Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Microbial ion-pumping rhodopsins are integral membrane proteins that actively transport ions across membranes upon light stimulation (1). Bacteriorhodopsin (bR) and halorhodopsin (HR) are well-known microbial ion-pumping rhodopsins found in halophilic archaea (2, 3). bR is a light-driven outward proton pump and HR is a light-driven inward anion pump, specific for chloride ion. Microbial ion-pumping rhodopsins possess common structural features consisting of seven α-helices with an all-trans retinal covalently bound to a lysine residue as the chromophore, despite the transport of different ions (4). The retinal undergoes photoisomerization from the all-trans to 13-cis configuration, which initiates the photocycle accompanied by several intermediates to export ions (4, 5). Its light-controllable function is suitable for optogenetics applications for manipulating cells, such as neurons, by changing the ion concentration inside or outside the membrane (6, 7). In fact, microbial rhodopsins, including channelrhodopsins and HRs, are employed as optogenetic tools (8–10).Nonlabens marinus rhodopsin-3 (NM-R3) is a light-driven chloride pump recently discovered in a marine flavobacterium (11). It is a distinct chloride pump from HRs and shows low amino acid sequence homology with HRs (11). To date, HR-type chloride pumps have been found in haloarchaea, marine bacteria, and cyanobacteria, including Halobacterium salinarum, Natronomonas pharaonis, and Mastigocladopsins repens, with sequence identities of 20%, 21%, and 20% to NM-R3, respectively (3, 12–15). Interestingly, NM-R3 has higher sequence identity (36%) to Krokinobacter rhodopsin 2 (KR2), a sodium pump found in Krokinobacter eikastus (16). NM-R3 possesses a unique NTQ motif (Asn98, Thr102, Gln109) in the third helix (helix C), which corresponds to key residues (DTD motif, Asp85, Thr89, Asp96) for proton transport in bR (11, 17, 18) (SI Appendix, Table S1). Asp85 acts as the primary proton acceptor of bR from the protonated Schiff-base (PSB), with assistance from Thr89 and Asp96, which is the proton donor (5, 17, 18). HRs from haloarchaea have a highly conserved TSA (Thr, Ser, Ala) motif, while the Ala residue is replaced by Asp in HR from cyanobacteria (19). In the X-ray crystal structure of NM-R3 (SI Appendix, Fig. S1A), a chloride ion located between the PSB and Asn98 (SI Appendix, Fig. S1B) is stabilized by the positive charge of the PSB (20). The position of this chloride ion is similar to those in the H. salinarum HR and N. pharaonis HR (NpHR) structures except for Thr and Ser, which correspond to Asn98 and Thr102 in NM-R3, respectively (20–22). Several amino acid residues around the retinal, including Arg95, Trp99, Trp201, and Asp231, are highly conserved among ion-pumping rhodopsins. Previous spectroscopic studies suggested that NM-R3 displays a similar sequence of intermediates, with K-, L-, N-, and O-like species, as in other HRs (23) (Fig. 1A). Recently, intermediate structures of NM-R3 obtained by low-temperature trapping X-ray crystallography and serial femtosecond crystallography (SFX) have been reported (24, 25). However, the detailed ion-pump mechanism still remains unclear, due to the lack of dynamic structures of anion transport at atomic resolution.Open in a separate windowFig. 1.TR-visible absorption spectroscopy for microcrystals. (A) Photocycle model of NM-R3 in the 1 M NaCl buffer solution (23). (B) TR difference spectra ΔA upon the 532-nm excitation. The difference was calculated by subtracting the spectrum of NM-R3. (C) Global fitting analysis with two exponentials. The A₁ and A₂ amplitude spectra correspond to the differences of [ΔA_O – ΔA_{10 µs}] and [ΔA_{200 ms} − ΔA_O], respectively. Here, ΔA_O represents the difference spectrum of the O intermediate minus NM-R3. (D) The isomeric forms of the retinal chromophore in bacterial-type rhodopsins.Time-resolved serial femtosecond crystallography (TR-SFX) is a powerful tool for visualizing reactions and motions in proteins at the atomic level (26–28). In SFX, myriads of microcrystals are continuously injected by a sample injector into an irradiation point of X-ray free electron lasers (XFELs) at room temperature, thus providing diffraction patterns before the onset of radiation damage by the intense X-ray pulse. Combined with a visible-light pump laser for reaction initiation, TR-SFX has been applied to light-driven ion pumps to observe the structural dynamics during the ion transfer. While TR-SFX has revealed femto-to-millisecond structural dynamics in light-driven cation pumps, including bR and KR2 (29–31), TR-SFX studies of anion pumps have been limited to early-stage structures adopted at picoseconds after light illumination (32). In addition, although NM-R3 pumps a chloride ion (Cl⁻) as a physiological substrate, it can also transport bromide (Br⁻), iodide (I⁻), and other anions from the extracellular side to the cytoplasmic side (23). I⁻ or Br⁻ serves as a marker for tracking the positions of ions, due to the greater number of electrons, whereas Cl⁻ is less distinguishable in X-ray crystallography. Therefore, TR-SFX experiments using I⁻ or Br⁻ are expected to directly visualize the process of ion transport.Here, we report the conformational alterations in NM-R3 during Br⁻ or I⁻ pumping, obtained by both TR-SFX and time-resolved spectroscopy of crystals. The resulting sequence of movements in NM-R3 demonstrates how the chloride pump transports anions with a large ionic radius and prevents the backflow of anions from the cytoplasmic side.     

