IgE is an immunoglobulin that plays a central role in acute allergic reactions and chronic inflammatory allergic diseases. The development of a drug able to neutralize this antibody represents a breakthrough in the treatment of inflammatory pathologies with a probable allergic basis. This review focuses on IgE-related chronic diseases, such as allergic asthma and chronic urticaria (CU), and on the role of the anti-IgE monoclonal antibody, omalizumab, in their treatment. We also assess the off-label use of omalizumab for other pathologies associated with IgE and report the latest findings concerning this drug and other new related drugs. To date, omalizumab has only been approved for severe allergic asthma and unresponsive chronic urticaria treatments. In allergic asthma, omalizumab has demonstrated its efficacy in reducing the dose of inhaled corticosteroids required by patients, decreasing the number of asthma exacerbations, and limiting the effect on airway remodeling. In CU, omalizumab treatment rapidly improves symptoms and in some cases achieves complete disease remission. In systemic mastocytosis, omalizumab also improves symptoms and its prophylactic use to prevent anaphylactic reactions has also been discussed. In other pathologies such as atopic dermatitis, food allergy, allergic rhinitis, nasal polyposis, and keratoconjunctivitis, omalizumab significantly improves clinical manifestations. Omalizumab acts in two ways: by sequestering free IgE and by accelerating the dissociation of the IgE-Fc In genetically susceptible individuals, exposure to specific allergens results in an increase of specific IgE, which can bind onto effector cells through a high affinity receptor known as Fcasophils .εRI increases cell survival and receptor upregulation [+ Th2 cells, which stimulate B-cell production of IgE antibodies that are also antigen-specific. During sensitization, the IgE antibodies bind to FcεRI on the surface of tissue MC and blood BS. Later exposure to the same allergen cross-links the bound IgE on sensitized cells, resulting in degranulation and secretion of preformed pharmacologically active mediators such as histamine. All of this occurs as an immediate reaction, starting within seconds. A late reaction caused by the induced synthesis and release of leukotrienes, chemokines, and cytokines by the activated mast cells allows the recruitment of other leukocytes, eosinophils, basophils, and Th2 lymphocytes to the site of inflammation. The allergic reaction includes symptoms like cough, bronchospasm, wheezing, diarrhea, and urticaria due to this process [IgE is very short-lived in plasma about 1 day), but receptor-bound IgE can remain fixed to cells in tissues for weeks or months. Moreover, IgE binding to Fcgulation , 3 and u day, but process .IgE-mediated chronic diseases have classically been treated with antihistamines, corticoids, and other anti-inflammatory medications, but a number of patients do not respond to these treatments. The discovery and characterization of the pathways that drive different asthma phenotypes and our growing understanding of the pathophysiology of chronic urticaria (CU) have opened up new avenues for their treatment. To target the IgE with biological drugs has been pursued in the treatment of more severe cases of these pathologies.The use of omalizumab (OmAb), an anti-IgE drug, is approved in severe allergic asthma not controlled by conventional treatment and in CU , 6. IgE At present, the most interesting feature of omalizumab is its efficacy in conditions in which no successful treatment has previously been reported . PerhapsIn recent years, a number of other anti-IgE drugs have been developed. They will be discussed in this review, but it is by no means clear whether they will eventually be used in humans.Asthma is a chronic inflammatory disease of the airways characterized by intermittent chest symptoms, variable airway obstruction, and bronchial hyperresponsiveness. In recent years, there has been a shift in the conception of asthma, which is no longer seen as a single disease but as a chronic condition with marked clinical heterogeneity over time . Today, Classically, asthma has been divided into extrinsic and intrinsic phenotypes. Extrinsic asthma is characterized by hypersensitivity to a foreign molecule  and is always associated with allergy. Intrinsic asthma covers all cases of asthma not attributable to allergies, such as asthma caused by sinus infections, chronic sinusitis, nasal polyps, acute bronchitis, colds, stress, or exercise. The attempts to understand the complexity of asthma presentation and the emergence of biological agents have led to a renewed interest in identifying clinically significant phenotypes. Currently, the study of asthma phenotypes is evolving, with a growing focus on their genetic base and corresponding biomarkers; but rather than creating an ever-expanding list of specific phenotypes, future research is likely to center on dissecting out the clinically relevant ones. The elucidation of asthma phenotypes has been further refined by the study of endotypes, which has provided information on the pathophysiological mechanisms present in different phenotypes , 12.Asthma has largely been viewed as a Th2-mediated process strongly linked to atopy and eosinophilic inflammation. However, a significant proportion of asthma cases do not present an increase in Th2 cytokines . Non-Th2Phenotyping studies have identified an early-onset allergic Th2-asthma phenotype  and several late-onset Th2-related phenotypes (often at the age of 20 or later). The clinical phenotype of exercise-induced asthma (EIA) is also likely to have a Th2 component, given its eosinophil- and mast cell-related profile .+ B-cells to become IgE plasma cells and to secrete soluble IgE against allergens. Soluble IgE, together with Th2 cells, return to the pulmonary tissue [εRI on the MC and BS cell surface. IgE-allergen complex promotes MC and BS degranulation of preformed mediators  and secretion of de novo soluble components, including leukotrienes, prostaglandins, and other Th2 cytokines, which contribute to the prolonged inflammation and to the recruitment of more immune cells [Early-onset allergic Th2 asthma is the most studied phenotype, accounting for 50% of subjects with asthma, and it is linked with other allergic diseases such as allergic rhinitis and atopic dermatitis. The impairment it causes ranges from mild to severe . This phy tissue . These Ine cells , 26. Th2ne cells .Secretion of IL4 by Th2 cells promotes ICAM-1 and VCAM-1 expression on the surface of blood vessels , which aLate-onset Th2 asthma is characterized by marked eosinophilia, less atopy, and recurrent exacerbations. This form of asthma is believed to be unrelated to allergic triggers. A family history of this asthma is also less commonly observed and the genetics of this phenotype have not been specifically studied. The lack of clinical allergy in this phenotype suggests that the Th2 process differs from and is probably more complex than the early-onset allergic phenotype . AspirinFurthermore, numerous environmental factors such as smoking, hormonal changes, infections, and obesity are comorbidities and confounders that can alter asthma phenotypes and influence the underlying immunoinflammatory process.Urticaria is characterized by pruritic wheals that develop quickly with a central edema and a surrounding area of erythema. The size of the wheals is variable and the lesions last from one to 24 hours. The disease may be accompanied by angioedema, defined as cutaneous or mucosal swelling that is generally nonpruritic but is painful and lasts from one to three days . UrticarChronic spontaneous urticaria may occur as a result of mast cell and basophil release of bioactive mediators. However, the mechanism of mast cell degranulation in urticaria patients remains unclear. Currently, we know that immunological and nonimmunological factors are involved. The key role in the pathogenesis of CSU is played by the vasoactive mediators released from dermal mast cells. Histamine is the most prominent of these mediators although there are others such as eicosanoids, cytokines, and proteases. Histamine acts on H1 receptors (85%) and on H2 (15%) in the skin. Histamine binding to H1 receptors provokes pruritus, vasodilatation, and edema . MechaniIn terms of pathophysiology, three categories of CSU have been defined . 1) Allergic. In this case, an allergen acts by stimulating the production of IgE, which binds to the FcεRI, leading to mast cell and basophil degranulation. (2) Autoimmune. An autoimmune etiology is suggested by several findings. Autologous intradermal injection of sera from patients with CSU causes wheal and flare reactions [εRI or to IgE itself [εRI induces degranulation of the mast cells and blood basophils, which is followed by the release of histamine [1 and IgG3 are the main anti-FcεRI autoantibody subclasses found in CSU [εRI autoantibodies in normal human mast cells and basophils in vitro [ Nonimmunological. The mechanisms are independent of IgE and FcεRI. This group includes inducible urticaria and urticaria secondary to drugs. Moreover, other CSU patients without autoimmunity or increased serum IgE could also be included: in these cases, the trigger is not known, but it may involve alterations in other unknown molecular mechanisms. is a recombinant humanized monoclonal antibody that was designed to bind to IgE on the Fc (constant fragment) portion, C epsilon 3 locus, in the same domain where IgE is bound to FcεRI , 45. ThiεRI+ or FcεRII+ (CD23) cells, B-cells, dendritic cells (DC), eosinophils (Eo), and monocytes. Interestingly, in recent years, the drug's action has been shown to go further, dissociating bound IgE from the IgE-FcεRI complex (mechanism (2) in εRI complex on the surfaces of mast cells and basophils in addition to its ability to neutralize the free IgE, leading to an impairment of the IgE-inflammatory signaling cascade [εRI expression on basophils, mast cells, and dendritic cells falls notably in patients receiving anti-IgE treatment within the first week of OmAb application [εRI expression has also been demonstrated in dendritic cells [It has been accepted for a long time that OmAb acts on the free IgE (mechanism (1) in  cascade . Moreovelication . This malication –50. All ic cells .Likewise, as a complementary mechanism, it has been proposed that OmAb-IgE complexes can bind to antigens and act as competitive inhibitors (mechanism (3) described in + B-cells, reducing IL4R expression and IgE synthesis and decreasing the number of these cells, possibly by causing B-cell anergy [It has also been published that OmAb may target membrane-IgE (mIgE) in IgEl anergy   compared with baseline after 16 weeks in patients with severe nonatopic asthma, as it does in severe atopic asthma [In an attempt to elucidate the drug's mechanism of action, another placebo-controlled trial was conducted in 41 adult patients with severe, nonatopic refractory asthma. Interestingly, OmAb regulated Fcc asthma . This fiThe standard duration of treatment with OmAb has not been established to date. A follow-up study showed that, after six years of OmAb treatment, most patients had mild and stable asthma in the ensuing three years after treatment discontinuation . It has Nowadays, several options are available for treating chronic urticaria. Practical measures include the avoidance of aggravating factors such as drugs , alcohol, stress, and local heat and friction. Currently, the guidelines recommend a stepwise approach, which includes nonsedating antihistamines as a first line of treatment or combinations with oral corticosteroids. Control is often insufficient and additional therapies have included leukotriene antagonists or cyclosporine, and more recently OmAb has been added if symptoms persist .