A receptors. Patients with schizophrenia show alterations in function, structure and molecular composition of the hippocampus, and a recent study demonstrated aberrant levels of hippocampal α5 subunit‐containing GABAA receptors. The purpose of this study is to investigate the effects of tricyclic compounds on α5 subunit‐containing receptor subtypes.Many psychotherapeutic drugs, including clozapine, display polypharmacology and act on GABAA receptor subtypes by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Computational structural analysis was employed to design mutated constructs of the α5 subunit, probing a novel binding site. Radioligand displacement data complemented the functional and mutational findings.Functional studies of effects by seven antipsychotic and antidepressant medications were performed in several GABAA receptor subtypes, including those containing α5‐subunits. Based on a chlorpromazine binding site observed in a GABA‐gated bacterial homologue, we identified a novel site in α5 GABAA receptor subunits and demonstrate differential usage of this and the orthosteric sites by these ligands.The antipsychotic drugs clozapine and chlorpromazine exerted functional inhibition on multiple GABAA receptor subtypes.Despite high molecular and functional similarities among the tested ligands, they reduce GABA currents by differential usage of allosteric and orthosteric sites. The chlorpromazine site we describe here is a new potential target for optimizing antipsychotic medications with beneficial polypharmacology. Further studies in defined subtypes are needed to substantiate mechanistic links between the therapeutic effects of clozapine and its action on certain GABA ECDextracellular domainELICErwinia ligand‐gated ion channelNAMnegative allosteric modulationPDBProtein Data BankPETpositron emission tomographyTBPSS]t‐butylbicyclophosphorothionate[35TEVCtwo‐electrode voltage clampTHDOCtetrahydrodeoxycorticosteroneClozapine and other tricyclic molecules reduce GABA effects at ionotropic GABA receptors.Chlorpromazine interacts with a novel site in a GABA‐gated bacterial homologue.The effects of clozapine on α5β3γ2 GABA receptors are consistent with orthosteric antagonism.3H]muscimol and interacts with a novel site in α5 subunits.Chlorpromazine does not displace muscimol in the absence or presence of 10 mM GABA . Membranes were filtered through Whatman GF/B filters and the filters were rinsed twice with 4 ml of ice‐cold 50 mM Tris/citrate buffer. Filters were transferred to scintillation vials and subjected to scintillation counting after the addition of 3 ml Rotiszint Eco plus liquid scintillation cocktail. The scintillation counter used is TriCarb 4910TR from Perkin Elmer.Frozen membranes were thawed, resuspended and incubated for 60 min at 4°C in a total of 500 μl of TC50/NaCl , various concentrations of the drug to be studied and 10 nM muscimol (NET574250UC) was purchased from PerkinElmer . All other chemicals were purchased from Sigma Aldrich.2.9http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22  for the initial functional assessment. Only current reduction was seen in the tested range with clozapine (1‐100 μM) and no enhancement or biphasic effects were observed  were known to interact with GABA3.3bicuculline, and by negative allosteric modulation from different allosteric sites such as the Bz‐site, for which a γ‐subunit is needed  in order to perform a computational exploration of the remaining candidate binding sites. GABAA receptors and the chlorpromazine‐bound ELIC β3γ2 receptors. The GABA dose response curves of α5F53Wβ3γ2, α5L222Wβ3γ2 as well as α5F53W;L122Wβ3γ2 were matching the wild‐type, in comparison to the other two that were right‐shifted Figure . Diazepan Figure . Effectsn Figure . The mosn Figure . Loxapinn Figure  and h–l.n Figure .50 of either compound  was right‐shifted in α5L222Wβ3γ2 and in the double mutant α5F53W;L122Wβ3γ2  and relatively weak for the antidepressants nortriptyline and imipramine, or levomepromazine (Figure Converging evidence points to pivotal alterations of GABA‐ergic signalling in schizophrenia (Charych et al., e Figure , which ie Figure .A receptor isoforms might separate these two drug classes.Accumulated evidence suggests a complex and probably multicausal, aetiology of the pathogenic mechanisms that drive schizophrenia symptoms, involving several neurotransmitter systems including dopamine, GABA and glutamate (Charych et al., A receptors are strong candidates, and strongly inhibited by the antipsychotics we tested. Molecules which hit ‘classical’ targets such as 2 receptorsD and GABAA receptors may thus be an attractive alternative to the strategy that drove the development of basmisanil, namely, to augment antipsychotics with GABA‐ergics. Our observation that the degree of functional inhibition we observe in vitro appears to correlate with antipsychotic efficacy is very exciting, but definitely requires systematic investigation with a larger number of compounds and with additional methods in order to substantiate a link between their GABAA receptor effects and their therapeutic benefits. The findings of this study further emphasize the need to identify and characterize allosteric sites which may potentially be targeted and prove useful to avoid the toxicological effects associated with the orthosteric site.In conclusion, existing evidence suggests a ‘therapeutic portfolio’ mode of action of antipsychotic medications. The exact configuration of an antipsychotic target portfolio remains to be elucidated and is likely to contain both metabotropic and ionotropic receptors Figure . HippocaK.B., M.W., and M.E. conceived the study. K.B. planned and supervised electrophysiological experiments. K.B., L.S., S.R., F.D.V., and F.Z. performed electrophysiological measurements and data analysis. P.S. performed radioligand assays and data analysis. F.K. performed structural analysis and computational docking. T.S., A.G., and T.L. performed similarity analysis, pharmacophore modelling and screening. K.B., M.E., and M.W. wrote the manuscript. K.B., F.K., and F.Z. prepared figures.The authors declare no competing interests.Design & Analysis and as recommended by funding agencies, publishers and other organisations engaged with supporting research.This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research, as stated in the BJP guidelines for Data S1. Supporting InformationClick here for additional data file.


The with-no-lysine (WNK) kinase family, comprising four serine-threonine protein kinases (WNK1-4), were first linked to hypertension due to their mutations in association with pseudohypoaldosteronism type II (PHAII). WNK kinases regulate crucial blood pressure regulators, SPAK/OSR1, to mediate the post-translational modifications (PTMs) of their downstream ion channel substrates, such as sodium chloride co-transporter (NCC), epithelial sodium chloride (ENaC), renal outer medullary potassium channel (ROMK), and Na/K/2Cl co-transporters (NKCCs). In this review, we summarize the molecular pathways dysregulating the WNKs and their downstream target renal ion transporters. We summarize each of the genetic variants of WNK kinases and the small molecule inhibitors that have been discovered to regulate blood pressure via WNK-triggered PTM cascades. Electrolyte homeostasis plays a pivotal role in blood pressure regulation . RegardiAberrant renal SGK1/WNK-SPAK/OSR1 regulation is involved in disease conditions such as Gordon’s Syndrome and Gitelman-like conditions ,8,9,10. WNK1, WNK4, KLHL3, and CUL3 have been identified to result in the overactivation of NCC and NKCC2, which boosts the sodium reabsorption capacity in the distal convoluted tubule (DCT) and decreases the secretion of K+ by ROMK in the following nephron segment (CCD), leading to PHAII  levels.As uromodulin is excreted in bulk to urine by the TAL, it is not surprising that it plays an essential role in the regulation of TAL-localized ion channels, such as NHE3, Naand ENaC ,193,194.and ENaC . In hypeand ENaC . In paraand ENaC . In contIn addition, the NKCC2 phosphorylation and activity was also regulated by the dDAVP-V2R-cAMP-PKA pathway; namely the dDAVP promotes salt reabsorption through the upregulation of p-NKCC2 . HoweverApart from NKCC2 regulation, there are studies suggesting that uromodulin regulates NCC phosphorylation in HEK293 cells possibly by modulating the SPAK/OSR1 signaling pathway. THP also increased the sensitivity of NKCC2 to intracellular chloride depletion in the presence of WNK3, a strong activator of NKCC2, which can also activate NCC .+ transport . Therefore, the large K+ influx stimulates the activity of K+-secreting channels, such as ROMK, to extrude K+, leading to hypokalemia [As sodium flushes into the aldosterone-sensitive epithelial cells through hyperactive ENaC, the basolateral Naokalemia . Then, eokalemia . Conversokalemia . + concentration  and L511Q mutations in the γ subunit (γL511Q) and β subunit V348M largely increase channel activity by reducing ENaC’s self-inhibiting effect, which is caused by an increased intracellular Nantration  [241,242Besides the mutations, the open probability of ENaC, its channel activity, and its protein levels are highly regulated by PTMs such as phosphorylation, ubiquitination, palmitoylation, and protease cleavage. The cAMP-PKA/PKC pathway has also been reported in vitro to increase ENaC internalization and degradation through phosphorylation of ENaC at βT613 and γT623 via ERK or cyclin-dependent kinases (CDKs) ,244. The+ self-inhibition [+ current when expressed in oocytes with WT β- and γ-ENaC, and a 28% reduction in surface expression compared with WT, as shown in recent studies [Protein cleavage plays an important part in ENaC maturation, cellular localization to the plasma membrane, subunit assembly, and activity. The three subunits of ENaC are transported to the plasma membrane, assembled, and get activated only after cleavage by proteases, such as furin and prostasin. Furin cleaves the extracellular domain of the ENaC α-subunit twice (after residue 181 and 204) and the γ-subunit once (after residue 138), thereby preventing the initiation of Nahibition ,246. Thehibition . On the  studies .+ self-inhibition [Moreover, other studies indicated that the co-expression of prostasin with ENaC increases both channel open probability and cleavage at the polybasic RKRK186 tract in the γ-subunit, showing that prostasin can also recognize ENaC at the RKRK motif and cleave at the K186 site, which is distal to the furin cleavage site. An ENaC mutant with a substitution of RKRK186 to QQQQ186 prevented ENaC activation, and, interestingly, prostasin kinase-dead mutant S238A could still facilitate ENaC cleavage and activation. However, the detailed mechanism is still unclear ,248. Prohibition . Other shibition . Howeverhibition ,251,252.+ and Li+ cause the self-inhibition of ENaC through the direct binding and saturation of ENaC transporting site capacity, which plays a significant role in ENaC inhibition [+ ions have also been demonstrated to block ENaC activation, the effect was much weaker and the mechanism of K+ inhibition was different from that of Na+ inhibition and has not been clearly defined [+ inhibits ENaC by binding to an extracellular cation sensing site at the acidic cleft without interacting with other associated proteins. Na+ and Li+ inhibit ENaC in a similar way, while Li+ functions at a slightly slower rate [+ binding sites. It was also shown that a polymorphism, W493R, in the WPSXXS motif of ENaC α-subunit was related to the loss of Na+ self-inhibition and an increased channel open probability, implying a possible cation binding site. Also, further studies of the crystal structure of ASIC1 (acid-sensing ion channels) determined that cation binding sites are located in the finger and thumb domains of ENaC, which is exactly the domain that undergoes furin or trypsin protease cleavage to prevent Na+ self-inhibition [+ self-inhibition, while αH282 in mouse ENaC (αH255 in human) inhibits Na+ self-inhibition, these two sites are shown to be related to the pH regulation of the channel [+ and H+ transport have synergistic effects. Increased Na+ absorption generates a transmembrane potential that boosts H+ secretion, and the ENaC current is also mediated by H+ concentration. The binding of H+ ions at αD365 attenuates the effect of ENaC Na+ self-inhibition, therefore increasing overall ENaC activity. αD365W could also impair the protonation of the ENaC H+ binding site, reversing the effect of acidification on the ENaC current. This implies the fact that pH regulates ENaC by changing the channel gating.Extracellular cations such as Nahibition . Though  defined . Extracewer rate . Recentlhibition ,256. Funhibition . Moreove channel . The rat+, K+, and Cl− gradients are also influenced by the ENaC sodium current. In DCT cells, ENaC absorbs Na+ ions from the lumen without carrying in negative ions, thereby causing an increased negative charge in the lumen and thus generating a negative membrane potential. With the accumulative effect of ENaC transport along DCT to CCD, this electrical