Prevalence of Burnout among Internal Medicine and Primary Care Physicians before and during the COVID-19 Pandemic in Japan

Objective To evaluate the change in the prevalence of burnout during the COVID-19 pandemic among internists and primary care physicians in Japan, and to identify factors associated with the exacerbation of burnout among these populations during this period. Methods This was a cross-sectional study based on two web-based surveys conducted in January 2020 (before the declaration of the COVID-19 pandemic) and June 2020 (during the pandemic). The participants were internists and primary care physicians of the Japanese Chapter of the American College of Physicians. The main outcome was the change in the prevalence of burnout between before and during the “first wave” of the pandemic. We also examined factors associated with the exacerbation of burnout during this period. Results Among the 283 respondents in the first survey and 322 in the second survey, 98 (34.6%) and 111 (34.5%) reported symptoms of burnout, respectively. In June 2020, 82 respondents (25.5%) reported that their level of burnout exacerbated compared to January 2020. Only the experience of self-quarantine was associated with the exacerbation of burnout [odds ratio (OR) 3.12; 95% confidence interval (CI) 1.49-6.50; p=0.002], while being a woman, being a resident physician, and an experience of having worked in a prefecture under a state of emergency were not. Conclusions No marked change in the prevalence of burnout among internists and primary care physicians in Japan was observed during the COVID-19 pandemic as a whole. However, self-quarantine was associated with the exacerbation of the burnout level.     

Pulmonary Artery Diameter (PAD) and the Pulmonary Artery to Aorta Ratio (PAD/AAD) as Assessed by Non-contrast Cardiac CT: The Association with Left Ventricular (LV) Remodeling and the LV Function