ƐRI [The use of OmAb in urticaria has focused mainly on CSU with autoimmune form . The effƐRI , 47. NevInterestingly, OmAb has also been reported to be effective in treating other forms of urticaria: cold urticaria, solar urticaria, cholinergic urticaria, delayed pressure, and symptomatic urticaria factitia, although the role of IgE in these urticarial conditions is unknown –71.εRI on circulating basophils and mast cells, it seems to lower their activity and thus reduces their potential reactivity [Mastocytosis is a heterogeneous disorder that results from abnormal proliferation and accumulation of mast cells in one or more organs. When this infiltration extends to extracutaneous organs such as bone marrow, liver, spleen, and gastrointestinal tract in spite of the cutaneous infiltration, systemic mastocytosis is diagnosed. Increased local concentration of soluble mast cell growth factors in lesions is believed to stimulate mast cell proliferation. Impaired mast cell apoptosis and interleukin-6 have also been implicated, as evidenced by BCL-2 upregulation and high IL6 levels in tissue. Most patients with the systemic form present an activating point mutation in the c-kit gene in codon 816 (D816V), which is thought to contribute to the abnormal proliferation of mast cells and enhanced mast cell survival . OmAb haactivity , 75. Curactivity . In anotactivity . The mecHyperimmunoglobulin E syndrome (HIES) is a heterogeneous group of immune disorders characterized by very high levels of serum IgE, dermatitis, and recurrent skin and lung infections. There are two forms of HIES: a dominant form caused by mutations in STAT3 and a recessive form for which a genetic cause is unclear. These syndromes have distinct presentations, courses, and outcomes but both present clear increases in IgE levels. Studies report clinical improvements in patients with high serum IgE levels and presenting severe atopic eczema and in patients presenting several other symptoms after OmAb treatment , 79.Eosinophilic gastroenteritis is characterized by patchy or diffuse eosinophilic infiltration of any part of the gastrointestinal tract. Anti-IgE treatment with OmAb is associated with a 35–45% drop in peripheral blood eosinophil count and decreases in duodenal and antral eosinophils. It effectively blocks CD23-mediated allergen binding to B-cells .There is a close relationship between asthma and allergic rhinitis. For this reason, OmAb was expected to be effective in the treatment of concomitant rhinitis in patients with asthma. Indeed, in one trial, the odds ratio for a positive effect on rhinitis was 3.56, indicating that the probability of improvement was three and a half times higher in subjects treated with OmAb . In a 20 Staphylococcus aureus enterotoxin acting as a superantigen rather than atopy. Interestingly, IgE antibodies against S. aureus enterotoxin were found in a significant higher concentration in severe asthma patients compared to controls suggesting a relationship between these antibodies and asthma severity [In nasal polyposis, the results of using OmAb are less obvious since this condition appears in nonallergic patients. Significantly, high levels of IgE in polyps have been related toseverity . In thisseverity , 85. Aspergillus characterized by high IgE levels, which usually occurs in combination with cystic fibrosis (CF) [Omalizumab has also demonstrated its clinical relevance in patients with allergic bronchopulmonary aspergillosis (ABPA), an allergic reaction tosis (CF) , 87. Sevsis (CF) , 89.Atopic dermatitis (AD) is one of the most frequent chronic inflammatory skin disorders associated with elevated serum IgE levels. Acute AD skin lesions are characterized by intensely pruritic, erythematous papules associated with epidermal intercellular edema as well as increased Langerhans cells, inflammatory dendritic epidermal cells, macrophages, eosinophils, and activated CD4-positive Th2 cells. The results with the use of OmAb for atopic dermatitis are controversial: several case reports investigating anti-IgE therapy in patients with AD have found symptomatic improvement , 91, butBullous pemphigoid (BP) is an acquired autoimmune disease presenting subepidermal blistering, eosinophilia, and severe itching. It is characterized by the presence of autoantibodies against the 230 kDa bullous pemphigoid antigen within basal keratinocytes and the 180 kDa type XVII collagen in the basement membrane zone lying between the epidermis and dermis. IgE-specific antibodies against type XVII collagen were detected in sera and biopsy samples from the majority of BP patients and these IgE autoantibodies have been shown to be pathogenic. Clinical trials with OmAb showed effectiveness in several cases , 94.OmAb induced a significant increase in the threshold dose for an oral food challenge with peanuts causing allergic symptoms . OmAb haAtopic keratoconjunctivitis is a severe ocular disorder of the cornea with immediate and delayed hypersensitivity reactions, which can produce loss of visual acuity and blindness. In an open-label trial, six patients treated with OmAb showed an improvement in their ocular symptoms .+ B-cells, the membrane-bound ε-chain exists predominantly in the long isoform, thus providing an attractive site for immunologic targeting of mIgE+ cells. CεmX-specific antibodies have proved potentially useful for targeting mIgE+ cells to control IgE production [Membrane-bound IgE (mIgE) is part of the IgE-BCR and is essential for generating isotype-specific IgE responses. On mIgEoduction . These woduction . Unfortuoduction . Nor didoduction .εRI and IgE. These effects allowed almost complete suppression of the skin prick response to the allergen, which was superior in extent and duration compared to the case with OmAb [Ligelizumab (QGE031) is a humanized IgG1 monoclonal antibody that binds with higher affinity to the C epsilon 3 domain of IgE. Designed to achieve greater IgE suppression than OmAb, it may overcome some of the limitations associated with OmAb dosing and achieve better clinical outcomes. Data from preclinical experiments and two phase I randomized, double-blind, placebo-controlled clinical trials showed QGE031 to be superior to OmAb in suppressing free IgE and basophil surface expression of Fcith OmAb .εRI and the low-affinity IgG receptor (FcγRIIb) on mast cells and basophils inhibits allergen-induced cell degranulation [Some studies have shown that the use of bispecific antibodies that cross-link Fcnulation , 105.γRIIb [εRI interactions and actively stimulates the dissociation of preformed ligand-receptor complexes [DARPins are genetically engineered proteins that typically exhibit highly specific and high affinity target protein binding. One study reported a specific anti-IgE DARPin (DE53-Fc) fused to the Fc part of a human IgG1, which inhibited allergen-induced basophil activation in samples of different donors via FcγRIIb . For itsomplexes .Bispecific antibodies and DARPins represent promising drug candidates for the treatment of IgE-related diseases but their potential for use in humans is still to be confirmed.εRI and FcεRII. The IgE-R419N-Fc3-4 mutant, in combination with OmAb, can effectively exchange cell-bound IgE for IgE-R419N-Fc3-4 and this dual inhibitor treatment blocks basophil activation more powerfully than either inhibitor alone. This approach, involving simultaneous depletion of antigen-specific IgE while engaging FcεRI and FcεRII receptors with an IgE variant, can be used to further test the role of IgE-dependent regulatory pathways during anti-IgE treatment and may provide a promising way forward for enhancing current anti-IgE therapies [Structural studies of OmAb have facilitated the design of an IgE-Fc3-4 mutant (IgE-R419N-Fc3-4) that is resistant to OmAb neutralization but is able to bind Fcherapies .A better understanding of asthma and chronic urticaria phenotypes and endotypes will allow us to select treatments based on the likelihood of response, thereby improving the control and quality of life of these patients. The number of biological treatments for these diseases continues to grow.Targeting IgE has proved to be a successful approach to IgE-related diseases with poor response to traditional treatments. Although the indications for OmAb are currently limited to allergic asthma and CSU, its potential in the treatment of other allergic comorbidities is becoming increasingly clear. In fact, the off-label uses of OmAb have shown promising results in a variety of diseases in which IgE has a limited or unconfirmed role. The knowledge of OmAb's mechanism of action may help to elucidate the relationships between all the factors interacting in IgE-mediated pathologies and may shed some light on some others that are not IgE-mediated.Thus, more studies are needed to uncover the molecular insights of these pathologies and the mechanism of action of anti-IgE biological drugs such as OmAb and to test the efficacy of new IgE-targeted drugs in humans.