gradient provides a driving force for the movement of other ions, such as K+ or Cl−, to downstream parts of distal nephrons [+ (BK) channel activation via an electrogenic effect is induced by ENaC functioning, and that multiple ion channels, including NCC and NKCC2, are all affected by the electrogenic effect on ENaC activity [As ENaC-mediated sodium transport is electrogenic, the Hnephrons . It was activity . + concentration in the body induces hyperaldosteronism. As for repressed ENaC activity in PHAII, it was reported to result from the upstream overactivation of NCC, largely decreasing Na+ concentration in the lumen, and therefore reducing the function of electric-gradient-driven ENaC. This could probably explain why ENaC was shown to have an increased effect in a previous study conducted on Xenopus oocytes with a WNK4 PHAII mutant, however, this statement still requires further research confirmation [Generally, WNK4 regulates NCC in a kinase-dependent manner, while its effects on ENaC and ROMK are kinase-independent . The incirmation . Anotherirmation ,19.WNK centers in the hub of the SPAK/OSR1 kinase network drive robust post-translational modification cascades and prime ion channels in the kidney for the balance of electrolytes together. WNK pathways were also reported as the primary responder to the fluctuation of extracellular chloride ions, neuro-endocrines , and hydrostatic signals. Proven by independent but intercorrelated studies from GWAS cohort analysis, genetic mutant animal models, and biochemical cell studies, WNK together with the SPAK/OSR1 kinase axis stand out as the upstream regulators of key renal ion channels . Overall, this evidence accentuates the deep involvement of the dysregulated WNK network in the pathophysiology of hypertension. In addition to hypertension, a wide spectrum of electrolyte disorders, such as hyperkalemia, hyperchloremia, and metabolic acidosis, were also found to occur with dysregulation of the WNK signaling network. While this review focused on the WNK-centric pathomechanisms in hypertension, we also surveyed the small molecule inhibitors that target the WNK-SPAK/OSR1 axis in the context of hypertension. Among the small molecule inhibitors mentioned in this article, only tenapanor has undergone clinical trials for its ability to block NHE3 from the gastrointestinal tract, and the investigation for novel kinase inhibitors of the WNK signaling pathway still provides the prospect for potential therapeutic applications in the future. By revisiting the genetic characters, molecular pathways, and pharmaceutical aspects of WNK-related hypertension, we provided an in-depth report for future therapeutic inventions and precision medicine workers to engage in the study of hypertension.


We evaluate marginal ice zone (MIZ) extent in a wave–ice 25 km-resolution coupled model, compared with pan-Arctic wave-affected sea-ice regions derived from ICESat-2 altimetry over the period December 2018–May 2020. By using a definition of the MIZ based on the monthly maximum of the wave height, we suggest metrics to evaluate the model taking into account the sparse coverage of ICESat-2. The model produces MIZ extents comparable to observations, especially in winter. A sensitivity study highlights the need for strong wave attenuation in thick, compact ice but weaker attenuation as sea ice forms, as the model underestimates the MIZ extent in autumn. This underestimation may be due to limited wave growth in partially covered ice, overestimated sea-ice concentration or the absence of other processes affecting floe size. We discuss our results in the context of other definitions of the MIZ based on floe size and sea-ice concentration, as well as the potential impact of wave-induced fragmentation on ice dynamics, found to be minor at the climate scales investigated here.This article is part of the theme issue ‘Theory, modelling and observations of marginal ice zone dynamics: multidisciplinary perspectives and outlooks’. In both hemispheres, MIZs are areas of particular importance for atmosphere–ocean–sea-ice processes e.g. ,2), incl, incl2])Sea ice inhibits wave generation and attenuates the waves propagating from open ocean. As a consequence, the presence of waves in sea ice has been suggested as a definition of the MIZ , since tA number of dedicated modelling studies e.g. ,13–17) a–17 a13–1The lack of climate-scale evaluation of different modelling studies derives from an absence of observational datasets on wave–ice interactions (i.e. wave properties in ice and sea-ice floe size) covering large geographical areas  over long time periods (more than a few days). Recently progress has been made by exploiting long-period moorings  and spacIn this study, we evaluate the extent of ice affected by waves in the neXtSIM–WAVEWATCH III –34 coupl. 2 (a)in situ data would be required to fully assess the uncertainties associated with wave detection using IS-2, recent studies [The IS-2 dataset and retrieval method are described in detail in . Briefly17 m. In  the segm studies ,37 have  studies  suggeste studies , where s studies . Summer a compared with figs S1 and S5 in the supplementary material of [WAF values are reported only when more than 1000 segments are recorded in a given month in each bin, making the 25 km grid significantly noisier than the 100 km grid . Yet gib). We use a higher threshold of 0.25 to delimit areas more confidently affected by waves. The observed wMIZ extent is relatively insensitive to further increases in this threshold. We also define a second high-confidence area by filtering out all cells with fewer than 30 000 segments observed monthly. This restriction reduces the total observed area by approximately c), and includes almost entirely observations of ‘no waves’ a), as we do not expect our model with a 25 km resolution to resolve wave growth and propagation in these areas.We use the coupled wave–ice model developed in . The wavfigure 1The main difference between the model used in this study and that of  is that We define a reference simulation, denoted by REF, where we do not activate processes  relatedWe also run sensitivity experiments to investigate the effects of the values of the inelastic dissipation of wave energy through the fined in  (eqn (20sipation  (eqns (Bn [σflex  (eqn (12n [σflex . Simulat (c)et al. [To evaluate simulated wave heights in open water, we use a wave-height dataset focusing on the Arctic compiled by Heorton et al. . It provFor evaluation of sea-ice properties, we follow the methods of  and use . 3 (a)b,c, electronic supplementary material). Wave heights tend to be underestimated within the Arctic Basin, such as in the Kara, Beaufort, Chukchi and East Siberian Seas, which are only ice free (and therefore usable for this comparison) for a short part of the year (approximately June to October). Wave heights are generally quite low in these regions, even when they are ice free .We first examine open-ocean wave conditions, with the results presented in figure S1 of the electronic supplementary material. Overall the model reproduces the observed wave energies, in particular with low significant wave-height bias in key Arctic regions of interest such as the Greenland, Barents and Bering Seas  and more compact than in observations . As a result, the model is likely to overestimate wave attenuation and underestimate the wMIZ extent in this region. Conversely, in summer, overestimation of wave height in ice may arise from an overestimation of the available fetch. Sea-ice thicknesses are underestimated, mainly in the central Arctic (not shown), where waves are not expected to be found, at least in winter. (b)As described in §2a, we seek to define a model wMIZ that can be compared with the region where the IS-2 WAF is higher than 0.1. The WAF provides information about the frequency of detection of waves with amplitude higher than 0.54 m (1.08 m wave height). In  this thrrid cell . Thus, aet al. [e in [This definition is simple, but the maximum ice edge ), the wat et al.  call ν (found in ), resultg. 3e in ), we defethod of , assumina) captures most of the observed locations with a WAF exceeding 0.1 in the vicinity of the ice edge, though a number of points with high WAF values, over 0.25, are not captured farther in the ice pack of the Arctic Basin. A similar pattern holds in April and May, though there are fewer observations of waves close to the ice edge and more of them in pack ice not captured by the model (not shown). From June to August (figure 2b), the model fails to capture most of the observations of waves. This means either that the model underestimates wave heights in ice or that the data are very noisy in this period, as suggested in [Maps of 2019 wMIZ extents are shown in figure 2ested in . In Novea (in orange) along with the wMIZ extent estimated using the binned 100 km IS-2 dataset in [b), as it contains more cells with fewer than 1000 segments per month. Note that in our analysis, we only sample model cells where IS-2 data are available, the problem being that wave observations by IS-2 are often scattered points between cells with no (or not enough) detected waves. The reasons behind this behaviour are discussed later in this section. Trying to extrapolate a continuous wMIZ from IS-2 observations is not straightforward given the difficulty in assessing uncertainties when computing the WAF. However, it is encouraging that the modelled wMIZ generally shows the same key features as the observed one: a decrease from winter to spring, a sharp rise in the autumn, and a rather stable extent in winter. Using the 90th percentile of The evolution of integrated wMIZ extent is shown in figure 3taset in . The maga and We next investigate some quantitative metrics for the modelled wMIZ extent. Define el score . In our a). This is illustrated in figure 3b, which shows the dependence of c, but allows us to define a more conservative version of b) and increases dramatically when the wMIZ extent becomes very large. This is because as the wMIZ extent keeps increasing, it ends up encompassing large areas of pack ice with very few waves observed. The area A consequence is that the ed waves a. This ia illustrates a second challenge in quantitative evaluation of the wMIZ extent. It is due to the high values of WAF that are not captured by the model in the central Arctic pack ice, particularly from May to November [figure 2b,c. It is unlikely that the model in its current configuration would be able to capture these waves, whether these observations are due to noise  or to wave generation in openings in pack ice [The monotonic increase of November , visiblepack ice . This ispack ice ; and (iipack ice .c). This metric only samples points with wave observations in the IS-2 dataset and checks whether they are captured by the modelled wMIZ. The model shows a clear seasonal pattern, performing well in the winter and less well in the summer. From December to March, we find that the modelled wMIZ captures about 70% of observations, with little dependence (about 10% maximum) on the definition of the wMIZ. This result varies little with the choice of the a. The model’s ability to capture observations drops from April to August (figure 2c), but increases again from September onwards. The results in summer are sensitive to the use of either the maximum value or the 90th percentile of d. The higher d). This means that to evaluate different simulations, one can compare the values of d). In winter, where the wMIZ extent seems reasonable in figure 2a, b). In general, the model is not too sensitive to the choice of the We propose separate evaluations of the ability of the model to capture the areas with observed waves, so as not to extend the wMIZ into areas where the IS-2 dataset is confident that there are no waves. First, we compute the fraction of observations captured by the modelled wMIZ by computing the evolution of  for REF c. This m (c)a. We also use this sensitivity experiment to investigate the reasons behind the underestimation of the autumn wMIZ extent in REF. This underestimation could be due to false positives in the observations of waves by IS-2. This is likely to be the case in summer (June–August), as the number of segments per cell tends to be lower and melt ponds could be mistaken for ocean surface points when computing the WAF [To investigate what the important factors are in simulating the wMIZ, we compute the quantities  the WAF . Because the WAF , giving We first look at DMG to check that activating the link between damage and fragmentation has little impact on the wMIZ extent. This is needed as this link is activated in other simulations investigating the sensitivity of other parameters compared to REF . The impa). This increase of the wMIZ extent in NIDIS results in an increase of a). Moreover, this increase of the wMIZ mostly corresponds to areas in the Greenland and Barents Seas, where we would expect an underestimation of the wMIZ given that the model consistently overestimates the ice extent in these regions. This increase is particularly visible when considering the upper bound of the wMIZ extent (using the threshold c), and the wMIZ extent does not appear to be overestimated, at least when using a).The NIDIS simulation changes the attenuation process by removing inelastic dissipation, which is efficient at attenuating waves with longer wave periods (greater than oken ice . Inelastoken ice . To compoken ice ). In wind to REF a. This iocations a. Moreovchi Seas c, and thFebruary a.FigureThe overestimation of the wave attenuation from inelastic dissipation in REF in autumn could also originate from an overestimation of the floe size. This is likely to be the case in our model, as we ignore the effects of processes other than waves  and assu used in   shows that the autumn sea-ice extent in the Beaufort and Chukchi Seas is slightly overestimated, but also that sea-ice concentration shows a clear positive bias , which likely explains part of the underestimated wMIZ extent. This is supported by the finding  that wav (d)et al. [We now investigate how the wMIZ definition would compare with a floe size-based definition. Such definitions of the MIZ have been suggested in other studies ,14,23, gb), with the exception of the months of September to November, where the area of broken ice exceeds the wMIZ extent by about 33% for REF. This is similar to the observed wMIZ, which also shows a clear peak in October. This similarity is not unexpected, as floe size in the current set-up is mostly influenced by wave break-up, and it is likely that more differences would emerge if other processes were also affecting the floe size. The dMIZ shows a rather high sensitivity to the choice of the value of et al. [Here, we use the same definition as in , using tthan 2 m , while 3es (e.g. ). For alp occurs . We havep occurs ), as Voe [et al.  note thaThe greater sensitivity of the dMIZ to wave attenuation processes than the wMIZ (when looking at REF and NIDIS) is possible because the wMIZ extent is constrained by the minimum  the ice , and obsa).While we do not expect the model to predict ch month a.b), which is a useful metric for climate model evaluation [a,c), except in limited areas where the wMIZ encompasses more compact ice with a concentration higher than 0.8, mostly in the Greenland Sea and north of the Barents Sea. It is only when the wMIZ (and hence the dMIZ) extent is overestimated in winter (in NIDIS) that these areas of pack ice affected by waves become really significant , i.e. the area with a sea-ice concentration between 0.15 and 0.8 ,50 figub, which aluation . Here, waluation . This icaluation . More imaluation . As notealuation , the wMI (e)One of the main goals of waves-in-ice modelling is to assess the potential of waves to affect sea-ice evolution, as was done with the model presented here and the sea-ice dynamics in , the latd). For a fair comparison between these four simulations, we define a fixed geographical area for each month that is independent of the simulation. We take it to be the area where b). We therefore limit our analysis to the months from October to March.Here, we compare the simulations DMG, LFXST and NIDIS, in which fragmentation lowers the ice strength of compact ice, with REF d. For a fined in , as it hWe find little difference in the average sea-ice drift in the selected area between the different simulations (not shown). A greater difference is visible when looking at the 20th percentile of drift speed, which is of the same order of magnitude as the average drift speed. This is consistent with , i.e. th. 4We have compared the wMIZ extent in a coupled wave–sea-ice model with observations from IS-2, showing satisfying results for our reference simulation. We have suggested two quantitative metrics, elopment , should elopment . Combinielopment  and analelopment  in ice cOur results suggest the need for a modelled mechanism to dissipate wave energy in compact, thick, solid ice. Sea-ice models including floe size distributions should therefore be careful to have strong wave attenuation in the winter so as to not strongly overestimate the impact of waves in this season. A consequence of strong attenuation in pack ice in winter is that even when the wMIZ extent peaks due to large open-ocean waves, the amount of thick, compact ice affected by waves in the Arctic remains small. This is likely not the case in the Southern Ocean, where large wave events and long-period waves are much more frequent ,36. Thiset al. assumed that the recovery time scale of ice strength after a fragmentation event is the same as for brittle fracture in the pack ice, but wave break-up is likely to create a larger number of cracks in the sea ice and may have a longer-lasting effect. Another limitation of their approach is that it neglects the impact of floe–floe interactions on internal stress, which can be significant [A consequence for sea-ice dynamics is that taking wave-induced fragmentation into account when computing the ice strength differs little from using classical concentration-based parametrizations . Howevernificant .


Pueraria lobata leaves contain a variety of phytoestrogens, including flavonoids, isoflavonoids, and coumestan derivatives. In this study, we aimed to identify the active ingredients of P. lobata leaves and to elucidate their function in monoamine oxidase (MAO) activation and Aβ self-aggregation using in vitro and in silico approaches. To the best of our knowledge, this is the first study to elucidate coumestrol as a selective and competitive MAO-A inhibitor. We identified that coumestrol, a coumestan-derivative, exhibited a selective inhibitory effect against MAO-A (IC50 = 1.99 ± 0.68 µM), a key target protein for depression. In a kinetics analysis with 0.5 µg MAO-A, 40–160 µM substrate, and 25 °C reaction conditions, coumestrol acts as a competitive MAO-A inhibitor with an inhibition constant of 1.32 µM. During an in silico molecular docking analysis, coumestrol formed hydrogen bonds with FAD and pi–pi bonds with hydrophobic residues at the active site of the enzyme. Moreover, based on thioflavin-T-based fluorometric assays, we elucidated that coumestrol effectively prevented self-aggregation of amyloid beta (Aβ), which induces an inflammatory response in the central nervous system (CNS) and is a major cause of Alzheimer’s disease (AD). Therefore, coumestrol could be used as a CNS drug to prevent diseases such as depression and AD by the inhibition of MAO-A and Aβ self-aggregation. Thus, MAOs play a key role in regulating monoamine levels in the central nervous system (CNS). Of the two isomers (A and B types) of MAO, MAO-A exhibits substrate selectivity for serotonin, melatonin, norepinephrine, and epinephrine, and MAO-B exhibits substrate selectivity for phenylethylamine and benzylamine. However, both isomers are known to non-selectively catalyze dopamine and tyramine [Monoamine oxidase (MAO) is a group of flavoenzymes that catalyze the oxidation of biogenic and xenobiotic amines including primary , secondary , and tertiary amines, producing Htyramine . Therefotyramine . In addityramine . Aggregatyramine . Thus, iPueraria lobata (Willd.) Ohwi belongs to the Leguminosae family and is one of the climbing plants that have been used as food or traditional medicine in many countries, including Korea, China, and Japan [P. lobata, it has been widely used in folk remedies or herbal medicines since ancient times; research on its active ingredients and pharmacological activity is in progress. The representative components of the roots and leaves of P. lobata are puerarin (daidzein-8-C-glucoside) and robinin (kaempferol 3-O-robinoside-7-O-rhamnoside), respectively. In addition, P. lobata contains many isoflavones, flavonoids, triterpenoids, and coumestan derivatives [P. lobata is rich in isoflavones that help relieve female menopausal symptoms; hence, it is widely used by women. The isoflavone-rich fraction of P. lobata exerts neuroprotective effects by enhancing the metabolism of neurotransmitters, and puerarin exerts anti-PD effects by protecting dopaminergic neurons [P. lobata roots exhibited anti-depressant activity in a cerebral ischemia reperfusion mouse model [P. lobata leaves and its component are still limited. The discovery of an active ingredient from P. lobata, which can be easily consumed as food, is of great nutritional and industrial importance. Therefore, in this study, we aimed to identify the active ingredients of P. lobata leaves and to elucidate their function in MAO activation and Aβ aggregation using the analytical technique HPLC–Q-TOF–MS, in vitro enzyme inhibitory and protein aggregation assays, and in silico computational approaches. In silico studies are useful to understand the role of the steric hindrance of the functional groups of phytochemicals in the active site of a target enzyme. In addition, a kinetic assay, which observes changes in enzyme activity according to the substrate and inhibitor (=active ingredient) concentration, was conducted to identify the inhibitory type of active ingredient. To further support their potential as a drug, the pharmacokinetic parameters of the active molecules were also predicted.nd Japan . Due to ivatives ,7. In pa neurons . In addise model . However25–35), hexafluoroisopropanol (HFIP), and selegiline hydrochloride were purchased from Sigma-Aldrich . All solvents used for high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (HPLC–Q-TOF–MS) were liquid chromatography–mass spectrometry grade and purchased from Merck .Human monoamine oxidase (hMAO) isozymes, robinin, rutin, nicotiflorin, daidzin, genistin, coumestrol, daidzein, genistein, curcumin, amyloid β peptide 25–35  in August 2021. The plant was authenticated by Dr. S. Han of Honam National Institute of Biological Resource (voucher no. shan2021-142). The 70% ethanol extract of P. lobata leaves (PL-L-70E) was obtained from the Bank of Bioresources from Island and Coast (BOBIC), Republic of Korea (registered no. HNIBR NP374).18 column (Cat. 25005-254630), equipped with the Chromeleon Software 7.3 , for component analysis. The mobile phase consisted of a gradient of 0.1% HCOOH (A) and acetonitrile containing 0.1% HCOOH (B) as follows: 0–60 min, B 10% to 45%; 60–60.1 min, B 45% to 100%; and 60.1–65 min, B 100%. The sample injection volume was 2.5 μL, column temperature was 25 °C, and flow rate was 0.5 mL/min. MS analysis was performed using Xevo G2-XS Q-TOF–MS with MassLynx v10 . ESI–MSe mode was used to obtain low- and high-resolution spectral data in the negative mode. MS conditions were as follows: acquisition mass range, 50 to 1500 Da with a 0.5 s scan time; cone voltage, 40.0 V; source and desolvation temperatures, 100 °C and 350 °C, respectively. Mass calibration was performed using 0.5 mM sodium formate. The mass was corrected during acquisition using 200 pg/mL leucine enkephaline.The PL-L-70E was separated on the Ultimate™ 3000 UHPLC system using the Hypersil GOLD™ CP. lobata-derived phytochemicals against MAO was evaluated through the luminometric method. Various doses of phytoestrogens in 0.1 M HEPES buffer (pH 7.5) with 5% 1,2,3-propanetriol for MAO-A and 5% 1,2,3-propanetriol and 10% dimethyl sulfoxide for MAO-B were mixed with the substrate (160 µM for MAO-A and 16 µM for MAO-B) and 0.5 µg of the enzyme and incubated at 25 °C. After 1 h incubation, reconstituted luciferin-detection reagent was added to the reaction mixture and incubated at 25 °C for 20 min. After that, luminescent signals were monitored using a microplate reader . Selegiline hydrochloride  was used as a positive control [The inhibitory potential of the  control .The type of inhibition exhibited by coumestrol was analyzed based on the changes in enzyme activity according to various concentrations of the substrate and the inhibitor . The expThe equation for the LB plot is as follows:The equations for the LB-2nd plots are as follows:25–35 self-aggregation assay was performed following the method described by Naldi et al. [25–35 solution in a 1:29 ratio (v/v) and incubated overnight at 4 °C. After incubation, the reaction mixture was supplemented with 0.025 mM thioflavin-T in 50 mM glycine–NaOH buffer (pH 8.5). The fluorescence emission was monitored at 490 nm with excitation at 446 nm using the Gemini XPS . Curcumin was used as the standard [The Aβi et al. . First, standard .The X-ray crystallographic structure of hMAO-A was obtained from the PDB (ID 2z5x) . Water ahttps://preadmet.bmdrc.kr/, accessed on 1 June 2022).The pharmacokinetic characteristics of coumestrol were calculated using Marvin Sketch (v17.1.30) and the PreADMET server v1.0 (50) calculated from the dose–inhibition curve is expressed as the mean ± standard deviation (SD) of three independent experiments. The statistical significance of the group treated with the tested compounds against the control group for the Aβ25–35 self-aggregation assay was calculated via Student’s t-test .The 50% inhibitory concentration (ICe (P. lobata leaves based on the comparison of their retention times (RT) with those of standard compounds and the analysis of their fragmentation patterns. In particular, robinin was identified as the most abundant component. In addition, flavan-3-ols , isoflavones , and the coumestan-derivative coumestrol were also present in this extract.The chemical constituents of the PL-L-70E were analyzed using HPLC–Q-TOF–MSe . We dete2+, Cd2+, and Al3+), pH, and temperature affect MAO activity [The protocol for the MAO assay was verified using selegiline hydrochloride as a positive control. In addition, since factors such as organic solvents, metal ions  = 0.02). As shown in 50 values of 4.77 and 3.42 μM for MAO-A and -B, respectively. On the other hand, daidzein, which has a structure similar to that of genistein without a hydroxyl group at the C5 position, showed a weak inhibitory effect against both MAO isozymes . Previously, Zarmouh et al. [The PL-L-70E exhibited dose-dependent inhibitory activity against MAO isozymes A, with hh et al.  also demTo the best of our knowledge, for the first time, the potent and selective inhibitory activity of coumestrol against MAO-A was elucidated. Therefore, an enzyme kinetic analysis was performed to elucidate the mode of inhibition of MAO-A by coumestrol. As in the previous studies, it was confirmed that the lower the concentration of the substrate, the lower the enzyme reaction rate of the control group (treated with only buffer) . As shown in 25–35/1–42 self-aggregation) were investigated to evaluate their inhibitory potential against Aβ25–35 self-aggregation based on the fluorescence of thioflavin-T  [Based on the chemical structure of coumestrol, the pharmacokinetic parameters were predicted. The pharmacokinetic analysis of coumestrol revealed that its blood–brain barrier (BBB) permeability score was 0.76, which indicated that coumestrol could pass across the BBB with moderate absorption by the CNS. In addition, coumestrol showed 93.51% human intestinal absorption (HIA), which implies that it can be easily absorbed by the human intestine and is suitable for oral delivery. According to the CMC-like rule , the lip values) . TherefoIn this study, we investigated the inhibition of MAO by the PL-L-70E and its constituents. We determined that coumestrol showed the best inhibitory effect against MAO-A, followed by genistein, daidzein, and rutin. In the case of MAO-B, genistein exhibited the strongest inhibition, followed by coumestrol and daidzein. However, the remaining compounds did not exhibit a significant inhibitory effect against either isozyme. Moreover, for the first time, we discovered that coumestrol acts as a selective and competitive MAO-A inhibitor.P. lobata, soybean, alfalfa (Medicago sativa), and Trifolium sp. [Psoralea corylifolia seeds, which has an additional prenyl moiety at the C2 position of coumestrol, did not show a significant inhibitory effect against either MAO-A or -B [Glycyrrhiza uralensis roots, which has an additional prenyl group at the C2 position and a methoxyl group at the C3 position of coumestrol, had an IC50 value of 29.5 μM against MAO-B (no activity below 40 μM for MAO-A) [max remained constant despite changes in substrate or coumestrol concentrations, which corresponds to a typical competitive inhibition pattern. Therefore, when coumestrol is consumed, there could be no side effects (ex. hypertension) linked to the ‘cheese effect’ caused by the consumption of nonselective MAOIs. However, an additional reversibility assay is needed to confirm whether coumestrol is a reversible inhibitor. In addition, it is needed to check the thermodynamic effect of the inhibitor on the enzyme through a scan of the changes in thermal stability in the free enzyme and inhibitor–enzyme complex.Coumestrol is a coumestan-derivative and is found in lium sp. ,24. Coumlium sp. ,26; it i-A or -B . In anotr MAO-A) . Unlike r MAO-A) . Further50 of 37.40 μM. In a previous study, it was reported that a nanomolar concentration of coumestrol exhibited neuroprotective effects in mouse astrocytes by significantly reducing Aβ-induced cytotoxicity and inflammatory cytokine levels [The thioflavin-T assay for inhibitory compounds  against MAO was used to investigate their inhibitory effect against the self-aggregation of Aβ, which is known to cause neuroinflammation and AD. Coumestrol exhibited the most potent inhibitory effects with an ICe levels . In addie levels . Thus, te levels . 1–40/1–42 reduced the expression of p-glycoprotein in the BBB, thereby interrupting the clearance of Aβ in the brain [In this study, the potential of coumestrol as a CNS drug for the treatment of AD and depression was confirmed via in vitro and in silico approaches, and its pharmacokinetic properties were also predicted to support its potential. Coumestrol was predicted to possess suitable lipophilicity as a neuronal drug and to have normal BBB permeability . Moreovehe brain ,35. TherP. lobata leaves, acts as a selective and competitive MAO-A inhibitor by competing with the substrate at the substrate-binding site and stably binding to key residues and FAD in the active site of the enzyme. In addition, coumestrol significantly reduced the self-aggregation of Aβ, which is known to be a major cause of AD. Taken together, this study suggests that coumestrol may act as a food-derived natural anti-depressant and anti-AD material that can treat or prevent diseases related to MAO-A activation and Aβ self-aggregation.The phytoestrogen coumestrol, present in


Corrosion prevention has been a global phenomenon, particularly in metallic and construction engineering. Most inhibitors are expensive and toxic. Therefore, developing nontoxic and cheap corrosion inhibitors has been a way forward. In this work, L-arginine was successfully grafted on chitosan by the thermal technique using a reflux condenser. This copolymer was characterized by Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and X-ray diffraction (XRD). The corrosion inhibition performance of the composite polymer was tested on mild steel in 0.5M HCl by electrochemical methods. The potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) results were consistent. The inhibition efficiency at optimum concentration rose to 91.4%. The quantum chemical calculation parameters show good properties of the material as a corrosion inhibitor. The molecular structure of the inhibitor was subjected to density functional theory (DFT) to understand its theoretical properties, and the results confirmed the inhibition efficiency of the grafted polymer for corrosion prevention. Corrosion is defined as the degradation of materials caused by chemical or electrochemical attacks within the working environment . It resuVarious approaches have been put in place to combat the persistent problem, with corrosion inhibitors being one of the most cost-effective and practical methods of preventing metallic corrosion in various corrosive media . CorrosiThe vast majority of conventional corrosion inhibitors are hazardous . Thus, gLonger hydrocarbon chains in amino acids have often shown stronger corrosion mitigation . As a reChitosan of medium molecular weight (190–310 kDa and 75–85% degree of deacetylation), L-arginine, hydrochloric acid (HCl) and acetic acid were purchased from Sigma Aldrich . Tert-Butyl hydroperoxide, 70% solution in water was purchased from Thermo Fischer Scientific . Other reagents such as acetone, ethanol was supplied by DKSH Specialty Chemicals . All the chemicals used were of analytical grade and used without further purification. Mild steel coupons and emery paper were collected from the KhonKaen University workshop, Thailand.L-arginine was grafted on chitosan by dissolving 2 g of chitosan in 100 mL of 0.01 M acetic acid solution, and 2 g of L-arginine in 50 mL of acetone. These two solutions were mixed in a beaker and 1 mL of Tert-Butyl hydroperoxide (THB) was then poured into the resulting mixture. This mixture was then taken into reflux at 120 °C for two hours. Cs-g-L-arginine was then precipitated by adding 20 mL of 0.5 M NaOH to the mixture. The solution was filtered through 11μ filter paper. The filtrated Cs-g-L-arginine was then extracted with deionized water using Soxhlet extraction at 90 °C for six hours to remove excess homopolymer. The extracted copolymer was then dried in an oven at 60 °C for 6 h and stored in a desiccator (at room temperature).Fourier transformed infrared (FT-IR) spectra were obtained using FTIR infrared spectrophotometer model; Tensor27 S/N; 3683 (broker Hong Kong limited). Thermal analysis (TGA) was carried out using Hitachi TG/DTA thermogravimetric analyzer instrument model no. STA7200 Hitachi, using a scan rate of 10 °C/min range 25 °C to 600 °C, and X-ray diffraction (XRD) spectra were analyzed using Empyrean X-ray Diffractometer from Malvern.2 exposed areas as a working electrode, and copper wire as a counter electrode. The open-circuit potential was set for one hour before any electrochemical measurement to maintain the steady-state potential. The electrochemical test was carried out using a Metron auto lab Potentiostat (company) equipped with NOVA 1.1 software. Three electrode systems consist of a saturated calomel electrode as a reference electrode, mild steel with 1 cm−1 and 303 K, the corrosion parameters were extrapolated from the Tafel plot fitting. The potentiodynamic corrosion inhibition efficiency (% IE) was calculated using Equation (1)pI E is potentiodynamic inhibition efficiency The potentiodynamic polarization was carried out within the potential range of −8 V to +2 V using the scan rate of 0.001 VsEIS) was analyzed with the frequency range from 100 Hz to 10 MHz at 303 K. The electrochemical corrosion circuit was used to fit the corrosion data. The electrochemical corrosion inhibition efficiency % EISIE was calculated by Equation (2)The electrochemical impedance spectroscopy  indicating that L-arginine was grafted on the chitosan [ch other ,19. The N-H bend . The ban and CH3 . The FTIchitosan . The appchitosan . In compmax) of 300 °C. However, Cs-g-L-arginine has a Tmax of around 400 °C. The DTG (The TGA curve of chitosan in  The DTG c maxima corr), and corrosion current (Icorr), were derived from Tafel plots by nova1.1 software. Tafel fit and percent inhibition efficiency (% IE) ) are shown in corr values towards the cathodic side. The corrosion inhibition mechanism is generally considered a cathodic inhibitor when the potential change exceeds 85 mV, while it is a mixed inhibitor when the potential change is less than 85 mV [In an 85 mV . This inThe Nyquist plots for the corrosion of mild steel surface in 0.5 M HCl, inhibited by Cs-g-L-arginine, are depicted in 2O and other ions that were initially adsorbed at the steel/solution interface, the inhibitive layer on the electrode surface controlled the mild steel dissolution. In the presence of uninhibited 0.5M HCl, the Fe-H2O complex is generated [The electrochemical impedance parameters were obtained by fitting various impedance profiles into an equivalent circuit, which is given in enerated . This coThe effectiveness of an inhibitor is often determined by its structure and molecular orbital distribution. Quantum and theoretical chemistry have been known to be efficient tools for understanding corrosion inhibition mechanisms of the compound. Proper models with computational simulations based on quantum chemistry can support and confirm experimental discoveries. An inhibitor’s effectiveness is determined by both its spatial and molecular electronic structures. The optimized structure of chitosan-graft- L-arginine and pure chitosan are presented in Quantum chemical parameters are presented in Since most corrosion inhibitors are hazardous and expensive, interest in using eco-friendly and affordable materials as corrosion inhibitors has increased significantly in recent years. This was expanded to include the utilization of natural substances to prevent metallic corrosion. Chitosan is a suitable option because of its desirable qualities; however, it was more effective at inhibiting growth when it was soluble in an acidic and aqueous environment. However, modifying chitosan with another molecule that can inhibit corrosion (such L-arginine) may increase the effectiveness of this inhibition. The L-arginine was successfully grafted on chitosan by thermal method; a modified copolymer was characterized by different characterization techniques. The corrosion inhibition efficiency of these polymers was tested on mild steel in 0.5 M hydrochloric acid by potentiodynamic polarization, electrochemical impedance spectroscopy and validated with DFT. The results show high inhibition efficiency by the modified polymer up to 91.4% at optimum conditions. The EIS analysis supported the inhibitory function of the co-polymer by demonstrating an increase in polarization resistance with increasing inhibitor concentration. A polarization analysis showed that the inhibitor molecule inhibits both cathodic and anodic corrosion processes, leading to its classification as a mix type corrosion inhibitor. The results of the inhibitor molecule’s quantum chemical calculations confirmed the findings of the experiment and offered proof of the interaction between the metal and the inhibitor. Hence, chitosan modified by L-arginine can serve as an alternative for corrosion mitigation methods.


