Mechanism of NMDA Receptor Inhibition and Activation.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that mediate synaptic transmission and underpin learning and memory. NMDAR dysfunction is directly implicated in diseases ranging from seizure to ischemia. Despite its fundamental importance, little is known about how the NMDAR transitions between inactive and active states and how small molecules inhibit or activate ion channel gating. Here, we report electron cryo-microscopy structures of the GluN1-GluN2B NMDA receptor in an ensemble of competitive antagonist-bound states, an agonist-bound form, and a state bound with agonists and the allosteric inhibitor Ro25-6981. Together with double electron-electron resonance experiments, we show how competitive antagonists rupture the ligand binding domain (LBD) gating "ring," how agonists retain the ring in a dimer-of-dimers configuration, and how allosteric inhibitors, acting within the amino terminal domain, further stabilize the LBD layer. These studies illuminate how the LBD gating ring is fundamental to signal transduction and gating in NMDARs.

Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states.

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain "layer" of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.

Mechanism of NMDA receptor channel block by MK-801 and memantine.

The NMDA (N-methyl-D-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1-GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer's disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.

Human endogenous retroviruses: our genomic fossils and companions.

Approximately 8% of the human genome, over four times more than its protein-coding regions, comprises sequences of viral origin that are known as human endogenous retroviral elements (HERVs). Present in the genome of all human cells, HERVs resulted from the integration of now-extinct exogenous retroviruses into mammalian ancestor germ cells or their precursors on several occasions, sometimes as long as tens of millions of years ago. Most HERVs have become silenced because of mutations such as substitutions, insertions, or deletions, and as a result of epigenetic changes, and are vertically transmitted in the population. Considered for a long time to be part of the "junk DNA," HERVs were shown, in more recent years, to perform critical functions in the host. Two of the very few HERVs known to encode functional proteins, syncytin-1 and syncytin-2, are critical during embryogenesis, when they contribute to the formation of the placenta and facilitate tolerance of the maternal immune system toward the developing fetus. Homologs of syncytin-encoding genes were described in several other species, and it appears that during evolution they were stably endogenized into the respective genomes on multiple occasions and became co-opted for critical physiological functions. The aberrant expression of HERVs has been linked to conditions that include infectious, autoimmune, malignant, and neurological diseases. HERVs, our genomic fossils and storytellers, provide a fascinating and somewhat mysterious insight into our co-evolution with viruses, and will undoubtedly offer many teachings, surprises, and paradigm changes for years to come.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4-9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.

Effects of Diet versus Gastric Bypass on Metabolic Function in Diabetes.

BACKGROUND: Some studies have suggested that in people with type 2 diabetes, Roux-en-Y gastric bypass has therapeutic effects on metabolic function that are independent of weight loss. METHODS: We evaluated metabolic regulators of glucose homeostasis before and after matched (approximately 18%) weight loss induced by gastric bypass (surgery group) or diet alone (diet group) in 22 patients with obesity and diabetes. The primary outcome was the change in hepatic insulin sensitivity, assessed by infusion of insulin at low rates (stages 1 and 2 of a 3-stage hyperinsulinemic euglycemic pancreatic clamp). Secondary outcomes were changes in muscle insulin sensitivity, beta-cell function, and 24-hour plasma glucose and insulin profiles. RESULTS: Weight loss was associated with increases in mean suppression of glucose production from baseline, by 7.04 µmol per kilogram of fat-free mass per minute (95% confidence interval [CI], 4.74 to 9.33) in the diet group and by 7.02 µmol per kilogram of fat-free mass per minute (95% CI, 3.21 to 10.84) in the surgery group during clamp stage 1, and by 5.39 (95% CI, 2.44 to 8.34) and 5.37 (95% CI, 2.41 to 8.33) µmol per kilogram of fat-free mass per minute in the two groups, respectively, during clamp stage 2; there were no significant differences between the groups. Weight loss was associated with increased insulin-stimulated glucose disposal, from 30.5±15.9 to 61.6±13.0 µmol per kilogram of fat-free mass per minute in the diet group and from 29.4±12.6 to 54.5±10.4 µmol per kilogram of fat-free mass per minute in the surgery group; there was no significant difference between the groups. Weight loss increased beta-cell function (insulin secretion relative to insulin sensitivity) by 1.83 units (95% CI, 1.22 to 2.44) in the diet group and by 1.11 units (95% CI, 0.08 to 2.15) in the surgery group, with no significant difference between the groups, and it decreased the areas under the curve for 24-hour plasma glucose and insulin levels in both groups, with no significant difference between the groups. No major complications occurred in either group. CONCLUSIONS: In this study involving patients with obesity and type 2 diabetes, the metabolic benefits of gastric bypass surgery and diet were similar and were apparently related to weight loss itself, with no evident clinically important effects independent of weight loss. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT02207777.).

Efficacy and safety of MAS825 (anti-IL-1ß/IL-18) in COVID-19 patients with pneumonia and impaired respiratory function.

MAS825, a bispecific IL-1ß/IL-18 monoclonal antibody, could improve clinical outcomes in COVID-19 pneumonia by reducing inflammasome-mediated inflammation. Hospitalized non-ventilated patients with COVID-19 pneumonia (n = 138) were randomized (1:1) to receive MAS825 (10 mg/kg single i.v.) or placebo in addition to standard of care (SoC). The primary endpoint was the composite Acute Physiology and Chronic Health Evaluation II (APACHE II) score on Day 15 or on the day of discharge (whichever was earlier) with worst-case imputation for death. Other study endpoints included safety, C-reactive protein (CRP), SARS-CoV-2 presence, and inflammatory markers. On Day 15, the APACHE II score was 14.5 ± 1.87 and 13.5 ± 1.8 in the MAS825 and placebo groups, respectively (P = 0.33). MAS825 + SoC led to 33% relative reduction in intensive care unit (ICU) admissions, ~1 day reduction in ICU stay, reduction in mean duration of oxygen support (13.5 versus 14.3 days), and earlier clearance of virus on Day 15 versus placebo + SoC group. On Day 15, compared with placebo group, patients treated with MAS825 + SoC showed a 51% decrease in CRP levels, 42% lower IL-6 levels, 19% decrease in neutrophil levels, and 16% lower interferon-γ levels, indicative of IL-1ß and IL-18 pathway engagement. MAS825 + SoC did not improve APACHE II score in hospitalized patients with severe COVID-19 pneumonia; however, it inhibited relevant clinical and inflammatory pathway biomarkers and resulted in faster virus clearance versus placebo + SoC. MAS825 used in conjunction with SoC was well tolerated. None of the adverse events (AEs) or serious AEs were treatment-related.

SPEACH_AF: Sampling protein ensembles and conformational heterogeneity with Alphafold2.

The unprecedented performance of Deepmind's Alphafold2 in predicting protein structure in CASP XIV and the creation of a database of structures for multiple proteomes and protein sequence repositories is reshaping structural biology. However, because this database returns a single structure, it brought into question Alphafold's ability to capture the intrinsic conformational flexibility of proteins. Here we present a general approach to drive Alphafold2 to model alternate protein conformations through simple manipulation of the multiple sequence alignment via in silico mutagenesis. The approach is grounded in the hypothesis that the multiple sequence alignment must also encode for protein structural heterogeneity, thus its rational manipulation will enable Alphafold2 to sample alternate conformations. A systematic modeling pipeline is benchmarked against canonical examples of protein conformational flexibility and applied to interrogate the conformational landscape of membrane proteins. This work broadens the applicability of Alphafold2 by generating multiple protein conformations to be tested biologically, biochemically, biophysically, and for use in structure-based drug design.

Sequence and structural determinants of ligand-dependent alternating access of a MATE transporter.

Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pyrococcus furiosus Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.

The far-reaching impact of abortion bans: reproductive care and beyond.

On 24 June 2022, the US Supreme Court overturned Roe v. Wade, a 49-year-old precedent that provided federal constitutional protection for abortions up to the point of foetal viability, returning jurisdiction to the individual states. Restrictions that came into effect automatically in several states, and are anticipated in others, will severely limit access to abortions in approximately half of the US. Even though every state allows for exceptions to the abortion bans, in some instances these exceptions can be used to preserve the health of a pregnant patient, while in other instances, only to preserve their life. The vague and confusing nature of the abortion ban exceptions threatens to compromise the standard of care for patients with pregnancy complications that are distinct from abortions, such as nonviable pregnancies, miscarriages, and ectopic pregnancies. Additionally, we envision challenges for the treatment of women with certain autoimmune conditions, pregnant cancer patients, and patients contemplating preimplantation genetic diagnosis as part of assisted reproductive technologies. The abortion ban exceptions will impact and interfere with the medical care of pregnant and non-pregnant patient populations alike and are poised to create a medical and public health crisis unlike any other one from the recent past.