Elucidating the source(s) of Meltwater Pulse 1a, the largest rapid sea level rise caused by ice melt , is important for understanding mechanisms of rapid ice melt and the links with abrupt climate change. Here we quantify how much and by what mechanisms the North American ice sheet could have contributed to Meltwater Pulse 1a, by driving an ice sheet model with two transient climate simulations of the last 21,000 years. Ice sheet perturbed physics ensembles were run to account for model uncertainties, constraining ice extent and volume with reconstructions of 21,000 years ago to present. We determine that the North American ice sheet produced 3–4 m global mean sea level rise in 340 years due to the abrupt Bølling warming, but this response is amplified to 5–6 m when it triggers the ice sheet saddle collapse. The cause of the largest rapid sea level rise ever recorded, Meltwater Pulse 1a (14.6 ka), is unresolvedWe quantify how much and by what mechanisms the North American ice sheet could have contributed to Meltwater Pulse 1aThe Bolling warming likely triggered the saddle collapse mechanism contributing 5‐6 m or more to MWP1a from the North American ice sheet In total, this produced around 130 m of global mean sea level rise (GMSLR) [Gomez et al., Gomez et al., Liu et al., The source of MWP1a has previously been assessed through fingerprinting the pattern of sea level rise/fall caused by the change in gravitational pull exerted by an ice mass on oceans. Although initial studies rejected a major North American ice sheet (NAIS) source, recent work overturned this finding and suggested it could actually have contributed up to 10 m to the total sea level change in 340 years [Weber et al., Golledge et al., Deschamps et al., Dyke, Gowan et al., Peltier et al., Tarasov et al., Gregoire et al. [For the Antarctic ice sheet, direct constraints on changes in thickness or extent around the time of MWP1a are limited and debated. Southern Ocean records of iceberg‐rafted debris [Dyke, Abdul et al., Dyke, From geological constraints, exactly when the separation of the two ice sheets took place is uncertain Dyke, . It is cCarlson et al. [Given their close timing, it is compelling to think that at least part of MWP1a could also have been linked to the abrupt Bølling warming in the Northern Hemisphere, through accelerated ice melt in North America and Europe. n et al.  suggesteHere we provide a mechanistically based statistical assessment of the possible range of North American ice sheet contribution to MWP1a from both the saddle collapse mechanism and the Bølling warming.2Rutt et al., We ran ensembles of model experiments simulating North American ice sheet evolution over the last deglaciation (21–7 ka), using the Glimmer‐CISM 3‐D thermodynamic ice sheet model version 1.14 [2.1Liu et al., The trace‐21 ka experiment run with CCSM3 (henceforth “T‐21k”) simulates a rapid warming similar in magnitude and spatial pattern to the Bølling warming event [Gregoire et al., The FAMOUS transient experiment as used in G12 (henceforth “F‐21k”) reproduces well the overall slow climatic change during the deglaciation but does not simulate millennial scale climate change, including the Bølling warming event [We forced the ice sheet model with transient simulations of the last 21 thousand years run with two different GCMs, FAst Met Office/UK Universities Simulator (FAMOUS) and Community Climate System Model 3 (CCSM3), allowing us to account for some degree of climate uncertainty. The climate experiments were forced with similar boundary conditions for changes in greenhouse gases, orbit, and ice sheets (see respective references below). However, they used different scenarios for how much, where, and when the melting from ice sheets freshened the ocean. As a result, the climate evolves differently in the two simulations:In both climate experiments, sudden and artificial surface temperature changes occur when and where the ice sheet extent is updated in the models, due to surface albedo shifts. This ice mask  is updated every 1000 years in the FAMOUS experiment and at irregular intervals in the Trace‐21k experiment. As in G12, we process the FAMOUS climatic fields to remove these stepped climate artifacts before they are input to Glimmer. This is done by averaging each monthly field over 1000 year intervals and interpolating linearly between these averages. We did not process the Trace‐21k data in the same way since we are specifically interested in the effect of the real abrupt climate changes in these experiments. Instead, we take the conservative approach of disregarding any abrupt change in ice sheet mass balance that occurs soon after a sudden change in the Trace‐21k ice mask.Fabs forcing). For the T‐21k transient climate, we adopt two forcing methodologies (see section Cabs forcing) and (ii) an anomaly forcing, where we correct for present‐day model biases in the GCM's climatology (Cano forcing).For the F‐21k forcing, we drive the Glimmer‐CISM ice sheet model directly using model output climate fields (absolute forcing), as in G12 .We select ensemble members based on their LGM ice volumes and their evolving extent throughout the deglaciation Dyke,  . The selected simulations have reasonable ice extents at the LGM  tends to smooth out the details and local features in the reconstructed ice extent. We consider that such errors in our simulations are acceptable and set the maximum acceptable extent error through an iterative process by evaluating ice extent maps of the selected simulations. We discarded simulations with a cumulative extent error of more than 23% corresponding to 225,500 cells over 13 reconstructed maps , we ran a perturbed physics parameter ensemble of 201 experiments, 603 simulations in total. After spin‐up, at 21 ka, we obtain ice volumes ranging from 0 to 70 × 103 Figure . MoreoveDyke [To select the most realistic simulations, we apply constraints on ice extent evolution from the reconstruction of Dyke  and on tDyke [Dyke, Peltier et al., Tarasov et al., Ice extent and thickness is similar between the Fabs and Cabs ensembles, as reflected in the NROY ensemble mean ice extent and thickness Figures  and S2. Dyke  than theke Dyke, , whereas3.22.The saddle collapse event occurs spontaneously during the North American deglaciation, when the Laurentide and Cordilleran ice sheets separate. To identify these events systematically, we detect the timing and amplitude of the largest peak in melt rate over the duration of each individual simulation. We then verified that this peak occurred during the separation of the Cordilleran and Laurentide ice sheet in the experiments by plotting the location of the thickness changes during the event. Finally, we calculated the GMSLR from ice volume change in the 340 years period centered around the time of the maximum melt rate , as its forcing was smoothed to remove such artifacts (see G12 methods).The timing of the saddle collapse meltwater pulse varies within the ensembles of Fabs NROY experiments but always occurs between 12.3 ka and 10.9 ka, with a maximum meltwater flux of 0.25 to 0.43 Sv producing 5.7 to 11.0 m GMSLR in 340 years. The exact timing and amplitude of the pulse is a result of many factors, and we have not found a clear relationship with ice sheet geometry or any specific parameter values.3.3The abrupt Bølling warming in the T‐21k forcing starts at 14.7 ka with maximum North American temperature at 14.3 ka. Following this, there is an acceleration in melt rate in the NROY Cano and Cabs ensembles . These are the simulations that produce the cluster of high‐end GMSLR (5.1 to 6.1 m) in the Cano ensemble  than other NROY experiments.Wickert, Wickert, Teller et al., In the simulations with an early (pre‐Bølling warming) separation of the Cordilleran and Laurentide ice sheets, the melt associated with the abrupt warming is located predominantly in the ice free corridor. Otherwise, the melt is mainly on the northern part of the saddle linking the two ice sheets. In both cases, this melt would be routed through the Mackenzie River toward the Arctic Ocean Wickert, . Melt alThe F‐21k (Fabs) forcing does not include a Bølling warming signal and so cannot be used to assess the climate event's influence on North American ice sheet evolution.4Dyke, Gregoire et al., Gregoire et al. [Our results suggest that the North American ice sheet could have contributed to MWP1a through two different mechanisms: the ice saddle collapse caused by the separation of the Cordilleran and Laurentide ice sheets (described in G12), which we associate with the timing of MWP1a, and accelerated melt from the abrupt Bølling warming in the Northern Hemisphere at 14.6 ka. The separation of the two ice sheets on its own can produce a meltwater pulse of 5.7–11.0 m GMSLR in 340 years associated with the saddle collapse. However, in these experiments, the separation of the two ice sheets occurs after 12 ka, whereas geological reconstructions indicate the separation occurred between 16 and 14 ka [e et al. . The sepWhen the Cordilleran and Laurentide ice sheets are already separated at the time of the Bølling, the abrupt warming produces 3–4 m GMSLR in 340 years. This response is amplified by 2/3 (5–6 m) when the Bølling warming triggers the separation . These simulations produce the best match to geological data. Thus, these results strongly suggest that the Bølling warming triggered the saddle collapse mechanism contributing 5–6 m or more to MWP1a from the North American ice sheet. Additional processes not included in this study, such as marine ice sheet instability, interaction with proglacial lakes, and “higher‐order” ice stream dynamics, could have enhanced ice loss leading to a potentially larger North American ice sheet contribution to MWP1a.Constraining the timing of the separation of the Cordilleran and Laurentide ice sheets with respect to the Bølling warming is crucial to narrowing down the potential North American contribution to MWP1a and to confirm the mechanisms that caused the most rapid sea level rise in our geological past.Supporting Information S1Click here for additional data file.