Objective Dilatation of the pulmonary artery itself (PAD: pulmonary artery diameter) or in relation to the ascending aorta (PAD/AAD: pulmonary artery diameter to ascending aortic diameter ratio) has been reported to be associated with pulmonary hypertension and with a prognostic outcome of either heart failure or cardiovascular events. We herein aimed to assess the correlations between pulmonary hypertension-related parameters PAD (or PAD/AAD) and left ventricular (LV) remodeling and LV function. Methods This retrospective study included 193 patients (ages: 67±12 years) who underwent both coronary CT angiography (CCTA) and echocardiography. The PAD and the AAD were measured on a transaxial non-contrast CCTA image at the level of the pulmonary artery bifurcation. Left ventricular mass (LVM), relative wall thickness ratio (RWT), left ventricular ejection fraction (LVEF), left atrial volume (LAV), and early mitral inflow velocity to mitral annular early diastolic velocity ratio (E/e'') were evaluated by echocardiography. The relationships between PAD (or PAD/AAD) and echocardiography parameters were assessed, and adjusted for the demographic data and cardiovascular disease (CVD) risk factors by a multivariable linear regression analysis. Results PAD (mean±SD: 2.6±0.4 cm) was positively correlated with LVM (r=0.34, p＜0.001), LAV (r=0.41, p＜0.001), and E/e'' (r=0.29, p＜0.001). PAD/AAD (mean±SD: 0.76±0.12 cm) was positively correlated with LVM (r=0.12, p=0.09), LAV (r=0.24, p＜0.001), and E/e'' (r=0.15, p=0.04). These correlations remained significant after adjusting for demographic data and CVD risk factors. PAD (or PAD/AAD) did not correlate with LVEF or RWT (p＞0.05). Conclusion Greater PAD or PAD/AAD is significantly associated with LV remodeling and an impaired LV function.     

Diagnostic accuracy of enhanced liver fibrosis test for nonalcoholic steatohepatitis-related fibrosis: Multicenter study

Aim

The enhanced liver fibrosis (ELF) test is a noninvasive method for diagnosing hepatic fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). This multicenter cohort study aimed to evaluate the accuracy of the ELF test and compare it with other noninvasive tests in Japan.

Methods

We analyzed 371 Japanese patients with biopsy-proven NAFLD. We constructed area under the receiver operator characteristic curves (AUROC) to determine the diagnostic accuracies of the ELF test, the Mac-2-binding protein glycosylation isomer (M2BPGi), the Fibrosis-4 (FIB-4) index, and combinations of these indices.

Results

In patients with F0/F1/F2/F3/F4 fibrosis, the median values of the ELF test were 8.98/9.56/10.39/10.92/11.41, respectively. The AUROCs of the ELF test for patients with F0 versus F1–4, F0–1 versus F2–4, F0–2 versus F3–4, and F0–3 versus F4 fibrosis were 0.825/0.817/0.802/0.812, respectively. The AUROCs of the ELF test were greater than those of the FIB-4 index and M2BPGi at each fibrosis stage. Respective low and high cut-off values yielded sensitivities and specificities for predicting advanced fibrosis (≥F3) of 91.1% and 50.8%, and 38.5% and 92.8%, respectively. For F3 or F4 fibrosis, the combined values from the ELF test and FIB-4 index showed a sensitivity of 98.5%, and the combined values from the ELF test and M2BPGi assay showed a specificity of 97.5%.

Conclusions

In Japan, the ELF test predicts NAFLD-related fibrosis from its early stages. The diagnostic ability of the ELF test was not inferior to that of other indices, and the combined values of ELF plus other indices were more accurate.     

Interaction of poly(styrene sulfonic acid) with the alternative pathway of the serum complement system

Bioartificial pancreas, in which the islets of Langerhans are enclosed in artificial membrane to be protected from the host immune system, is expected to be a promising medical device to treat patients who suffer from insulin-dependent diabetes. Our strategy for preparation of a bioartificial pancreas involves utilizing a membrane including polymeric materials that can inhibit the complement reaction. In this study, we examined the effects of poly(styrene sulfonic acid) (PSSa) on the alternative pathway of the serum complement system to identify the mechanism(s) involved. PSSa was dissolved in pooled normal human serum (NHS), and the mixtures were incubated at 37 degrees C for 30 min. Complement activities in sera were determined by hemolytic assays. Amounts of complement activation products released were determined by ELISA. Interactions of PSSa with complement components and fragments were examined with electrophoresis and immunoblotting. From these examinations, it appeared that the manner of PSSa effects on the alternative pathway (AP) highly depends on its concentration. PSSa seemingly acted as an activator when its concentration was 0.005 g/dl to 0.05 g/dl, while it acted as an inhibitor when its concentration was more than 0.1 g/dl. In terms of activation or inhibition of the AP, forming complex of PSSa with factor H induced activation, and that with factor D induced inhibition.