration (BUN, p = 0.013), triglyceride concentration (p < 0.001), and hematocrit (p = 0.006). These relationships were independent associations. Endothelial glycocalyx injury, which is reflected by serum syndecan-1 concentrations, is related to age, hematocrit, AST concentration, BUN concentration, and triglyceride concentration.

Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc.

The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.

Neutrophil Elastase Damages the Pulmonary Endothelial Glycocalyx in Lipopolysaccharide-Induced Experimental Endotoxemia.

Neutrophil elastase (NE) is necessary for effective sterilization of phagocytosed bacterial and fungal pathogens; however, NE increases alveolocapillary permeability and induces proinflammatory cytokine production in sepsis-induced acute respiratory distress syndrome. Under septic conditions, the pulmonary endothelial glycocalyx covering on the healthy endothelium surface is injured, but the contribution of NE to this injury remains unknown. Our aim was to examine whether NE-induced pulmonary endothelial injury is associated with endotoxemia. Lipopolysaccharide (LPS; 20 mg/kg) was injected intraperitoneally into 9- to 12-week-old granulocyte colony-stimulating factor knockout (G-CSFKO) mice, which harbor few neutrophils, and littermate control mice; in a second assay, mice were injected with the NE-inhibitor sivelestat (0.2 mg/kg) at 3, 6, 9, and 12 hours after LPS administration. Subsequently, vascular endothelial injury was evaluated through ultrastructural analysis. At 48 hours after LPS injection, survival rate was more than threefold higher among G-CSFKO than control mice, and degradation of both thrombomodulin and syndecan-1 was markedly attenuated in G-CSFKO compared with control mice. Ultrastructural analysis revealed attenuated vascular endothelial injury and clear preservation of the endothelial glycocalyx in G-CSFKO mice. Moreover, after LPS exposure, survival rate was approximately ninefold higher among sivelestat-injected mice than control mice, and sivelestat treatment potently preserved vascular endothelial structures and the endothelial glycocalyx. In conclusion, NE is associated with pulmonary endothelial injury under LPS-induced endotoxemic conditions.

Crystal structure of the red light-activated channelrhodopsin Chrimson.

Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Red-light-activated channelrhodopsins are of particular interest, because red light penetrates deeper into biological tissues and also enables dual-color experiments in combination with blue-light-activated optogenetic tools. Here we report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama, Chrimson, at 2.6 Å resolution. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for Chrimson's red light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, we engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics.

Three-dimensional ultrastructure of capillary endothelial glycocalyx under normal and experimental endotoxemic conditions.

BACKGROUND: Sugar-protein glycocalyx coats healthy endothelium, but its ultrastructure is not well described. Our aim was to determine the three-dimensional ultrastructure of capillary endothelial glycocalyx in the heart, kidney, and liver, where capillaries are, respectively, continuous, fenestrated, and sinusoidal. METHODS: Tissue samples were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx. RESULTS: Scanning and transmission electron microscopy revealed that the endothelial glycocalyx layer in continuous and fenestrated capillaries was substantially thicker than in sinusoids. In the heart, the endothelial glycocalyx presented as moss- or broccoli-like and covered the entire luminal endothelial cell surface. In the kidney, the glycocalyx appeared to nearly occlude the endothelial pores of the fenestrated capillaries and was also present on the surface of the renal podocytes. In sinusoids of the liver, glycocalyx covered not only the luminal side but also the opposite side, facing the space of Disse. In a mouse lipopolysaccharide-induced experimental endotoxemia model, the capillary endothelial glycocalyx was severely disrupted; that is, it appeared to be peeling off the cells and clumping. Serum concentrations of syndecan-1, a marker of glycocalyx damage, were significantly increased 24 h after administration of lipopolysaccharide. CONCLUSIONS: In the present study, we visualized the three-dimensional ultrastructure of endothelial glycocalyx in healthy continuous, fenestrated, and sinusoidal capillaries, and we also showed their disruption under experimental endotoxemic conditions. The latter may provide a morphological basis for the microvascular endothelial dysfunction associated with septic injury to organs.

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.

Initial central venous pressure could be a prognostic marker for hemodynamic improvement of polymyxin B direct hemoperfusion: a retrospective cohort study.

BACKGROUND: Direct hemoperfusion with polymyxin B-immobilized fiber column (PMX-DHP) could improve the hemodynamic status of septic shock patients. As PMX-DHP is an invasive and costly procedure, it is desirable to estimate the therapeutic effect before performing the therapy. However, it is still unclear when this therapy should be started and what type of sepsis it should be employed for. In this study, we retrospectively examined the clinical effect of patients treated with PMX-DHP by using central venous pressure (CVP). METHODS: Seventy patients who received PMX-DHP for septic shock during the study period were recruited and divided into a low CVP group (n = 33, CVP < 12 mmHg) and a high CVP group (n = 37, CVP≧12 mmHg). The primary endpoint was vasopressor dependency index at 24 hours after starting PMX-DHP, and the secondary endpoint was the 28-day survival rate. Additionally, we performed a multivariate linear regression analysis on the difference in the vasopressor dependency index. RESULTS: The vasopressor dependency index significantly improved at 24 h in the low CVP group (0.33 to 0.16 mmHg-1; p < 0.01) but not in the high CVP group (0.43 to 0.34 mmHg-1; p = 0.41), and there was a significant difference between the two groups in the index at 24 h (p = 0.02). The 28-day survival rate was higher in the low CVP group (79 vs. 43 %; p < 0.01). Multivariate linear regression analysis showed that CVP (p = 0.04) was independently associated with the difference in the vasopressor dependency index. CONCLUSIONS: Our study indicates that the clinical effect of PMX-DHP for septic shock patients with higher CVP (≧12 mmHg) might be limited and that the initial CVP when performing PMX-DHP could function as an independent prognostic marker for the hemodynamic improvement.