The integration of excitatory and inhibitory synaptic inputs is fundamental to neuronal processing. In the mammalian auditory brainstem, neurons compare excitatory and inhibitory inputs from the ipsilateral and contralateral ear, respectively, for sound localization. However, the temporal precision and functional roles of inhibition in this integration process are unclear. Here, we demonstrate by in vivo recordings from the lateral superior olive (LSO) that inhibition controls spiking with microsecond precision throughout high frequency click trains. Depending on the relative timing of excitation and inhibition, neuronal spike probability is either suppressed or—unexpectedly—facilitated. In vitro conductance-clamp LSO recordings establish that a reduction in the voltage threshold for spike initiation due to a prior hyperpolarization results in post-inhibitory facilitation of otherwise sub-threshold synaptic events. Thus, microsecond-precise differences in the arrival of inhibition relative to excitation can facilitate spiking in the LSO, thereby promoting spatial sensitivity during the processing of faint sounds. Binaural cue processing in auditory brainstem relies on the precise temporal relationship between excitatory and inhibitory inputs. Here the authors provide direct evidence that sub-millisecond precise inhibition can tune sensitivity to input processing in the lateral superior olive via post inhibitory facilitation. While excitatory inputs cause neurons to generate action potentials , inhibitory inputs typically lower neuronal excitability8. Thus, the particular temporal relationship of the inputs defines a time window for signal propagation and modulation12. This processing motif is particularly prominent in auditory brainstem circuits dedicated to the processing of binaural cues for the localization of sound sources  and are first computed by neurons in the lateral and the medial superior olive 17. In both nuclei, the computation is based on precise interactions of glutamatergic excitation and glycinergic inhibition in response to sounds arriving at the two ears19  can tune ITD-sensitivity by modulating the timing of the net excitatory post-synaptic potentials (EPSPs). The relevance of these mechanisms in the intact brain is, however, discussed controversially28, as mechanistic evidence in vivo is lacking. This is partially attributable to the fact that both inhibition and excitation are activated by either ear31, which hinders the interpretation of in vivo data.Despite these evident specializations for precision, it remains controversial to what extent the precise timing of MNTB-mediated inhibition is a necessary part of the binaural computations31. Thus, investigating binaural interactions in the LSO allows a direct assessment of the roles of timed inhibition in synaptic integration and spike generation during spatial processing19. Indeed, beyond the well-known gauging of input strength, the comparison of the timing of the excitatory and inhibitory inputs in the LSO is an integral part of ILD computation: any changes in the ILD  entail a change in the relative arrival times of the respective inputs at the LSO32 42, and similar properties may be present in LSO neurons43.Thus, ILD processing by LSO neurons consists of the gauging of both input strength (amplitude) as well as timing. The importance of timing is further emphasized by the fact that LSO neurons are also sensitive to ITDsTo gain better insight into the functional relevance of precisely timed glycinergic inhibition for binaural spatial processing, we performed single-cell recordings in vivo in the LSO. By disentangling amplitude effects from effects that are specific to input latency, we demonstrate that inhibition sustains microsecond temporal precision throughout high frequency click trains, resulting in input timing-specific modulation of spike timing. Importantly, our data reveal prominent PIF of spiking for a highly specific relative timing between inhibition and excitation. The data suggest that PIF facilitates ILD coding precision of weak excitatory inputs. In vitro whole-cell recordings in mature LSO neurons confirm that these inhibitory functions occur during the binaural integration process and establish that a reduction in the firing threshold due to prior hyperpolarization contributes to PIF. Consequently, we provide direct evidence for the significance of microsecond precise glycinergic inhibition to tune sensitivity to input timing in the auditory brainstem.33. We used trains consisting of six identical clicks, with individual clicks being separated by an inter-click-interval (ICI) varying from 1–5 ms in 1 ms steps  < 0.0001, F = 22.48; P(cTD) < 0.0001, F = 16.16; Interactions: P < 0.0001, F = 9.59]. As expected for the stimulation of the inhibitory ear, min-rates for all ICIs were consistently lower compared to excitatory only stimulation . During monaural stimulation (excitation only), a progressive decline in spike rate probability to clicks later in the train with decreasing ICI became apparent  < 0.0001, F = 124.1; P(ICI) = 0.017, F = 3.2; interactions: P < 0.0001, F = 2.76). Interestingly, this decline was partially counteracted by the presence of inhibitory inputs during binaural stimulation at max-cTDs  = 0.032, F = 2.47; P(ICI) = 0.49, F = 0.87; Interactions: P = 0.28, F = 1.16). Thus, dependent on the relative timing, contralateral inputs in the LSO decreased or facilitated spiking to excitatory inputs in the LSO.The unexpected effect of contralateral inputs enhancing spiking in a timing-specific manner may not be equally pronounced for all spikes elicited during a click train. Specifically, spikes elicited by later clicks may be affected differently than early spikes because of the relative time course of inhibition and potential summation effects. To test this, we analyzed response dynamics for each click individually by binning spikes according to their occurrence . Based on the resulting ∆t-spike rate functions , we analyzed the change in spike rate during binaural stimulation compared to excitation only. We focused our analysis on the “best ICI” of each neuron, i.e., the ICI that elicited maximal response rate modulation as a function of cTD .What mechanism could account for increased spiking by addition of inhibition? Our results so far are reminiscent of facilitatory effects of timed inhibition that have been observed in slice recordings from juvenile MSO 44 Fig. , i.e., tt . In particular, compared to the timing during excitation only, spikes occurred significantly earlier when inhibition led excitation by 600 µs  to 800 µs , coinciding with the delay of highest spike facilitation  to mimic the six click trains performed in vivo . We next paired the excitatory conductance with a corresponding inhibitory conductance train with a fixed amplitude  at relative time differences (rTDs) spanning an entire event cycle (0.25 ms steps from 0 to –4.75 ms). Inhibition at certain rTDs faithfully reduced spiking , and examined temporal interactions between excitation and inhibition at LSO somata using conductance-clamp. To this end, we measured excitatory and inhibitory synaptic conductance waveforms in voltage-clamp to obtain conductance templates that reflect natural synaptic kinetics in adult gerbil LSO neurons . Furthermore, because in vivo spike rate enhancement was only statistically significant on the last four clicks  for each event in the train illustrates the Ge sensitivity  revealed the exquisite sensitivity of the timing window of PIF on Ge , whereas at +3 nS this value increased to 2.21 ± 0.25 ms , PIF functions for each of the last four events were extremely stable  enforced by inhibition , albeit with considerable variability in the magnitude of boosts . These findings suggest that despite the hyperpolarization evoked by inhibition, a relief from Nav channel inactivation lowers the effective AP voltage threshold and could explain the ability of inhibition to promote repetitive spiking throughout our six-event trains.What biophysical mechanism could be underlying PIF? One possibility is that the hyperpolarization itself effectively relieves Naion Fig. . To testion Fig. . This reion Fig. , bottom,ion Fig. , top. AcIh) promotes rebound spiking activity in the neighboring superior paraolivary nucleus46. Although the PIF we report here is not a rebound spike per se (because it requires excitation), we evaluated the involvement of Ih in PIF  revealed a robust current with a fast-activating time constant of just 43 ms. Because the Ih block also hyperpolarized the membrane potential and increased membrane resistance . Although ZD did influence AP shape, PIF persisted in the absence of Ih  and PIF function half-widths  tended to be larger in the presence of ZD  channel, whose current  over a range of stimulus intensities by boosting responsiveness to weaker signals. Without PIF, one would assume that for contralateral stimulus locations, the large increased inhibitory inputs would effectively prevent any spiking arising from the weaker excitatory inputs, resulting in a steepening of the slopes of the ILD-spike rate functions and effectively diminishing the dynamic range of the neuron. PIF may counteract this inhibitory dominance exactly at ILDs at which weak excitation is paired with stronger—and thus faster and putatively preceding—inhibition Fig. . We test48, we provide direct evidence that in the intact brain, this high temporal acuity of inhibition is not limited to the first spike. Rather, it is precisely maintained and effective throughout click-trains. Whole-cell in vitro recordings reproduced several features of PIF and provided mechanistic insight into the underlying biophysical properties.We determined a fundamental divergence from the typically assumed subtraction mechanism in the interaction between glutamatergic excitation and glycinergic inhibition in binaural auditory brainstem neurons. Synaptic inhibition decreased or increased the spike rate of LSO neurons depending on the particular sub-millisecond time difference relative to excitation. The increase was mediated by PIF to otherwise sub-threshold excitatory inputs, thereby maintaining high ILD sensitivity over a wide range of sound intensities. Contrary to current models of inhibitory function for processing microsecond differences in input timing38. Our observation that this temporal specificity of inhibitory effectiveness was maintained throughout the click train even at 500 Hz demonstrates its ability influence excitation on a cycle-by-cycle basis for relatively high click frequencies. These data thus challenge previous hypotheses on the role of MNTB-mediated inhibition for spatial processing, which suggested that inhibition was insufficiently fast to interact in a