Do lifestyle factors and quality of life differ in people with metabolically healthy and unhealthy obesity?

BACKGROUND AND OBJECTIVES: Although obesity is typically associated with metabolic co-morbidities, some people with obesity do not develop metabolic abnormalities. We evaluated whether modifiable lifestyle factors (i.e., physical activity, dietary composition, and sleep characteristics) can help explain why some people with obesity are metabolically healthy (MHO) and whether metabolically unhealthy obesity (MUO) affects quality of life (QOL). SUBJECTS/METHODS: Physical activity and sleep characteristics were assessed by using tri-axial accelerometers and dietary intake, sleep quality, and QOL were evaluated by using validated questionnaires in people stratified into three groups: (1) lean with normal glucose tolerance, plasma triglyceride (TG) concentration and intrahepatic TG (IHTG) content (metabolically healthy lean [MHL]; n = 20); (2) obesity and normal glucose tolerance, plasma TG concentration and IHTG content (MHO; n = 36); and (3) obesity with abnormal glucose metabolism and hepatic steatosis (MUO; n = 43). RESULTS: People with MHO performed ~45-min more light-intensity physical activity/day than the MHL and MUO groups (P < 0.05). QOL, particularly the physical function domain, was higher in the MHO than the MUO group (P < 0.05). Although self-reported intake of starch, dairy, and cured meats were higher in the MUO than the MHO group (P < 0.02), the absolute differences were small and unlikely to have metabolic effects. No differences were found in sleep duration or quality between groups. CONCLUSIONS: These data suggest physical activity, but not sleep or dietary intake, contribute to better metabolic health in people with MHO than those with MUO, and that QOL is lower in people with MUO than those with MHO.

Mifepristone: A Safe Method of Medical Abortion and Self-Managed Medical Abortion in the Post-Roe Era.

BACKGROUND: The U.S. Supreme Court's Dobbs v. Jackson Women's Health Organization decision on June 24, 2022 effectively overturned federal constitutional protections for abortion that have existed since 1973 and returned jurisdiction to the states. Several states implemented abortion bans, some of which banned abortion after 6 weeks and others that permit abortion under limited exceptions, such as if the health or the life of the woman is in danger. Other states introduced bills that define life as beginning at fertilization. As a result of these new and proposed laws, the future availability of mifepristone, one of two drugs used for medical abortion in the United States, has become the topic of intense debate and speculation. AREAS OF UNCERTAINTY: Although its safety and effectiveness has been confirmed by many studies, the use of mifepristone has been politicized regularly since its approval. Areas of future study include mifepristone for induction termination and fetal demise in the third trimester and the management of leiomyoma. DATA SOURCES: PubMed, Society of Family Planning, American College of Obstetricians and Gynecologists, the World Health Organization. THERAPEUTIC ADVANCES: The use of no-touch medical abortion, which entails providing a medical abortion via a telehealth platform without a screening ultrasound or bloodwork, expanded during the COVID-19 pandemic, and studies have confirmed its safety. With the Dobbs decision, legal abortion will be less accessible and, consequently, self-managed abortion with mifepristone and misoprostol will become more prevalent. CONCLUSIONS: Mifepristone and misoprostol are extremely safe medications with many applications. In the current changing political climate, physicians and pregnancy-capable individuals must have access to these medications.

Emergency Contraception: Access and Challenges at Times of Uncertainty.