G protein–coupled receptor (GPCR) kinases (GRKs) and arrestins mediate GPCR desensitization, internalization, and signaling. The spatial pattern of GPCR phosphorylation is predicted to trigger these discrete GRK and arrestin-mediated functions. Here, we provide evidence that distal carboxyl-terminal tail (C-tail), but not proximal, phosphorylation of the chemokine receptor CXCR4 specifies βarrestin1 (βarr1)-dependent signaling. We demonstrate by pharmacologic inhibition of GRK2/3-mediated phosphorylation of the chemokine receptor CXCR4 coupled with site-directed mutagenesis and bioluminescence resonance energy transfer approaches that distal, not proximal, C-tail phosphorylation sites are required for recruitment of the adaptor protein STAM1  to βarr1 and focal adhesion kinase phosphorylation but not extracellular signal–regulated kinase 1/2 phosphorylation. In addition, we show that GPCRs that have similarly positioned C-tail phosphoresidues are also able to recruit STAM1 to βarr1. However, although necessary for some GPCRs, we found that distal C-tail sites might not be sufficient to specify recruitment of STAM1 to βarr1 for other GPCRs. In conclusion, this study provides evidence that distal C-tail phosphorylation sites specify GRK–βarrestin-mediated signaling by CXCR4 and other GPCRs. GRKs phted pits . Arrestited pits , 6, 7. Tted pits . Althoug2-adrenergic receptor (β2AR), whereas GRK6 phosphorylates proximal Ser/Thr sites  , 10. Indhr sites . Importahr sites . In thishr sites .Arrestins typically engage with phosphoresidues found within the C-tail of GPCRs, and they may also engage with phosphoresidues located on the intracellular loops . The numvia balanced contributions from β-arrestin1-dependent  ependent , 22 and ependent . We haveependent , 25, 26.ependent  or overeependent , 22. STAIn this study, we addressed the receptor determinants specifying STAM1 recruitment to βarr1 following CXCR4 activation and the broad applicability to other GPCRs. Here, we provide evidence that ligand-stimulated phosphorylation of the distal C-tail of CXCR4 is required for βarr1-mediated signaling by STAM1. We show that CXCL12-stimulated phosphorylation of FAK, but not ERK1/2, requires GRK2/3-promoted phosphorylation of phosphosites located at the distal C-tail of CXCR4 but not membrane proximal phosphosites. Importantly, the distal phosphosites are required to recruit the adapter protein STAM1 to βarr1. This information is transposable because swapping the C-tail of a GPCR that does not recruit STAM1 to βarr1 with the C-tail of CXCR4 is sufficient to confer the ability of this GPCR to recruit STAM1 to βarr1, indicating the information dictating this interaction resides solely within the C-tail. We found that other GPCRs, but not all, that contain potential phospho-Ser or phospho-Thr residues at their distal C-tail are also able to recruit STAM1 to βarr1. Our data suggest that distal, but not the proximal, C-tail phosphosites on CXCR4 and other GPCRs facilitates STAM1 recruitment to βarr1 and FAK activation.A and B). In contrast, CXCL12 promoted that ERK1/2 phosphorylation was elevated by treatment with compound 101 but not treatment with Gö6983 (A and C). These data suggest that GRK2/3 phosphorylation of CXCR4, but not PKC, is required for FAK signaling promoted by CXCL12, whereas GRK2/3 suppresses ERK1/2 signaling. This effect on ERK1/2 signaling is similar to what has been previously observed with RNAi against GRK2 –tagged STAM1 and acceptor GFP-tagged βarr1 (βarr1-GFP) treated with varying doses of CXCL12 (A), in agreement with our previous report indicating that the interaction between βarr1 and STAM1 is promoted by stimulation of CXCR4 (A), whereas the PKC inhibitor had no effect on the BRET response (B). We also examined the effect of the GRK2/3 or PKC inhibitor by BRET on the recruitment of βarr1-GFP to Rluc3-tagged CXCR4 in HEK293 cells following stimulation with CXCL12. CXCL12 promoted a dose-dependent increase in BRET between βarr1-GFP and CXCR4-Rluc3 that was shifted to the right in the presence of the GRK2/3 inhibitor (C). The EC50 values in the presence of vehicle or GRK2/3 inhibitor are 0.30 nM  and 7.79 nM (95% CI = 2.89–20.31), respectively. In contrast, the PKC inhibitor had little effect on the BRET response (D). These data suggest that βarr1 recruitment to CXCR4 and STAM1 recruitment to βarr1 requires GRK2/3 phosphorylation of CXCR4 but not phosphorylation by PKC.Previously, we reported that FAK phosphorylation by CXCL12 stimulation of CXCR4 is mediated by a complex formed by βarr1 and STAM1 , 22. To f CXCL12 . Stimulaf CXCL12 A, in agrof CXCR4 A, wherearesponse B. We alsnhibitor C. The ECresponse D. These A). The GRK2/3 inhibitor significantly reduced phosphorylation at Ser-346/7  or Ser-324/5  upon CXCL12 stimulation. Previous studies have shown that CXCR4 phosphorylation is hierarchical in nature whereby phosphorylation of Ser-324/5 is dependent upon initial phosphorylation at Ser-346/7 by GKR2/3 , located at the distal end of the C-tail, and Ser-324/Ser-325 (Ser-324/5), located more membrane proximal on the C-tail , CXCL12 Fig. 3y GKR2/3 , which mD). The BRET response was effectively blocked in cells expressing Ser-346/7A but not in cells expressing Ser-324/5A (D). We also examined the BRET response between the variant receptors and βarr1. Previously, we have shown that CXCR4-YFP, but not variant Ser-324/5A-YFP, is robustly phosphorylated at Ser-324/5 (E), whereas the BRET response between βarr1-Rluc8 and Ser-346/7A-YFP was shifted to the right (F), suggesting reduced βarr1 recruitment to CXCR4. The relative surface expression of HA-CXCR4 (1.00 ± 0.22) to HA-Ser324/5A (1.28 ± 0.15) and HA-Ser-346/7A (1.01 ± 0.05) was similar as determined by ELISA. Taken together, these data suggest that phosphorylation of Ser-346/7 at the distal C-tail of CXCR4 promotes βarr1 recruitment to CXCR4, which we propose is necessary for STAM1 recruitment to βarr1.We next examined whether STAM1 is recruited to βarr1 by BRET in HEK293 cells expressing CXCR4 phospho-deficient receptors in which Ser-324/5 or Ser-346/7 are substituted to Ala residues (Ser-324/5A or Ser-346/7A). There was a dose-dependent BRET response between Rluc8-STAM1 and βarr1-GFP in cells expressing wildtype CXCR4 D. The BRr-324/5A D. We alser-324/5 ; therefoer-324/5 E, whereahe right F, suggesvia endogenous CXCR4 in transiently transfected HeLa cells with empty vector (A and B), possibly because of a dominant negative effect on endogenous CXCR4 signaling, similar to what has been observed in other cells when transfected with other CXCR4 variants (A and B). Importantly, CXCL12-stimulated ERK1/2 phosphorylation in Ser-324/5A or Ser-346/7A transfected cells was similar to wildtype receptor or empty vector (A and C), indicating that CXCR4 signaling was not globally impacted.Phosphorylation of Ser-324/5 by GRK6 is required for CXCR4 ubiquitination and lysosomal trafficking , 40, whey vector , similary vector . Phosphoy vector , likely y vector , A and Bvariants . Althougvariants , which cvariants , A and By vector , A and CA). There was no dose-dependent BRET response between Rluc8-STAM1 and βarr1-GFP in HEK293 cells transiently expressing FLAG-CCR5 treated with CCL5, the cognate ligand for CCR5 . However, in HEK293 cells transiently expressing chimeric receptor CCR5 with the C-tail of CXCR4 (CCR5-R4 tail), CCL5 stimulation dose-dependently increased the BRET response between Rluc8-STAM1 and βarr1-GFP . In contrast, in HEK293 cells transiently expressing chimeric receptor CXCR4 with the C-tail of CCR5 (CXCR4-R5 tail), there was no BRET response between Rluc8-STAM1 and βarr1-GFP following dose-dependent stimulation with CXCL12 when compared with CXCR4 . The CCR5-R4 tail chimera was phosphorylated following stimulation with CCL5, as determined by loss of immunoreactivity of an antibody that specifically recognizes an epitope of the unphosphorylated C-tail of CXCR4 that includes Ser-346/7 (D–F). The relative surface expression of HA-CCR5 (1.00 ± 0.18) to HA-CCR5-R4 tail (0.87 ± 0.18) and HA-CXCR4 (1.00 ± 0.05) to CXCR4-R5 tail (1.38 ± 0.24) was similar as determined by ELISA. These data indicate that the C-tail of CXCR4 is necessary and sufficient for STAM1 recruitment to βarr1, likely reflected by the presence of the distal C-tail Ser/Thr phosphoclusters.Our data suggest that the position of the phosphoresidues at the distal C-tail of CXCR4 is necessary to promote the interaction between βarr1 and STAM1. The chemokine receptor CCR5 has been shown to be phosphorylated and to recruit βarr1 following agonist activation , 44; howFig. 5A). We examined the ability of agonist stimulation of CXCR5 to promote recruitment of STAM1 to βarr1 by BRET. In HEK293 cells transiently expressing FLAG-CXCR5, CXCL13 dose-dependently increased the BRET response between Rluc8-STAM1 and βarr1-GFP, consistent with agonist-dependent recruitment of STAM1 to βarr1 (B). In addition, CXCL13 dose-dependently increased the BRET response between CXCR5-Rluc8 and βarr1-GFP, consistent with agonist-dependent βarr1 recruitment to CXCR5 (C). Substitution of the distal Ser residues to Ala residues (CXCR5-4S/A) (A) completely blocked the BRET response between Rluc8-STAM1 and βarr1-GFP (B) and between βarr1-GFP and CXCR5-4S/A-Rluc8 (C). The relative cell surface expression of HA-CXCR5 (1.00 ± 0.22) to HA-CXCR5-4S/A (0.89 ± 0.21) was similar as determined by ELISA. Because STAM1 is likely recruited to βarr1 when it is bound to the C-tail of the GPCR, we examined whether we could observe BRET between CXCR4 or CXCR5 and STAM1. We detected ligand-induced BRET between CXCR4-RLuc3 and STAM1-GFP and between CXCR5-Rluc8 and STAM1-GFP in HEK293 cells cotransfected with βarr1 (D), suggesting that STAM1 and βarr1 form a complex with the agonist-activated GPCR.To determine whether this is generalizable to other GPCRs, we examined a GPCR that contains potential phosphorylation sites at their distal C-tail. The chemokine receptor CXCR5, whose cognate ligand is CXCL13, has Ser/Thr phosphoclusters at the analogous position to CXCR4 in its C-tail A. We exato βarr1 B. In addto CXCR5 C. SubstiR5-4S/A) A completarr1-GFP B and bet/A-Rluc8 C. The reth βarr1 D, sugges1), angiotensin II type 1A receptor (AT1a), and β2AR have Ser/Thr residues at the distal C-tail position, similar to CXCR4 and CXCR5 (A). We performed BRET experiments with each FLAG-tagged GPCR transiently coexpressed in HEK293 cells with Rluc8-STAM1 and βarr1-GFP. Neurotensin stimulation of NTS1 promoted a dose-dependent increase in