timing-dependent manner and rather functioned as a constant suppressor of excitation48. Our patch clamp data suggests that the response facilitation via inhibition is generated by hyperpolarizing the membrane potential prior to arrival of the excitatory inputs, which increased the likelihood of this input to trigger a spike. PIF is supported by a prior report of OFF-responsiveness in the LSO49, and we have established that it is at least partially mediated by a reduction in the voltage threshold for spike initiation52.Previous studies showed that the effectiveness of inhibition to suppress spiking in response to short stimuli in LSO cells is restricted to specific phases of the IPSPBy varying stimulus settings, we found the temporal width of PIF in vitro to be highly sensitive to the event number (single event vs. train) and the applied excitatory conductance. This highlights a delicate balance in the interplay of inhibitory and excitatory inputs and the recruitment of associated cellular mechanisms that is required for PIF. Nonetheless, when matching both the stimulus paradigm (six-event train) and analysis, we observed a close match of the facilitation time window between the in vivo and in vitro data.Ih currents, which are involved in the classical post-inhibitory rebound spiking after long-lasting hyper-polarization46, were too slow to underlie the facilitation itself, but may modulate its timing window. Contrary to post-inhibitory rebound spiking, PIF has, to our knowledge, so far only been observed in vitro in the MSO of juvenile mice. In these experiments, the deactivation of low-threshold potassium channels55 resulted in response facilitation by leading inhibition42. Previous MSO data also explained how the interplay between synaptic inhibition and low-threshold potassium channels sharpens the temporal sensitivity of the binaural integration mechanism, particularly during ongoing activity56.The entire mechanism of the facilitation we report here is likely to be complex, as we have shown that additional ion channels and conductances might modulate PIF. In particular, 59, and consequently PIF represents a potential underlying mechanism.The effects of inhibition on synaptic integration we report here have important implications for its role in binaural processing in the auditory brainstem. We found evidence that the inhibitory input can increase the separability of ILDs for weak stimuli. Human ILD separability has been shown to be robust against absolute sound level18, intensity (and thus latency)-dependent effects will dominate the input timing at the LSO relative to the ITD-dependent changes  the increase in sound intensity shortens the neuronal latency, and (b) the relative traveling time of the sound  is shorter to the inhibitory ear. For high frequency sounds, where ILDs are largeges Fig. . However26. In particular, we showed that synaptic inhibition tunes ITD sensitivity in MSO cells by shifting the effective timing of the excitatory inputs24. Thus, our in vivo findings further strengthen the functionality of these mechanisms in the intact sound localization circuit.Next to response facilitation, we also provide evidence that both the absolute spike timing of LSO neurons and its jitter are significantly modulated by the relative arrival time of inhibition and excitation in vivo and in vitro, paralleling previous MSO in vitro resultsTogether, we have shown that fast synaptic inhibition originating from the MNTB provides important functions during binaural processing other than a reduction of spiking. Its evidently exquisite temporal precision persists after the onset of stimulation and profoundly affects crucial aspects of neuronal spatial sensitivity.All experiments were approved in accordance with the stipulations of the German animal welfare law (Tierschutzgesetz) (55.2-1-54-2532-53-2015). Animals were housed in groups of 4 to 6 individuals with 12 h light/dark cycles.Meriones unguiculatus) aged 31–38 days . Gerbils were anesthetized with isoflurane   and decapitated. Brains were removed and placed in ice-cold dissecting solution containing (in mM): 93 N-methyl-d-glucamine, 93 HCl, 30 NaHCO3, 25 glucose, 20 HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 10 MgCl2, 5 l-ascorbic acid, 3 myo-inositol, 3 Na-pyruvate, 2.5 KCl, 1.2 NaH2PO4, 0.5 CaCl2 (pH 7.4 when oxygenated with 95% O2 and 5% CO2). Two hundred micrometer-thick transverse brainstem slices were cut using a VT1200S vibratome (Leica). They were incubated at 35 °C for 30 min in dissecting solution and for another 30 min in an oxygenated perfusion saline containing (in mM): 125 NaCl, 25 NaHCO3, 25 glucose, 3 myo-inositol, 2.5 KCl, 2 Na-pyruvate, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, and 0.4 l-ascorbic acid (pH 7.4). Recordings were performed in perfused oxygenated saline (1 ml min−1). Temperature was measured near the slice and maintained at 35 ± 1 °C by an SF-28 in-line heater (Warner Instruments) and a PH-1 bath chamber heater . Tissue was visualized under an Axioskop upright microscope (Zeiss) equipped with infrared Dodt gradient contrast optics. Whole-cell recordings from principal neurons visually confirmed to be within the LSO were obtained with borosilicate glass electrodes using an EPC10/2 patch-clamp amplifier (HEKA Elektronik).LSO slice preparation and electrophysiology: In vitro experiments were conducted on male and female Mongolian gerbils  triethylammonium chloride], 4 MgCl2, 4 Na2-ATP, 3 Na-l-ascorbate, 0.2 Cs-EGTA (cesium ethylene glycol tetraacetic acid), 0.4 Na2-GTP, and 0.03 Alexa Fluor 633, adjusted to pH 7.25 and 297 mOsm. Voltage-clamp recordings at –70 mV were made with electrodes of 2–3 MΩ tip resistances, and series resistance (4–7 MΩ) was compensated to a residual of 1.5–2 MΩ on the amplifier. Experiments were terminated if the uncompensated series resistance changed by >10%. Synaptic inputs were stimulated with borosilicate glass electrodes (of 3–4 MΩ resistance) filled with saline and placed in the vicinity (50–150 µm) of the recorded neuron. Lateral (excitatory) and medial (inhibitory) inputs were activated every 2 s with brief (0.2 ms) 10–50 V bipolar pulses generated by a Model 2100 isolated pulse generator (A-M Systems).Synaptic stimulation: For synaptic stimulation recordings  was 100% balanced on the bridge of the amplifier. The liquid junction potential was estimated to be 15 mV and subtracted offline. All reported membrane voltages reflect this subtraction.Conductance-clamp: For conductance-clamp experiments, the internal solution contained the following (in mM): 145 K-gluconate, 15 HEPES, 5 Nat)] by equation (Erev) = 5 mV] and inhibitory (Erev = –85 mV) synaptic conductance waveform templates [G(t)], while simultaneously measuring the membrane potential [V(t)].Selected synaptic conductance waveforms as measured in Supplementary Fig.  equation .1\documeGe) templates were applied to determine the spike (AP) conductance threshold. Then the same conductance ramps were performed in the presence of an inhibitory conductance (Gi) template of 20, 50 or 80 nS, varying the relative timing difference (rTD) of inhibitory to excitatory event onset. Templates were delivered as single events in 0.5 ms rTD steps between –1 to 10 ms  or as six events summated at 5 ms inter-stimulus intervals (ISIs) in 0.25 ms rTD steps between 0 and 4.75 ms . These protocols were repeated six times for each condition tested.Ramps (1 nS increments) of excitatory conductance  was applied . ZD-sensitive currents were measured using the K-based internal solution . In a subset of recordings (n = 4 cells), conductance-clamp PIF protocols  in voltage-clamp. Using IPSP waveform templates approximated from conductance-clamp experiments in the same recording, a command template of a portion of the IPSP waveform that abruptly depolarized to a suprathreshold voltage (–45 to –15 mV) after 0 (no inhibition) to 10 ms of IPSP waveform (IPSP time) in 0.25 ms increments. This causes neurons to attempt an AP, which under our recording conditions was reflected by a sharp inward current. This protocol was repeated six times for each recording  or Matlab (The Mathworks) software. Example synaptic stimulation traces represent the average of 50–100 trials. Example conductance-clamp traces are raw traces. Example AP current traces are an average of six repetitions. Averaged data points indicate mean ± s.e.m, and linear correlation values were obtained from Pearson’s correlation calculations).Ge ramp step that failed to return an AP on any trial for excitation alone. Because recordings exhibited diverse rTDs of maximal facilitation, the average AP probability function for each recording was fitted with a Gaussian, from which the peak was determined to be the Best rTD. For all subsequent analyses, rTD values were subtracted from this value. To quantify the magnitude of inhibition-induced facilitation, an AP conductance threshold was determined as the interpolated Ge value that returned 50% AP probability. Values for each rTD were then subtracted by the corresponding value for excitation alone. To compare AP properties, analysis was performed at the Ge ramp step, which produced an AP on at least one trial for excitation alone. AP timing and voltage threshold were determined at the peak time of the voltage double-derivative. For each rTD these values were subtracted from those obtained for excitation alone. AP jitter was determined as the s.d. of spike timing for all six at each rTD and was normalized to values obtained in excitation alone conditions. Data were only accepted for timing conditions in which minimally three spikes were elicited.For single event experiments, analysis of AP probability was performed at the largest e ramp steps relative to the condition that caused 100% AP probability (“Ge rel. to ceil.”) for at least two rTDs on the last four events. This allowed an assessment of the influence of excitatory drive on PIF function half-width and restricted data analysis to the same clicks that exhibited PIF in vivo.For six-event PIF experiments, analysis of AP probability was performed at GFor AP current experiments, currents were calculated from averaged current traces  as peak-to-peak values. The IPSP time that returned the largest current was then normalized to IPSP time = 0 (no inhibition). Analysis was performed only on recordings where inhibitory boost was also measured.