BACKGROUND: The UN Commission on Life-Saving Commodities for Women and Children identified emergency contraceptive pills as 1 of the 13 essential underused, low-cost, and high-impact commodities that could save the lives of millions of women and children worldwide. In the US, 2 emergency contraceptive regimens are currently approved, and their most plausible mechanism of action involves delaying and/or inhibiting ovulation. AREAS OF UNCERTAINTY: Abortion and contraception are recognized as essential components of reproductive health care. In the US, in the wake of the Dobbs v. Jackson Women's Health Organization Supreme Court decision on June 24, 2022, 26 states began to or are expected to severely restrict abortion. It is anticipated that these restrictions will increase the demand for emergency contraception (EC). Several obstacles to EC access have been described, and these include cost, hurdles to over-the-counter purchase, low awareness, myths about their mechanisms of action, widespread misinformation, and barriers that special populations face in accessing them. The politicization of EC is a major factor limiting access. Improving sex education and health literacy, along with eHealth literacy, are important initiatives to improve EC uptake and access. DATA SOURCES: PubMed, The Guttmacher Institute, Society of Family Planning, American College of Obstetricians and Gynecologists, the World Health Organization, The United Nations. THERAPEUTIC ADVANCES: A randomized noninferiority trial showed that the 52 mg levonorgestrel intrauterine device was noninferior to the copper intrauterine device when used as an EC method in the first 5 days after unprotected intercourse. This is a promising and highly effective emergency contraceptive option, particularly for overweight and obese patients, and a contraceptive option with a different bleeding profile than the copper intrauterine device. CONCLUSIONS: EC represents an important facet of medicine and public health. The 2 medical regimens currently approved in the US are very effective, have virtually no medical contraindications, and novel formulations are actively being investigated to make them more convenient and effective for all patient populations. Barriers to accessing EC, including the widespread presence of contraception deserts , threaten to broaden and accentuate the already existing inequities and disparities in society, at a time when they have reached the dimensions of a public health crisis.

Time matters - in vitro cellular disposition kinetics help rationalizing cellular potency disconnects.

Loss in potency is commonly observed in early drug discovery when moving from biochemical to more complex cellular systems. Among other factors, low permeability is often considered to cause such potency disconnects.We developed a novel cellular disposition assay in MDCK cells to determine passive uptake clearance (PSinf), cell-to-medium ratios at steady-state (Kp) and the time to reach 90% steady-state (TTSS90) from a single experiment in a high-throughput format.The assay was validated using 40 marketed drugs, showing a wide distribution of PSinf and Kp values. The parameters generally correlated with transcellular permeability and lipophilicity, while PSinf data revealed better resolution in the high and low permeability ranges compared to traditional permeability data. A linear relationship between the Kp/PSinf ratio and TTSS90 was mathematically derived and experimentally validated, demonstrating the dependency of TTSS90 on the rate and extent of cellular accumulation.Cellular disposition parameters could explain potency (IC50) disconnects noted for seven Bruton's tyrosine kinase degrader compounds in a cellular potency assay. In contrast to transcellular permeability, PSinf data enabled identification of the compounds with IC50 disconnects based on their time to reach equilibrium. Overall, the novel assay offers the possibility to address potency disconnects in early drug discovery.

Relation of social network support to child health behaviors among children in treatment for overweight/obesity.

PURPOSE: Little is known about the influence of social network support on child health behaviors in the context of weight-loss interventions. This study examined the associations between a child's co-participation (i.e., network support) in weight-related health behaviors (i.e., physical and sedentary activity, eating behavior) and the child's own health behaviors during family-based behavioral treatment (FBT). METHODS: Children (n = 241) with overweight/obesity (mean age = 9.4 ± 1.3y; 63% female) completed semi-structured interviews assessing network support for healthy/unhealthy eating and physical/sedentary activity, and a 3-day dietary recall. Physical activity was assessed with accelerometry, and sedentary activity was measured via parent-reported child screen time use. All assessments were taken at baseline and after 4 months of FBT. Hierarchical linear regressions examined changes in network support as they related to changes in health behaviors from baseline to the end of FBT. RESULTS: Changes in network support for healthy eating were related to changes in vegetable, but not fruit, intake across FBT, while changes in network support for unhealthy eating were negatively related to changes in diet quality. Changes in network support for sedentary activity were negatively related to changes in minutes of physical activity and positively related to changes in screen time. CONCLUSION: The present findings suggest that a child's network support for health behaviors may relate to behavior change among children during FBT and provide opportunities for targeted intervention. LEVEL OF EVIDENCE: III. cohort study.

Perturbation biology links temporal protein changes to drug responses in a melanoma cell line.