the BRET response between βarr1-GFP and Rluc8-STAM1 (E), consistent with STAM1 recruitment to βarr1. The potency  was similar to the potency of βarr1-GFP recruitment to NTS1-Rluc8  as determined by BRET (F). Angiotensin II stimulation of FLAG-AT1a also promoted a dose-dependent BRET response between βarr1-GFP and Rluc8-STAM1 with a potency of 0.07 μM (95% CI = 0.02–0.20), although above the potency for βarr1 recruitment to AT1a as measured by BRET , despite the fact that isoproterenol stimulation promotes a BRET response between β2AR-Rluc8 and βarr1-GFP (F). The relative cell surface expression of the receptors FLAG-β2AR (0.69 ± 0.18), FLAG-AT1a (0.68 ± 0.06), and FLAG-NTS1 (2.37 ± 0.34) were similar to FLAG-CXCR4 (1.00 ± 0.05) as determined by ELISA. These data suggest that at least for some GPCRs, distal phospho-sites along the C-tail are not sufficient to specify STAM1 recruitment to βarr1, although necessary for others.We next examined whether other GPCRs are able to recruit STAM1 to βarr1. The neurotensin receptor 1 . Remarkably, we show for the first time that phosphorylation of Ser-346/7 is necessary for STAM1 recruitment to βarr1 . Lack of STAM1 recruitment to βarr1 is consistent with the fact that GRK2/3-mediated phosphorylation of distal Ser-346/7 is also required for FAK phosphorylation (B), suggesting a common mechanism of FAK signaling by GPCRs. Because these sites on CXCR5 are also necessary for βarr1 recruitment (B), these data further suggest that STAM1 is recruited to receptor-bound βarr1. Consistent with this, we observed STAM1-GFP recruitment to CXCR4-Rluc3 or CXCR5-Rluc8 by BRET following CXCL12 or CXCL13 stimulation, respectively (D). Despite the fact that βarr1 is likely recruited to CXCR4 or CXCR5 at the plasma membrane, the location where STAM1 is recruited remains to be fully established. Previously, we have shown that βarr1 and STAM1 colocalize on early endosome antigen-1 (EEA1)–positive early endosomes with CXCR4  following agonist stimulation (A and D). CCR5, which is unable to recruit STAM1 to βarr1 (D), has a single Ser residue at the analogous position to Ser-346/7 of CXCR4, suggesting that two phosphosites are required. However, the β2AR also has double Ser/Thr at the distal C-tail (A), and yet it is unable to recruit STAM1 to βarr1 (B and 6D). However, Ser-346/7 on CXCR4 are part of a larger phosphocluster of Ser/Thr residues and negative charged amino acids (A). Similarly, CXCR5, NTS1, and AT1A, but not CCR5 or β2AR, have large phosphoclusters in the analogous region of the distal C-tail (A). Therefore, it is possible that GPCRs with phosphoclusters at the distal C-tail may specify βarr1 binding and STAM1 recruitment, although additional GPCRs will have to be examined before we can make this generalization with confidence. It is important to note that the phosphorylation status of these GPCRs was not examined experimentally in this study. Recently, a predicted phosphorylation pattern  (A) was defined based on the crystal structure of rhodopsin in complex with arrestin-1 (2AR (A), have this phosphorylation pattern at the distal C-tail; therefore, this phosphorylation pattern per se may not be driving STAM1 recruitment to βarr1, and other determinants and/or structural features are required. Furthermore, how the βarr1–STAM1 complex subsequently leads to activation of FAK remains to be investigated.Our study also provides evidence for the first time that STAM1 recruitment to βarr1 is mediated by other GPCRs. GPCRs that have double Ser/Thr residues at their distal C-tail are able to recruit STAM1 to βarr1  A was defrestin-1 . The sparestin-1 . All then-1   (Sigma) supplemented with 10% fetal bovine serum (FBS) (Omega). Rabbit monoclonal anti-FAK  and mouse monoclonal anti-GAPDH  antibodies were from Proteintech. The rabbit polyclonal anti-pTyr-397-FAK antibody  was from Life Technologies. The rabbit monoclonal anti-ERK1/2  and anti-pERK1/2  antibodies were from Cell Signaling Technology. The mouse monoclonal anti-HA antibody  was from BioLegend. The rabbit polyclonal anti-pS346/7-CXCR4 , anti-pS324/5-CXCR4 , and anti-CXCR4  antibodies were from 7TM Antibodies. The mouse monoclonal anti-GAPDH antibody  was from Abcam. The mouse monoclonal anti-FLAG antibody , neurotensin , and isoproterenol  were from Sigma–Aldrich. CXCL12, CXCL13, and CCL5 were from Protein Foundry. Compound 101  was from Hello Bio. PKC inhibitor Gö6983 , angiotensin II , and cilengitide  were from Tocris Bioscience. Coelenterazine H  and coelenterazine 400a  were from Biotium. Polyethylenimine  was from Polysciences, Inc.10 plasmids were kind gifts from Nikolaus Heveker  and Michel Bouvier , respectively. Rluc3 was previously described by Leduc et al.  by removing the modules for the tobacco etch virus protease cleavage site, the tetracycline transactivator protein, and the vasopressin V2 receptor tail and recircularization with the KLD enzyme mix . The NTS1-Rluc8 plasmid was made using NEBuilder HiFi DNA assembly with a fragment of Rluc8, amplified by PCR from pTRE-Tight-Rluc8 plasmid and the NTS1-coding region, amplified by PCR from the NTS1-Tango plasmid. The β2AR-Rluc8 plasmid was made using NEBuilder HiFi DNA assembly with an Rluc8 fragment amplified by PCR from pTRE-Tight-Rluc8 plasmid and a fragment of the β2AR containing the pcDNA backbone amplified by PCR. The four most C-terminal putative phosphorylation sites on the C-tail of CXCR5  were substituted for alanine residues (FLAG-CXCR5-4S/A and CXCR5-RLuc8-4S/A) using nonoverlapping back-to-back mutagenic primers encoding the alanine substitutions were used to amplify template DNA encoding FLAG-CXCR5 or CXCR5-RLuc8 using Platinum SuperFi DNA polymerase and circularized with KLD enzyme mix. The new plasmids described in this study and primers used to make new plasmids are listed in The CXCR4-Rluc3 and βarrestin1-GFPc et al.  and is ac et al. . The HA-c et al. . The Rluc et al. , 31. HA-eviously . FLAG-CX2, HEK293 cells grown on 6-cm or 10-cm dishes were cotransfected with GPCR (2–3 μg), Rluc8-STAM1 (200–400 ng), and βarr1-GFP10 (600 ng–1.2 μg) using PEI. To measure βarr1 recruitment to the GPCR by BRET2, HEK293 cells grown on 10-cm dishes were cotransfected with GPCR-Rluc8 (100 ng) and βarr1-GFP10 (300 ng). To measure βarr1 recruitment to CXCR4 or variant receptor by BRET1, HEK293 cells grown on 6-cm dishes were cotransfected with HA-CXCR4-YFP or variants HA-CXCR4-YFP-S324/5A or HA-CXCR4-YFP-S346/7A (30 ng) and βarr1-RLuc8 (5 ng). Twenty-four hours later, cells were seeded into 96-well white microplates at a density of 20,000 to 50,000 cells/well in DMEM containing 5% FBS. The next day, cells were washed with Dulbecco’s PBS supplemented with 20 mM Hepes, pH = 7.4 and then replaced with 80 μl of the same buffer. For inhibitor treatment, cells were pretreated with either 30 μM compound 101 for 30 min or 1 μM Gö6893 for 15 min before stimulation with increasing concentrations of ligands for 15 min at 37 °C. After incubation with ligands, 5 μM luciferase substrate coelenterazine 400A (DeepBlue C) or 50 μM luciferase substrate coelenterazine H was added for BRET2 or BRET1, respectively. BRET measurements were performed with a BioTek Cytation 5 cell imaging multimode reader or a BMG Labtech LUMIstar OMEGA microplate reader. For BRET2 readings, Rluc donor emission was detected at 440 ± 40 nm and acceptor GFP emission was detected at 515 nm ± 15 nm bandwidth. For BRET1 readings, Rluc donor emission was detected at 470 nm ± 15 nm and YFP acceptor emission was detected at 530 nm ± 15 nm. For each BRET experiment, RLuc plasmid–transfected cells were included as an internal background control. BRET ratios were calculated by dividing the acceptor emission light intensity by the donor emission light intensity, as we previously described , and equal amounts were analyzed by 10% SDS-PAGE, transferred to nitrocellulose, and blocked for 1 h in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% milk (w/v). Blots were incubated overnight at 4 °C with primary antibodies against CXCR4 (nonphospho) and (phosphosensitive) pSer346/7 or pS324/5. Blots were washed with TBS-T and incubated with anti-rabbit horseradish peroxidase–conjugated secondary antibody for 1 h at room temperature. Blots were washed extensively with TBS-T and developed with Promega ECL solution  on a ChemiDoc imaging system (Bio-Rad). Data were analyzed using ImageJ software . Phosphorylated  for 30 min, followed by stimulation with vehicle or 50 nM CXCL12 for 5 min or 15 min at 37 °C. For receptor variants, HEK293 cells grown on 6-cm dishes were transiently transfected with empty vector (pCMV10), HA-CXCR4, HA-CXCR4-S346/7A, HA-CXCR4-S324/5A, or HA-CCR5-R4 tail using PEI. Twenty-four hours post-transfection, cells were seeded onto 6-well plates. The next day, cells were serum starved in DMEM supplemented with 20 mM Hepes for 3 h. Cells were stimulated with vehicle, 50 nM CXCL12, or 500 nM CCL5 for 15 min at 37 °C. Cells were washed once with ice-cold PBS and lysed in 250 μl lysis buffer  containing protease inhibitors and phosphatase inhibitors . Samples were sonicated and cleared by centrifugation at 13,000orylated  or nonphorylated  CXCR4 noAgonist-induced FAK or ERK1/2 MAP kinase phosphorylation was performed as previously described . HeLa cel-lysine . The next day, cells were rinsed once with ice-cold TBS buffer  and immediately fixed with 3.7% formaldehyde  for 15 min. After three washes with TBS, cells were incubated for 45 min at room temperature in TBS containing 1% bovine serum albumin (BSA) and then incubated with an anti-FLAG alkaline phosphatase–conjugated antibody at a dilution of 1:1000 in TBS–1% BSA for 1 h at room temperature. Cells were washed three times with TBS followed by incubation with developing solution until the appearance of a light yellow color. For detection of HA-tagged receptors, cells were incubated with an anti-HA antibody at a dilution of 1:1000 in TBS–1% BSA for 1 h at room temperature. Cells were then washed three times with TBS followed by an incubation with TBS containing 1% BSA for 45 min. Then, cells were incubated with an alkaline phosphatase–conjugated mouse antibody at a dilution of 1:5000 in TBS–1% BSA for 1 h at room temperature. Cells were washed three times with TBS followed by incubation with developing solution until the appearance of a light yellow color. Reactions were quenched by transferring 100 μl of this solution to a 96-well plate containing 100 μl of 0.4 M NaOH. The 96-well plate was read at 405 nm single end-point reading. The relative