–1 body weight). Anesthesia was maintained with a continuous subcutaneous application of the same solution (2.4 µl per 100 g body weight per minute) using a syringe pump (Univentor Ltd). Anesthesia was routinely monitored by checking the hind leg withdrawal reflex.Surgical procedures: In vivo experiments were conducted on Mongolian gerbils (Meriones unguiculatus) 3–7 months of age and of either sex . Gerbils were anesthetized by an intraperitoneal injection of ketamine  and xylazine  diluted in 0.9% NaCl solution . The head of the animal was stereotactically aligned relatively to lambda. The tragus on both ears was incised to ease access to the ear canal and custom-made electrostatic headphones were placed into the ear canals.The animal was placed on a thermostatically controlled heating pad (Fine Science Tools GmbH) to maintain the body temperature at 38 °C that was monitored using a rectal probe. The scalp was cut to reveal the dorsal part of the skull. Anterior to bregma, a metal rod was glued onto the skull using UV-sensitive dental-restorative material . The neck muscles at the recording side were partially removed to reveal the skull posterior to lambda. The animal was then transferred to a sound-attenuated chamber onto another thermostatically controlled heating pad on a custom-made stereotactic setupFrequency responses between 15 to 90 kHz were calibrated for each animal and each speaker. Acoustic stimuli were generated digitally, converted to an analog signal  at 200 kHz sampling rate, attenuated  and conveyed to the headphones. White noise bursts  were presented monaurally to the ipsilateral ear to find responsive neurons. LSO neurons were identified by an EI response . ILD-response functions (data not shown) were recorded using a cassette of binaural correlated noise stimuli with varying ILDs for each side respectively . The CF of the neuron was determined audiovisually using tonal stimuli 20 dB above threshold. To quantify the temporal resolution of binaural processing, a train of six clicks with a single-click-duration of 50 μs was presented binaurally. These click-trains were presented at five different ICIs  and five different cTDs . All stimulus combinations were presented in a pseudo-randomized order. The relative intensity on each ear  was individually adjusted for each neuron according to two measures of responsiveness: (I) robust responsiveness during monaural ipsilateral stimulation, and (II) significant modulation of response rate by cTD during binaural stimulation (see below). The resulting mean ILD ± s.e.m. for the neurons included in the dataset was 0.7 ± 6.1 dB. To test the influence of changes in the ipsilateral intensity on ILD coding, response rates of 7 LSO neurons  to a white noise stimulus (50 ms duration) of various contralateral intensity were compared for three different ipsilateral intensities. Contralateral intensities were selected for each cell so that the resulting three ILD functions were positioned along the slope of the ILD-response function.Extracellular single-cell recordings: APs of single LSO neurons were extracellularly recorded using glass electrodes filled with 5 units/µl horseradish peroxidase (Sigma-Aldrich Corp.) diluted in a 10% NaCl solution (resulting in tip resistances of ~8–12 MΩ). Using a motorized micromanipulator  for remote control, the recording electrode was lowered into the brain tissue at an angle of 20°. Neuronal responses were measured by a pre-amplifier , amplified , filtered  and delivered to the computer via a real-time processor . Here, neuronal responses were analyzed online with BrainWare  allowing audiovisual control and refinements of the recordings. Single-neuron responses were isolated by visual inspection and online and offline spike sorting. A signal-to-noise ratio of the spike waveform of > 5 was required for recorded neurons to be included for further analysis.−1) intraperitoneally (2 µl g−1). The thorax was opened, and a cannula was inserted into the left ventricle while the right atrium was cut to cause blood efflux. The animal was perfused with Ringer-solution (containing NaCl (0.9%), heparin (100 µl ml−1) and 5 mM PBS in H2O) for 10 min followed by a perfusion with 4% paraformaldehyde (PFA in PBS pH 7.4) for another 10–25 min. For fixation, the brain was removed from the skull and incubated in 4% PFA for 1–2 days at 4 °C. After fixation, the brain was washed three times for 10 min in PBS (0.02 M) and was then embedded in 4% agarose to maintain stability during brain slicing. Coronal brainstem slices of 50–80 µm thickness were prepared. Labeling of the recording site was accomplished using a 3, 3′-diaminbenzidine (DAB) substrate kit for peroxidase . For counterstaining, an additional neutral red staining was accomplished. The brain slices were transferred onto glass objective slides and covered using DePeX (Serva Electrophoresis GmbH). Overview images of respective recording sites were acquired using an Olympus virtual slide flurorescence microscope . In total, 13 of 17 recording sites could be confirmed histologically. In the remaining cases, no distinct DAB counterstaining was found but recording sites could be reconstructed from the track of the recording electrodes.Histology: To mark recording sites, horseradish peroxidase was deposited iontophoretically by applying a current of 1 µA for 8 min. After conclusion of an experiment, the animal was injected with a lethal dose of Narcoren  in the LSO with CFs between 15–36 kHz ),where mu_n + 1 and mu_n are the mean values of the responses to two ILD values while sigma_n + 1 and sigma_n are their standard deviation. Depending on normality of the distribution, population average data are shown by the mean ± s.e.m., or the median and the 25 and 75% confidence intervals (interquartile range). Accordingly, parametric or nonparametric tests were used to determine statistical significances (see text and figure legends).The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Simian virus 40 large T-antigen (SV40 LT-Ag) is a 708 amino acid nuclear phosphoprotein. Among many functions of LT-Ag is itsability to perform as an ATPase-helicase, catalyzing the unwinding of viral genome during replication. The LT-Ag has been employedin the studies of helicase structure and function, and has served as a model helicase for the screening of antiviral drugs that target viralhelicase. In this study, using in vitro enzyme assays and in silico computer modeling, we screened a batch of 18 fluoroquinolones toassess their potential as antivirals by virtue of their inhibition of the LT-Ag helicase. We found all fluoroquinolones to be inhibitory tothe helicase activity of LT-Ag. In our docking analysis, most of these tested drugs showed similarity in their interactions with LT-Ag.Our study shows the potential of fluoroquinolones as antiviral drugs and of SV40 LT-Ag as a model protein for screening drugsagainst viral helicases. Simian virus 40 (SV40) is a small, non-enveloped DNA virus .The virAmong many functions of LT-Ag is also its ability to perform asan ATPase-helicase, catalyzing the unwinding of viral genomeduring replication, harvesting the energy required for thisprocess by means of ATP hydrolysis . The SV4Purified SV40 LT-Ag was purchased from CHIMERx, USA. Asdescribed previously , the helPercentage of unwound substrate = [(y-x)/y]*100Where x and y represent the fluorescence of reaction,respectively, with and without any added helicase.Inhibition assays for helicase activity were performed as reportedpreviously , 10. EigTo analyze the interactions between LT-Ag and the selectedfluoroquinolones, crystal structure of SV40 LT-Ag (PDB ID:1SVO) was obtained from Protein Data Bank(http://www.rcsb.org), while the SDF files of thefluoroquinolones were obtained from the Pubchem library of theNCBI database (http://www.ncbi.nlm.nih.gov/pccompound).The SDF files were subsequently converted to mol2 format byusing OpenBabel GUI . The binThe LT-Ag helicase activity was measured in the presence of 0.1,1.0,10 and 100μM fluoroquinolone. Percent inhibition of LT-Aghelicase activity by all 18 fluoroquinolones was observed tofollow almost a linear trend . At 100μThe model of SV40 LT-Ag (PDB ID: 1SVO) was dockedindividually with each of the above-mentioned 18fluoroquinolones. Each fluoroquinolone was docked deep intothe active site of LT-Ag helicase protein molecule. Coordinates ofthe LT-Ag binding sphere were designated between Glu473 andLys445. Based on the lowest docking energy, 6 fluoroquinolones,namely, Balofloxacin, Ofloxacin, Pefloxacin, Lomefloxacin,Ciprofloxacin, and Levofloxacin, were selected for detailedanalyses of their interactions with LT-Ag . Except In this study we have used SV40 LT-Ag as a model viral helicaseand have explored the inhibitory interactions offluoroquinoloneson with this enzyme using in vitro as well as insilico approaches. We tested the activity of a panel of 18fluorquinolones using a helicase assay and found all of them toinhibit the helicase activity of LT-Ag. Docking of thesefluoroquinolones on LT-Ag was also performed using in silicomethods. Based on the free energy values, 6 fluoroquinoloneswere found to interact most strongly with LT-Ag, with theirbinding affinities in the following order:Balofloxacin>Pefloxacin>Lomefloxacin>Ciprofloxacin>Levofloxacin>Ofloxacin. Docking analysis identified certain amino acids inthe LT-Ag active site, namely, Gly445, Glu473, and Leu440 thatwere found to interact frequently with most of the six selectedfluoroquinolones. For this study, we employed the nonradioactiveassay that we have previously published . In thisAlthough all fluoroquinolones were found inhibitory to both theviral helicases, as expected, the individual fluoroquinolonesbehaved differently with respect to the level of inhibitionobserved on HCV NS3 and SV40 LT-Ag. Since the architecture ofthe helicase active site represents diversification among differentviral helicases, the nature of their interaction withfluoroquinolones was expected to differ accordingly. A deeper understanding of the differential interaction offluororoquinolones on different helicases may be obtained byanalyzing crystal structures of helicase-fluoroquinolonecomplexes and/or by studying the effect of amino acid mutationsin the active site of LT-Ag. For the current study, we adopted anin silico approach to further dissect the interactions offluoroquinolones at the LT-Ag active site.Computer docking models of fluoroquinolone-LT-Ag helicasealso revealed certain key functional groups of fluoroquinolonethat were involved in their mutual interaction with LT-Aghelicase. The docked models of fluoroquinolones and LT-Agshowed that the piperazinyl moiety at position 7, carboxylic acidat position 3 and oxoquinoline at position 4 in thefluoroquinolone molecule were mostly involved in interactionwith the amino acid residues of LT-Ag. The docking outputreflected binding efficiency of LT-Ag and fluoroquinolones. Thebinding affinity of Balofloxacin was the highest followed byaffinities for Pefloxacin, Lomefloxacin, Ciprofloxacin,Levofloxacin, and Ofloxacin , as predicted by FlexX . ThesamThe authors declare that they have no competing interests.