Cancer cells have genetic alterations that often directly affect intracellular protein signaling processes allowing them to bypass control mechanisms for cell death, growth and division. Cancer drugs targeting these alterations often work initially, but resistance is common. Combinations of targeted drugs may overcome or prevent resistance, but their selection requires context-specific knowledge of signaling pathways including complex interactions such as feedback loops and crosstalk. To infer quantitative pathway models, we collected a rich dataset on a melanoma cell line: Following perturbation with 54 drug combinations, we measured 124 (phospho-)protein levels and phenotypic response (cell growth, apoptosis) in a time series from 10 minutes to 67 hours. From these data, we trained time-resolved mathematical models that capture molecular interactions and the coupling of molecular levels to cellular phenotype, which in turn reveal the main direct or indirect molecular responses to each drug. Systematic model simulations identified novel combinations of drugs predicted to reduce the survival of melanoma cells, with partial experimental verification. This particular application of perturbation biology demonstrates the potential impact of combining time-resolved data with modeling for the discovery of new combinations of cancer drugs.

General and Eating Disorder Psychopathology in Relation to Short- and Long-Term Weight Change in Treatment-Seeking Children: A Latent Profile Analysis.

BACKGROUND: Concurrent general psychopathology (GP) and eating disorder psychopathology (EDP) are commonly reported among youth with overweight/obesity and may impact weight change. PURPOSE: We identified patterns of GP and EDP in children with overweight/obesity and examined the impact on weight change following family-based behavioral obesity treatment (FBT) and maintenance interventions. METHODS: Children (N = 172) participated in 4 month FBT and subsequent 8 month weight maintenance interventions. GP and EDP were assessed prior to FBT (baseline). Child percentage overweight was assessed at baseline, post-FBT (4 months), and post-maintenance (12 months). Latent profile analysis identified patterns of baseline GP and EDP. Linear mixed-effects models examined if profiles predicted 4- and 12-month change in percentage overweight and if there were two-way and three-way interactions among these variables, adjusting for relevant covariates. RESULTS: Results indicated a three-profile structure: lower GP and EDP (LOWER); subclinically elevated GP and EDP without loss of control (LOC; HIGHER); and subclinically elevated GP and EDP with LOC (HIGHER + LOC). Across profiles, children on average achieved clinically meaningful weight loss (i.e., ≥9 unit change in percentage overweight) from baseline to 4 month FBT and sustained these improvements at 12 month maintenance. There was no evidence that latent profiles were related to percentage overweight change from baseline to FBT (p > .05) or baseline to maintenance (p > .05). There was no evidence for two-way or three-way interactions (p > .05). CONCLUSION: Concurrent GP and EDP do not portend differential short- or long-term weight change following FBT and maintenance. Future research is warranted on the durability of weight change among youth with GP and EDP. TRIAL REGISTRATION: NCT00759746.

Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile.

The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens. Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens. Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients. Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear. Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile. Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans. Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion. Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency. These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.

Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae.

Secondary active transporters belonging to the multidrug and toxic compound extrusion (MATE) family harness the potential energy of electrochemical ion gradients to export a broad spectrum of cytotoxic compounds, thus contributing to multidrug resistance. The current mechanistic understanding of ion-coupled substrate transport has been informed by a limited set of MATE transporter crystal structures from multiple organisms that capture a 12-transmembrane helix topology adopting similar outward-facing conformations. Although these structures mapped conserved residues important for function, the mechanistic role of these residues in shaping the conformational cycle has not been investigated. Here, we use double-electron electron resonance (DEER) spectroscopy to explore ligand-dependent conformational changes of NorM from Vibrio cholerae (NorM-Vc), a MATE transporter proposed to be coupled to both Na+ and H+ gradients. Distance measurements between spin labels on the periplasmic side of NorM-Vc identified unique structural intermediates induced by binding of Na+, H+, or the substrate doxorubicin. The Na+- and H+-dependent intermediates were associated with distinct conformations of TM1. Site-directed mutagenesis of conserved residues revealed that Na+- and H+-driven conformational changes are facilitated by a network of polar residues in the N-terminal domain cavity, whereas conserved carboxylates buried in the C-terminal domain are critical for stabilizing the drug-bound state. Interpreted in conjunction with doxorubicin binding of mutant NorM-Vc and cell toxicity assays, these results establish the role of ion-coupled conformational dynamics in the functional cycle and implicate H+ in the doxorubicin release mechanism.