cell surface levels ± the standard deviation from multiple independent experiments performed in triplicate are reported in the relevant text and figure legends.Surface expression of receptors was examined by whole-cell ELISA assay, as previously described . Brieflyp < 0.05 was considered significant). Differences between the two groups were tested with a Student’s t test, and the differences between three or more groups were tested by one-way or two-way ANOVA. For BRET data analysis, data were averaged from at least four independent experiments and fit to the nonlinear regression curve. BRET data were transformed using GraphPad Prism by minimum–maximum normalization to preserve the original relationships based on the equation X′ = (X − Xmin)/(Xmax − Xmin), where X′ is the newly transformed value. The EC50 values before and after transformation were similar.Data were analyzed using GraphPad Prism, version 9.3.1 software . Densitometric analysis of immunoblots was performed using ImageJ. The results from three or four independent experiments were analyzed by one-way ANOVA with Bonferroni’s multiple comparison test (All data associated with this study are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Arabidopsis thaliana. CDC48A hexamers are well preserved under negative staining conditions but disassemble during grid freezing using the classical blotting method. Vitrification of grids using the blot-free Chameleon method preserved the integrity of particles but resulted in their strong preferential orientation. We then used a strategy where we improved in parallel the purification of CDC48A and the conditions for cryo-EM data acquisition. Indeed, we noted that images taken from thicker ice presented an even distribution of intact particles with random orientations, but resulted in a lower image resolution. Consequently, in our case, distribution, orientation, image resolution, and the integrity of particles were tightly correlated with ice thickness. By combining the more homogeneous and stable CDC48A hexamers resulting from our improved purification protocol with an iterative search across different ice thicknesses, we identified an intermediate thickness that retained sufficiently high-resolution structural information while maintaining a complete distribution of particle orientations. Our approach may provide a simple, fast, and generally applicable strategy to record data of sufficient quality under standard laboratory and microscope settings. This method may be of particular value when time and resources are limited.Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric AAA ATPase from  Owing to the “resolution revolution,” cryogenic electron microscopy (cryo-EM) has become one of the major structural biology methods . The numEspecially multi-domain, and multi-protein complexes often require additional steps to prepare them for structural characterization. In particular, two challenges frequently occur when working with such complexes: 1) the complex is structurally heterogeneous, and/or dissociates during cryo-EM grid preparations, and 2) samples in optimally thin ice show a strong preferred orientation, and tend to dissociate or unfold at the air-water interface during grid preparation.A. thaliana cell division cycle 48 A (CDC48A) protein complex. Being targeted to specific substrates by adaptor proteins, CDC48A extracts proteins from their cellular location, unfolds them, and routes them for degradation by the proteasome. By rapidly modifying the protein composition in specific subcellular loci, CDC48A regulates a large variety of fast-response mechanisms in plants  cells containing an N-terminal GST tag with a PreScission protease cleavage site. The E. coli cells were cultured at 37°C in 2% LB Broth containing 100 µg/ml ampicillin, shaking at 200 rpm until the optical density at 600 nm reached 0.6–0.8. Subsequently, expression of CDC48A was induced using 150 µM of IPTG, and allowed to continue overnight, shaking at 200 rpm at 16°C. Cells were then pelleted by centrifugation at 7,500 rpm  for 15 min at 4°C, and stored at −80°C.The cDNA for CDC48A (AT3g09840) were purchased from Integrated DNA Technologies (IDT), and then subcloned to the pGEX6P-1 vector between BamHI, and NotI restriction sites. Proteins were expressed in CDC48A was purified using two methodologies. The standard method is as follows: frozen cell pellets were thawed on ice, and resuspended in lysis buffer containing . Cells were lysed by sonication  on ice for 8 min at 35% amplitude with a pulse rate of 1 s on, and 1 s off. The lysate was pelleted by centrifugation at 27,000 rpm  for 30 min at 4°C. The supernatant was added to a GST-affinity column containing glutathione sepharose beads (Cytiva) for 2 h, shaking at 4°C. The flowthrough was discarded, and the column washed with 3 column volumes (CV) of wash buffer containing  followed by 1 CV of an elution buffer containing  or  . CDC48A was cleaved overnight, shaking at 4°C in 15 ml of elution buffer with 3C protease. For samples analyzed at ACSMB, and eBIC, the cleavage solution additionally contained 15 units of apyrase (NEB). The eluate was then concentrated to a volume of 5 ml using an Ultra-15, 50 kDa MWCO, Amicon filtration device (Millipore), and purified using HiLoad 16/600 Superdex 200 pg  size-exclusion chromatography (SEC). Protein purity was analyzed using SDS-PAGE gels, and the relevant fractions were pooled, and stored at −80°C or used fresh, as described.The optimized purification was carried out as above, with the following changes: one tablet of complete EDTA-containing protease inhibitor was used (Roche) per 50 ml preparation, and the cell lysate was treated with 2.5 µl of benzonase nuclease (Millipore), and incubated for 20 min at room temperature. During GST-affinity chromatography, the supernatant was added to the column for 15 min at 4°C. The flowthrough was retained, and the column washed with 4 CV of high-salt wash buffer containing . The retained flowthrough was then added to the column for an additional 15 min, followed by an identical washing step. The flowthrough was discarded, and the column washed with 1 CV of elution buffer. CDC48A was similarly cleaved overnight, further purified using SEC, then stored at −80°C, or used fresh, as described.https://github.com/strubelab/multiprot) in several configurations, and the best fitting models were overlaid on the SAXS profile.SAXS data were recorded at the SWING beamline  using a wavelength of 1.03 Å. Scattering data were collected from a 5 mg/ml sample of CDC48A (from the standard purification after freeze-thawing once) saturated with 2 mM of ATP (Sigma-Aldrich), with the sample passed through Bio SEC-3 HPLC (Agilent) SEC prior to analysis. The distance of the sample to the detector was 1.8 m, resulting in the momentum transfer of a range of 0.1 1/Å < q < 0.5 1/Å. Buffer data were calculated from the buffer  eluted before proteins, and subtracted from the protein data using SWING’s on-site software. Data were analyzed using PRIMUS, as part of the ATSAS software package , and modFor comparison of the two methods of purification, an equimolar concentration of cleaved eluate after each method of purification was analyzed using Superdex 200 Increase 10/300 GL (Cytiva) SEC with the elution buffer. The absorbance at 280 and 260 nm were recorded. The protein purity of the optimized method was assessed using SDS-PAGE gels.To detect the presence of nucleotides, the peak hexameric fraction (A11) from both analytical SEC experiments was analyzed using only the ADP detection step of the ADP-Glo assay . LuminesFor the initial EM experiments, CDC48A samples were prepared fresh after SEC from a fraction eluting after the hexamer peak (peak 2) to limit contamination with aggregates eluting in the neighboring peak 1 . When foFor negative stain grid preparation, 7 µl of CDC48A (0.1 mg/ml) was placed onto glow discharged grids  for 1 min, then blotted with Whatman blotting paper. Next, ∼5 µl of 2% uranyl acetate was added to the grid, and immediately blotted. Then, ∼5 µl of 2% uranyl acetate was added again, and left to stain for 1 min. Grids were then blotted, and air-dried.For cryogenic grid preparation at KAUST, samples were prepared using the Vitrobot Mark IV (Thermo Fisher Scientific) at 22°C with 100% humidity. CDC48A samples were prepared as previously described, applying 2 µl of sample, and blotting for 3 s. Samples from the initial CDC48A dataset, and optimized datasets 1 and 2 were prepared on glow discharged Cu grids , and samples from optimized datasets 3 and 4 were prepared on glow discharged Au grids . For cryogenic grid preparation at ACSMB, samples were prepared using the Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C in 95% humidity, with a blot time of 3 s. Grids were prepared using 2 mg/ml of CDC48A by applying 3 µl of sample to glow discharged Quantifoil Cu R1.2/1.3 grids (EMS). Cryogenic blot-free grids were prepared using the Chameleon instrument (SPT Labtech) at Rosalind Franklin Institute (RFI), United Kingdom . Self-wiNegative stain samples were screened on a Titan 80-300 (Thermo Fisher Scientific) operated at an acceleration voltage of 300 kV. Images were taken at different magnifications with a US4000 CCD camera (Gatan). For the 2D analysis of the negative stain sample, 75 images were recorded on Titan ST (Thermo Fisher Scientific) with a OneView camera (Gatan) at a magnification of 69k, and a pixel size of 1.7 Å.2. At ACSMB, cryo-EM data were recorded on a Titan Krios G2 (Thermo Fisher Scientific) operated at 300 kV equipped with K2 summit direct detector, and GIF BioQuantum967 Imaging Filter (Gatan company). A total of 1,311 movies were collected at 130k magnification in electron counting mode at a pixel size of 1.07 Å/pixel. The total exposure time was 10 s, dose-fractionated into 60 frames (0.167 s per frame) with a total dose of 75 e/Å2. At eBIC, Diamond Light Source, cryo-EM data were recorded on a Glacios operated at 200 kV equipped Falcon IV direct detector (Thermo Fisher Scientific). A total of 1,472 movies were collected at 120k magnification in electron counting mode at a pixel size of 1.192 Å/pixel. The total exposure time was 10 s, dose-fractionated into 50 frames (0.200 s per frame), with a total dose of 48.1 e/Å2. A comparison of the data collection conditions across all cryo-EM datasets can be found in Cryo-EM data were recorded in multiple facilities. At KAUST, cryo-EM data were recorded on a Titan Krios G1 (ThermoFisher Scientific) operated at 300 kV, equipped with a K2 Summit direct detector, and GIF Quantum968 Imaging Filter (Gatan company). Movies were collected at 130k magnification in super-resolution mode yielding a pixel size of 0.52 Å/pixel at specimen level. The initial dataset had 1,242 movies, optimized dataset 1 had 1,073, dataset 2 had 797, dataset 3 had 2,118, and dataset 4 had 2,284. The total exposure time was 5.6 s, dose-fractionated into 32 frames (0.175 s per frame) with a total dose of 50 e/Åhttps://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/). A total of 74k particles were picked from 75 images, and extracted with a box size of 180 pixels in Relion-4.0-beta-1-commit-11e38ba  parameters were estimated by Gctf , and par-11e38ba , and 2D ab initio reconstruction was computed using the filtered particles through the ab Initio Reconstruction job in cryoSPARC v3.3.1. Default reconstruction settings were used, aside from 500 extra final iterations in all, and C6 symmetry was imposed where noted. Details including the number of movies, and particles picked can be found summarized in Data collected at KAUST were motion-corrected using Warp  with a bAs an indirect measure of ice thickness, all images corresponding to each of the cryo-EM datasets were plotted according to their relative ice thickness, determined using the relative ice thickness parameter in the Manually Curate Exposure option, following patch CTF estimation (multi), in cryoSPARC v3.3.1.