Palaeo data suggest that Greenland must have been largely ice free during Marine Isotope Stage 11 (MIS-11). However, regional summer insolation anomalies were modest during this time compared to MIS-5e, when the Greenland ice sheet likely lost less volume. Thus it remains unclear how such conditions led to an almost complete disappearance of the ice sheet. Here we use transient climate–ice sheet simulations to simultaneously constrain estimates of regional temperature anomalies and Greenland’s contribution to the MIS-11 sea-level highstand. We find that Greenland contributed 6.1 m  to sea level, ∼7 kyr after the peak in regional summer temperature anomalies of 2.8 °C (2.1–3.4 °C). The moderate warming produced a mean rate of mass loss in sea-level equivalent of only around 0.4 m per kyr, which means the long duration of MIS-11 interglacial conditions around Greenland was a necessary condition for the ice sheet to disappear almost completely. The most pronounced deglaciation of Greenland of the last several hundred thousand years occurred during Marine Isotope Stage 11. Here, the authors show that it was the exceptional duration of the interglacial period that allowed Greenland to melt even though temperatures were only moderately warmer than today. The globally averaged MIS-11 sea level is estimated to have reached between 6–13 m above that of todayAncient DNA has been identified in the silt beneath the DYE-3 ice core pertaining to several boreal forest species including Alder, Spruce and Pine, as well as that of some insectsThis finding is further supported by oceanic sediment records. Pollen obtained from cores off the southern coast of Greenland corroborate the existence of a pronounced increase in abundance of several species of boreal vegetationMeanwhile, air trapped in the basal layers of the GRIP ice core (drilled at the present-day summit of the ice sheet) has been estimated to date to around 1 Myr BPThe above palaeo information provides complementary constraints on the Greenland climate and ice-sheet extent during this time. The DNA and pollen records provide information about local climatic conditions over southern Greenland, while the sediment records, ice core dating and the summit landscape dating give lower and upper bounds, respectively, to changes in ice area in key locations.et al.Indeed, Reyes et al.Most importantly, what also remains unclear is the key factor responsible for the decline of the ice sheet. Insolation was not anomalously high compared to today throughout most of MIS-11, and temperatures in the region were likely not as high as during MIS-5e ref. —the mostIn order to better understand this time period, we use the above palaeo information to constrain an ensemble of simulations of the GrIS through MIS-11. The coupled climate–ice-sheet model REMBO-SICOPOLIS9The MIS-11 regional positive summer (June–July–August mean) temperature anomalies in our simulations are coincident with positive anomalies in summer insolation peaking near 411 kyr BP . The appThe majority of the ice-sheet response lags the temperature forcing. Retreat begins around the time that temperatures above those of present day are reached in the model. When the summer temperature anomaly reaches its maximum at around 411 kyr BP, the GrIS has already lost between 1–3 m s.l.e. of ice. This is also when peak rates of mass loss of around 0.8 m s.l.e. per kyr are seen . The max8The simulated magnitude of warming during MIS-11 compares well with proxy data for sea surface temperatures just off of the Southern Coast of Greenland. De Vernal and Hillaire-Marcel6 km2 land area south of 69 °N (demarcating the southern bedrock terranes), a minimum of 19% (8–37%) ice-covered area was reached, with only the high elevation mountainous regions remaining ice covered. Such a significant deglaciation in the South is consistent with the cessation of proglacial sediments for several kiloyears during this time periodThe valid simulations show that southern Greenland was ice free for several kiloyears. For example, DYE-3 was most likely ice free for around 10 kyr ago between 410 and 401 kyr BP. Of a total of 0.6 × 10et al.A greatly reduced central dome of ice remains intact throughout MIS-11 for the valid simulations. This is consistent with the constraint of an ice-covered GRIP locationThe modelled climatic conditions during MIS-11 are also consistent with the growth of boreal forest in southern Greenland, particularly at the time of the maximum summer temperature anomaly . Most siδ18O record with the global δ18O stack of Lisiecki and RaymoOur simulations thus indicate that most boreal forest growth would have occurred at the peak of regional summer insolation (and summer temperatures) at around 411 kyr BP. This result is so far inconsistent with the timing of the peak pollen deposition about 5–15 kyr later in the ocean sediment recordet al.Our best estimate of MIS-11 regional summer warming of 2.8 °C is lower than recent estimates for MIS-5e refs , 15, 16 et al.This difference can be attributed to the unusually long duration of positive temperature anomalies during MIS-11, which allowed the ice sheet enough time to react more significantly to the climatic forcing. As hypothesized by Reyes 2 and the timing of the Northern Hemisphere deglaciation. The latter, in particular, is poorly constrained for MIS-11, though it is unlikely that the interglacial period would be shorter than that presented here given other information from δ18O , consistent with the ensemble presented by Robinson Ms is the melt rate, ρw and Lm are the density of water and the latent heat of melting, respectively, τa is atmospheric transmissivity, αs is surface albedo, S is insolation at the top of the atmosphere and c+λT is a linear parameterization of the long-wave radiation and turbulent and latent heat fluxes as a function of near-surface temperature T. The parameter c was assigned a Gaussian prior of −55±2 W m−2, while the modelled sensitivity of precipitation to temperature change was assigned a prior of dP/dT=6.3±3.3% °C−1 and scaled internally in the REMBO climate model. The priors were determined through comparison with regional and global GCM simulations under present-day and global warming scenarios8where As mentioned above, the regional climate model is driven by a time series of monthly temperature anomalies over the surrounding ocean calculated in a global transient glacial-interglacial simulationfs, was used to modify the original temperature anomaly time series proportionally to its maximum value, dTorig,max, such thatWe thus accounted for this climatic uncertainty in the simulations by incorporating a scaling factor applied to temperature anomalies during the interglacial period. The scaling factor, tstart and tend are the times of the beginning and the end of the interglacial time window. In this case we applied this scaling to the period with summer temperature anomalies above −2.0 °C in the original time series, corresponding to times between −424 kyr BP and −395 kyr BP. The width of this window and the cosine scaling was chosen to avoid artifacts in the time series related to the application of the scaling perturbation only during the period of positive temperature anomalies . The scaling factor fs was given an even prior . The parameter value combinations used in the simulations were then obtained using Latin Hypercube sampling with a total ensemble size of 300 members.where The ensemble of simulations was constrained by first eliminating any simulation from consideration that did not satisfy the hard constraints of an ice-covered GRIP location at all times and an ice-free DYE-3 location at some point during the simulation. Based on palaeo dataw is the non-normalized weight of the simulation and Asouth is the ice-covered land area below 69 °N (%). The weight is 1 when Asouth=0 and it descends towards zero as the ice coverage increases. The exponential coefficient of 0.1 was chosen such that the weight approaches zero at around 50% southern Greenland ice coverage, which is consistent with the ice-free DYE-3 constraint. The weighting function is shown in the inset of where The probabilistic information presented in this paper was calculated from a weighted Kernel density estimate (KDE) of the probability distribution, given the prior and posterior weighting as described above. We used the R package ‘ks’All numerical code used in this study, analysis scripts and the model results are available from the authors upon request.How to cite this article: Robinson, A. et al. MIS-11 duration key to disappearance of the Greenland ice sheet. Nat. Commun.8, 16008 doi: 10.1038/ncomms16008 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Cooperativity is a feature many multimeric proteins use to control activity. Here we show that the bacterial heptose isomerase GmhA displays homotropic positive and negative cooperativity among its four protomers. Most similar proteins achieve this through conformational changes: GmhA instead employs a delicate network of hydrogen bonds, and couples pairs of active sites controlled by a unique water channel. This network apparently raises the Lewis acidity of the catalytic zinc, thus increasing the activity at one active site at the cost of preventing substrate from adopting a reactive conformation at the paired negatively cooperative site – a “half-site” behavior. Our study establishes the principle that multimeric enzymes can exploit this cooperativity without conformational changes to maximize their catalytic power and control. More broadly, this subtlety by which enzymes regulate functions could be used to explore new inhibitor design strategies. Cooperativity critically allows enzymes to make step-like rate responses to substrate concentration changes, and so offer rapid responses to changing cellular conditions. It is a key to link enzymes – the molecular building blocks of life – to the system-level events, such as signaling pathways and cellular responses. The effects of cooperativity have been characterized structurally in many systems4. Despite the simplicity of the underlying principle, different enzymes seem to adopt different strategies to accomplish cooperative conformational changes. Understanding how cooperativity is manifested in individual enzymes offers the opportunity to further understand the molecular basis of enzyme catalysis and facilitate drug discovery.Cooperativity is a fundamental feature of many multi-subunit enzymes5. Positive cooperativity amplifies the sensitivity of the enzyme’s ligand binding capacity upon increase of ligand concentration and is important for maintaining the responsiveness of biological systems6. In negatively cooperative enzymes, binding of the first ligand reduces the affinity of binding of subsequent ligands, of particular relevance to ligand binding in signaling networks7.Both positive and negative homotropic cooperativity have been observed in a wide range of enzymes+ 8. This was used as a partial proof for the validity of the “sequential” (Koshland-Némethy-Filmer) model of cooperativity9. Similarly, both positive and negative cooperativity has been observed in DNA binding proteins10. The extreme form of negative homotropic cooperativity is the so-called “half-site” reactivity, where enzyme active sites are paired, and only one can be active at a time12. Many enzymes consisting of tetramers or higher order multimers are made from two or more pairs of such coupled sites. These enzymes can then also display positive cooperativity towards the same ligand.Furthermore, it is not uncommon for proteins to display both positive and negative cooperativity to the same ligand. For example, yeast glyceraldehyde-3-phosphate dehydrogenase has long been studied as a model of cooperativity, as it has both positive and negative cooperativity for its substrate NAD11. However, in some cases of half-site occupancy the conformational changes are too subtle for this definitive change to be observed. In these cases, molecular and quantum mechanics must be used to explain the half-site reactivity13.This half-site effect has generally been explained by an allosteric conformational change upon ligand binding that prevents binding at the paired site. Indeed, observation of half occupancy in a crystal structure is considered definitive of enzymes following the sequential model14. They generally bind to sugar phosphates, using the phosphate to provide affinity, and catalyzing reactions at other positions in the sugar16. This SIS family of enzymes show an interesting range of active site chemistries: the same evolutionarily conserved scaffold is used to coordinate different active site side chains to drive a variety of reactions throughout the family. This wide range of potential routes to control activity, and the differing numbers of subunits in different proteins , offer evolution the opportunity to fine-tune the activity of SIS enzymes to cellular need.The sugar isomerase (SIS) family of enzymes catalyze a wide range of isomerase reactions involved in sugar interconversions17. These tetrameric proteins catalyze the conversion of the pentose phosphate pathway intermediate sedoheptulose-7-phosphate (S7P) into d-manno-heptopyranose-7-phosphate 24. Among these, the structure with the most interesting features is GmhA from B. pseudomallei, a tetramer with a zinc ion at the heart of the active site, coordinating by the sidechains of H64, E68, Q175 and H183. All of these side chains are completely conserved amongst GmhA orthologues, and mutations in all of them significantly reduce activity. GmhA defines a distinct class of SIS domains in Pfam25. Unlike other SIS domains, which generally form dimers with active sites at considerable distances from one another (e.g.28), GmhA forms pairs of adjacent actives sites separated by 15 Å, linked by a solvent filled channel  with substrate bound. This structure is almost indistinguishable from the previously solved unliganded, or product bound structures, precluding an allosteric conformational change that is often associated with enzyme cooperativity. Conversely, quantum mechanical (QM) calculations using active site models revealed that GmhA achieves negative cooperative control of its paired active sites through the solvent-filled channel that bridges these two active sites. Only one of such active site is able to bind to the substrate after energy optimization. Modeling of the global hydrogen bonding network suggested that positive cooperativity occurs between the two pairs of half-sites, with defined active sites showing cooperativity for each other. The half-site reactivity of GmhA gives it distinct advantage to control this step in heptose biosynthesis, hence the knowledge of cooperativity could be explored in inhibitor discovery.20  to the sample with all active sites intact ; and the sample with only one active subunit showed approximately one-third of the wild-type activity . A drop in activity would be expected on the loss of one active site, especially in the light of the positive cooperativity observed, as indicated by a Hill coefficient greater than 1. These results suggested that, as well as the positive cooperativity observed, there might be negative cooperativity occurring between pairs of active sites, so that loss of one active site would not affect the rate.Comparison of these chimeras to the wild-type enzyme showed that all three samples had a very similar  μM Fig. ; Table 122. A substrate bound structure was obtained, albeit in an “open”, inactive conformation22. To assist in modeling the catalytic complex of GmhA, we determined in the present study the crystal structure of four active site mutants shown to affect activity , soaked with the reaction substrate S7P , we reasoned that GmhA may use the short-range hydrogen bonding network between the water molecules in this channel, D61 and H64 (which coordinates to the zinc in the active site) to enable communication between the two paired active sites. This may lead to either the positive or negative cooperativity.Surprisingly, the structure of the E68Q Fig.  mutant s1 and O2 of the substrate making coordination bonds with zinc : here, the Zn-O1 distance is significantly increased from ~2.16 Å as seen from Model 2 and 2.19 Å in Model 4 to 3.14 Å in Model 5water. The difference is even more pronounced in Model 5, in which the water molecule bridging between Q175 and D61 in Model 5water is removed and the sidechain of Q175 forms hydrogen bond directly to D61. There is a strong preference not to bind to substrate at the second active site , in a manner reminiscent of the substrate observed in the structure of the E68Q mutant  than Model 1 (0.498), indicating that the paired active site environment is better at attuning Zn’s Lewis acidity by maintaining its net charge. It is conceivable that the more positively charged zinc in the paired active sites would make the intermediate (probably a negatively charged ene-ol) more favorable, and so may increase the rate of the reaction. On the other hand, loss of the coupling between the paired active site by means of the D61A mutation leads to an enzyme that is still active, and has no half-site effect, but is less active overall (i.e. D61A mutant reduces enzyme activity to 6% of wild-type).Zinc’s charge is related to its electron-accepting ability as a Lewis-type catalyst in metalloenzymes. Interestingly, both zinc ions within the unliganded paired active site models exhibit slightly increased charges (1.292 and 1.302 in Model 3) compared to their counterpart in the unliganded single active site model , consistent with negative cooperativity. In contrast, when the second substrate molecule binds on the opposite face of the GmhA tetramer , as expected for positive cooperativity. Figure The QM calculations clearly explain the basis for negative cooperativity in GmhA. However, they cannot explain the positive cooperativity observed. The two pairs of active sites with positive cooperative effect are too far apart to construct a QM model of both pairs. Therefore, we instead considered the global hydrogen bonding network of the enzyme upon substrate binding, expecting that cooperative binding events would alter this network. The global hydrogen bonding network of GmhA is surprisingly delicate. The main hydrogen bonding network Fig. , red29 lmer Fig. , there i−1), there is likely to be significant binding and release of substrate before a catalytic event takes place. The H-bond modeling observations therefore strongly support the biochemical observation of positive cooperativity with a Hill coefficient of 1.5–2, supporting a robust cooperativity between non-neighboring active sites. The data therefore suggest a model whereby alternating pairs of active sites on opposite sides of the molecule will engage with substrate and form a productive enzyme-substrate complex. The other pairs of active sites will not productively engage substrate, but instead provide catalytic power through the coupling of the active sites that exceeds the loss of activity from the non-use of these active sites. This enzyme therefore employs an elegant mixture of positive and negative cooperativity to tune its catalysis using subtle alterations to its hydrogen bonding network.These data are consistent with both the positive cooperativity and negative cooperativity demonstrated by the quantum mechanical calculations: they suggest a model in which binding of substrate at one active site deters binding at either of the neighboring active sites, but promotes binding at the non-neighboring active site. The coupled paired active site is deterred by the zinc ion in this active site strongly preferring the tetrahedral coordination; whilst the other neighboring active site is deterred by the unfavorable effects on the global hydrogen bonding network. In contrast, both of these effects will favor binding at the non-neighboring active site, as the global hydrogen bond network shows a substantial improvement (releasing energy for binding), and the coupling of the active sites will favor binding of a substrate molecule to the zinc ready for catalysis. As GmhA is a slow enzyme  and d,d-heptose-1,7-bisphosphate 7-phosphatase (WcbN) from B. pseudomallei strain K96423  were prepared as previously described20. Mutants of GmhA were prepared using the QuikChange Lightning kit (Agilent), and confirmed by sequencing. Hybrids of E115K and D98N were prepared by subcloning the D98N mutant into the pCDF-Duet vector (Novagen), using the NcoI and HindIII sites . pNIC28-GmhA E115K and pCDF-Duet-GmhA D98N were transformed into Rosetta (DE3) cells (Merck), and grown in ZYM-5052 media31 supplemented with 100 μg/mL kanamycin, 25 μg/mL spectinomycin, and 10 μg/mL chloramphenicol. Cells were grown, harvested, lysed and initially purified as described for wild-type GmhA20. For preparation of hybrid GmhA, the size exclusion column was eluted isocratically with 10 mM Tris pH 8.0, 50 mM NaCl (buffer A). The hybrid proteins were separated using a 1 mL HiTrap Q HP column. The column was equilibrated in buffer A, and the protein eluted using a gradient to 288 mM NaCl over 50 mL. Pooled fractions were dialysed twice against a 50-fold excess of 10 mM Hepes pH 7.0, 0.5 M NaCl (buffer B). GmhA for enzyme assays was concentrated as necessary using a Vivaspin centrifugal concentrator (Vivascience), and stored at −20 °C with 20% (v/v) glycerol added.Full length sedoheptulose-7-phosphate isomerase .All crystals were grown using the microbatch method, and were prepared using an Oryx6 crystallization robot (Douglas Instruments). Tag-removed GmhA mutants at 10 mg/mL were mixed with an equal volume of crystallization solutions, and crystals were grown at 4 °C. Prior to flash freezing, crystals were soaked in cryoprotectant solutions containing S7P  for final concentrations ranging from 0 to 1 mM . Samples were incubated at 37 °C for 10 min, and the reaction was terminated by heating to 70 °C for 5 minutes. The phosphate concentration was determined by the addition of 17 μL of Pi Colorlock Gold (Innova Biosciences) to 68 μL of reaction product. After 5 minutes, 6.8 μL of stabilization reagent was added to each sample. The absorbance at 635 nm was then determined using a Tecan M200 Infinite plate reader. The rate of GmhA was determined by comparison to a phosphate standard curve ranging from 0–70 μM phosphate, and by subtracting the background rate from negative controls containing no GmhA. All reactions were performed in triplicate, and a total of eight substrate concentrations were tested for each sample. The kinetic parameters of each GmhA preparation were determined by fitting the data to Equation GmhA activity was monitored by coupling product formation to WcbL and WcbN and monitoring PThe co-ordinates and structure factors for the structures described in this paper have been submitted to the Protein Data Bank with accession numbers 5LU7, 5LU6, 5LU5 and 5LTZ for the D61A, H64Q, E68Q and Q175E structures respectively.39. Ten variants of each structure were produced by adding random shifts with an average of 0.058 Å to the heavy atoms using PDBSET40, and adding hydrogens using YASARA. Hydrogen bonding network analysis was performed using Proflex29. Hydrogen bond dilution was performed using default settings. The strength of the hydrogen bonding network was determined automatically using HETHER41. HETHER aberrantly assessed approximately one-quarter of the networks with GmhA added to active sites A and B; these replicates were removed and replaced with additional replicates.Samples were prepared for computational analysis by setting all residues with alternate conformations to the most favored conformation using PHENIX. Structure with S7P molecules in defined locations were prepared by adding S7P molecules as appropriate from the Q175E structure to the wild-type structure (PDB ID: 2XBL). Explicit hydrogens were added to the structure, and the structure optimized, using the online version of YASARASub_A and Sub_B) and a water molecule link the paired active sites. Specifically, Model 3 contains two unliganded paired active sites; Model 4 contains one unliganded active site and one substrate-bound active site S7P, while Model 5 includes two substrate-bound paired active. In addition, two water molecules are incorporated into Model 5 (labelled as Model 5water) to mimic water molecule or the sidechain of S58, which are within hydrogen bonding distances to both D61 and Q175 during our preliminary molecular dynamics calculations. The initial structures of these models were taken from the apo enzyme (PDB ID: 2X3Y) and the Q175E mutant. Only the sidechains of the active site residues and the aldehyde and ketone moiety at position 1 and 2 of S7P were kept in the models. The methyl groups (−CH3) of the sidechains of active site residues were fixed during QM optimisation, so that the residues didn’t move too far away from their positions in the crystal structures. The models were optimized at M05 2X/6–31 + G* level, using program G0942 to local minima and verified by frequency calculations. M05 2X level has been shown to be able to model the geometry and binding energy of zinc complexes well44. Zinc’s electron accepting ability (Lewis acidity) was estimated by the condensed Fukui indices f+znqzn(n) and qzn(n + 1) represent, respectively, zinc’s charge in the model system (containing n electrons) and its charge upon adding one electron (n + 1) to the model systems (one Zn2+ in Model 1) and two electrons (n + 2) (two Zn2+ in Model 3)46. The natural population analysis was used to calculate the charge of zinc ions at the M05 2X/6–31 + G* level.A total of five model systems were set up. Model 1 and Model 2 are single active site models. Model 1 contains the zinc and the coordinating H64, E68, Q175 and H183 (unliganded). Model 2 is Model 1 with addition of the substrate S7P (substrate-bound). Models 3, 4 and 5 are all paired active site models, in which D61 from the two subunits (labelled as The research materials supporting this publication can be accessed by contacting the corresponding author.Supplementary figuresdataset 1