λ = 322 nm) on a Titan Krios operated at 300 kV using 100 micron Objective Aperture, and equipped with Gatan’s GIF/K2 detector. Ice thickness was measured from one thin ice region, one thick ice region, and two regions of intermediate ice thicknesses. For each ice thickness, five measurements were averaged.As a direct measure of ice thickness, frozen aliquots of cleaved CDC48A (following the optimized methodology) were further purified using SEC. Fresh sample corresponding to the peak hexameric fraction was used to prepare cryogenic grids under the conditions used for optimized datasets 3 and 4. Ice thickness was measured using the aperture limited scattering method of A. thaliana CDC48A in E. coli and purified the protein according to our standard protocol  showed that CDC48A persists as hexamers during gel filtration, and that hexamers are the prevailing species in solution, as homo-hexameric models best fitted the scattering curve (We recombinantly expressed ng curve . These eWe first performed a negative stain (NS) screening to probe the suitability of CDCD48A hexamers for cryo-EM studies. We iteratively optimized the protein concentration, and staining protocol to obtain NS-EM images of densely packed but well-separated particles (see Methods). NS-EM revealed our sample to be sufficiently monodispersed, and mostly hexameric . Indeed,Methods), and collected the initial cryo-EM images at the Imaging, and Characterization Core Lab (IAC) in KAUST. We observed a sufficiently dense distribution of particles throughout grid regions. However, the particles did not appear homogeneous in size, and shape and differed markedly from the NS-EM images  using a Vitrobot . In the section thereafter, we report our concurrent improvements to the protein production strategy. The benefits of the improvements of grids, and samples were additive, as demonstrated in the final section that reports on the data obtained from the optimized samples with the improved data collection strategy.Methods), and used them for a subsequent round of cryo-EM screening at the Astbury Centre for Structural Molecular Biology (ACSMB), University of Leeds, United Kingdom. Inspection of the images confirmed that hexamers largely dissociated in optimally thin ice regions, evidenced by an increase in background noise, and a reduction in visible hexamers. However, when screening regions of the grids that had thicker ice we noted the substantially increased prevalence of intact hexamers , instead of Vitrobot blotting. The faster spray-on method is expected to limit the number of interactions of the protein with the air-water interface prior to vitrification, and thus preserve the integrity of proteins . Additiotruction . Therefotruction . Using tIn conclusion, using the standard Vitrobot protocol, we could either observe a dissociated complex in optimally thin ice or an intact complex in thicker ice at the cost of resolution. Conversely, blot-free vitrification by the Chameleon preserved the sample integrity but introduced a strong preferential orientation due to the asymmetrical shape of our complex that compromises its 3D reconstruction. To be able to have a rapid workflow using our in-house Vitrobot, we decided to explore optimizing the ice thickness jointly with improving the intrinsic stability of the protein sample.Methods). This protocol quickly separated the GST-fused protein from contaminants such as proteases and reduced the overall time for the purification  affinity column, and then a SEC. With this protocol we observed that the resulting samples displayed an absorption ratio of 260–280 nm (A260/280) of above 1 indicating strong contamination of the sample by nucleic acids and/or bound nucleotides . Therefofication . The higfication ; Table 2fication . Moreovefication . ATP is fication . Hence, Next, we wanted to assess the impact the new protocol had on the quality of the EM grids using NS-EM. We found that grids prepared from the peak hexameric CDC48A fractions that were pooled, concentrated, and frozen contained large amounts of aggregate clusters and indiscernible individual particles . ConversBecause our initial cryo-EM work had shown disassembly and preferential orientation in thin ice for our hexameric CDC48A sample, we first investigated the behavior of our biochemically optimized protein sample in thin ice. In line with the improvements we saw in NS-EM, the optimized sample preparation increased the integrity of the sample in cryo-EM, as homogeneous and well-dispersed hexameric particles were observed under thin ice conditions following Vitrobot blotting. However, our screening of thin ice grid regions revealed a high degree of preferential orientation with particles oriented primarily in a top or bottom view. The first movies recorded from grid regions with thick ice showed a homogeneous distribution of orientations, but at the cost of high-resolution information. Therefore, we decided to start from thick ice that displays a wide distribution of particle orientations, and record datasets at different ice thicknesses to identify the optimal compromise between particle resolution, and orientation. In line with our previous observations, we did not record movies in holes presenting the thinnest ice.Using the image grayscale as a proxy for ice thickness, we collected four separate datasets from grid regions of decreasing ice thickness and processed each dataset individually. Utilizing CTF parameter estimation in cryoSPARC, the relative ice thickness and CTF fit resolution was compared for each dataset . The estab intio reconstructions were computed for each dataset , and preferential particle orientation. We demonstrate that in our case, the hexameric ATPase CDC48A, these problems could be solved through the combination of efficient but non-conventional methods that led to higher oligomer stability, and orientational homogeneity.Indeed, we initially observed that purified CDC48A displayed a hexameric organization when observed in negative stain but appeared dissociated in cryo-EM images, likely indicating that the freezing step dramatically impaired the integrity of CDC48A. It is well-described that the interaction of proteins with the air-water interface during the vitrification step can have a strong effect on the integrity of protein complexes. We showed that the Chameleon’s blot-free and spray-on method  preserveWe then decided to develop a strategy where we improved in parallel the purification of CDC48A, and the conditions for cryo-EM data acquisition. One part of our strategy was to improve the workflow from cell lysis to grid preparation. Simple improvements of the handling speed, buffers, and protocols sufficiently enhanced the protein stability to enable data collection without particle dissociation, and that from ice thickness suitable for high resolution acquisition. In our case study we achieved both a higher purity and conformational homogeneity of CDC48A by removing DNA, and nucleotides from our sample. Other means for removing DNA contamination exist but may need to be assessed for unwanted side effects. For example, polyethyleneimine (PEI) treatment may lead to a significant “collateral” precipitation of the protein of interest.ab initio structure modeling.A second part of our strategy was the selection of the most suitable ice thickness. It is not unexpected that thicker ice may improve the integrity of particles and their angular distribution on cryo grids. However, these effects of ice thickness are normally not systematically assessed. Moreover, in most standard settings, obtaining high-resolution data is the main focus, leading to an automated recording of movies in grid regions presenting the thinnest ice layers. In our case, we show that ice thickness correlates positively with the particle orientational coverage , and witThere are, of course, also other, more sophisticated ways of achieving a more homogeneous particle orientation . In our Several other biochemical solutions have been reported to increase the particle orientation coverage in optimally thin ice, such as the use of detergents, biotinylation, antibodies, or DNA origami, in varying the pH and/or the ionic strength . But theIn summary, although more sophisticated alternative methods exist to increase particle stability, integrity, and orientation, our parallel approach has the advantage of not requiring specific chemicals, expertise, or instruments, being based on off-the-shelf ingredients and standard procedures . Hence, our approach provides a robust and fast method applicable to all standard laboratory settings and cryo-EM facilities. Although our dual strategy was only tested on one project, the method is general enough to apply to other non-covalently linked oligomeric samples, samples with DNA contamination, and/or samples that show a preferential orientation in thin ice. In particular, our approach can help “salvage” grids for achieving sufficient data quality without further delays. Indeed, speed may have become a more important parameter than maximum resolution in the highly competitive cryo-EM field.