Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function. Proteins perform many important jobs in each of the cells in our bodies, such as transporting other molecules and helping chemical reactions to occur. The part of the protein directly involved in these tasks is called the active site. Other areas of the protein can communicate with the active site to switch the protein on or off. This method of control is known as allostery.Switching proteins on and off could help us to develop treatments for certain diseases. For example, a protein called PTP1B reduces how well cells can respond to insulin. Switching this protein off could therefore help to treat diabetes. However, much like it’s hard to guess how a light switch is wired to a light bulb without seeing behind the walls, it is hard to predict which remote areas of a protein are ‘wired’ to the active site.Keedy, Hill et al. have now used two complementary methods to examine the structure of PTP1B and find new allosteric sites. The first method captured a series of X-ray images from crystallized molecules of the protein held at different temperatures. This revealed areas of PTP1B that can move like windshield wipers to communicate with each other. The second method soaked PTP1B crystals in trays with hundreds of drug-sized molecules and assessed which sites on the protein the molecules bound to. The molecules generally bound to just a few sites of the protein. Further tests on one of these sites showed that it can communicate with the active site to turn the protein on or off.Further work will be needed to develop drugs that could treat diabetes by binding to the newly identified allosteric sites in PTP1B. More generally, the methods developed by Keedy, Hill et al. could be used to study allostery in other important proteins. Proteins are collections of atoms that are mechanically coupled to one another, which gives rise to coordinated motions within the constraints of the folded structure. These motions are critical for many processes in molecular biology, including small-molecule and protein:protein binding interactions, catalytic cycles in enzymes, and allosteric communication between active sites and distal regulatory sites. Allostery in particular is now recognized to occur not only in classical oligomeric proteins like hemoglobin but also in monomers -- and indeed may be inherent to nearly all protein structures . HoweverOne system that would benefit immensely from an improved mechanistic understanding of allostery is the archetypal protein tyrosine phosphatase, PTP1B . PTP1B is highly validated as a therapeutic target for diabetes  and cancBy contrast, an allosteric inhibitor that binds to a less-conserved and less-polar surface site could bypass the limitations of active-site inhibitors. Two classes of compounds have been identified that allosterically inhibit PTP1B, although each has limitations. The first class of compounds are based on a benzbromarone (BB) scaffold and inhibit allosterically by binding to the space normally occupied by the regulatory C-terminal α7 helix . Recent Here, we have addressed the challenge of discovering unique opportunities for allosteric inhibition of PTP1B by taking advantage of two new techniques in X-ray crystallography that reveal minor conformational states of protein and ligands. First, multitemperature crystallography  can reveOverall, by highlighting promising allosteric sites and ligands that bind to them, our work may aid future development of potent non-covalent small-molecule allosteric inhibitors for PTP1B. More broadly, we illustrate a generalizable approach to characterizing and exploiting coupled conformational heterogeneity to enable long-range control of protein function.To identify allosteric sites in PTP1B that can communicate with the active site, we searched for regions of the protein whose conformational heterogeneity is coupled to that of the active site. We began by examining the conformational heterogeneity of the active-site WPD loop. Transition of this loop from the open to the closed state is rate-limiting for catalysis . In the Surprisingly, upon closer inspection, the electron density strongly suggests a significant population for the open state as well , left. OTo better characterize the conformational heterogeneity of the WPD loop in apo PTP1B, we collected X-ray datasets at several elevated temperatures including 180 K, 240 K, and 278 K (‘room temperature’) in addition to the 100 K (‘cryogenic’) model from the PDB, all at better than 2 Å resolution . Each coThe WPD loop adopts both the open and closed conformations across this range  and the Overall, we also observed temperature-dependent conformational heterogeneity for several other regions of PTP1B, including the previously characterized BB allosteric site, plus additional sites we refer to as the ‘197 site’ and the ‘loop 16 (L16) site’. These regions are all contiguous in the structure , suggestTo connect these multitemperature structures to known allosteric regulatory mechanisms, we first turned to a benzbromarone derivative compound (here referred to as BB2) that binds to an allosteric site >12 Å away from the active site and inhibits enzyme activity . The autWe additionally solved a high-resolution 1.80 Å,  structurSecond, at 273 K, we observe significant electron density just above BB3 . ModelinWe also observed temperature-dependent ordering in a loop  that sits underneath the α6-α7 junction just beyond the BB binding site. By contrast to lower temperature , the eleThe L16 site is seemingly coupled to the α6 helix: Lys239 from L16 H-bonds with Ile281 from α6 in the global closed state, but not in the global open state in which L16 adopts its alternative conformation. Because α6 is directly coupled to the α7 order-disorder transition, we therefore propose that the L16 site is a component of the collective allosteric network in PTP1B.The L16 site was not identified as part of the allosteric network in PTP1B based on a study using mutagenesis, NMR, and MD . HoweverIn addition to the temperature-dependent conformational heterogeneity observed at the BB site and L16 site, we observed residues with temperature-sensitive conformational heterogeneity in the ‘197 site’ . MoreoveA major link between the WPD loop and the 197 site is Tyr152. When the WPD loop is closed and the α7 helix is ordered, Tyr152 adopts a ‘down rotamer’ , red. ByIn our datasets at temperatures above 100 K, the electron density suggests a complex interplay between alternative conformations for Asn193 on the α3 helix and Tyr152 on loop 11 (L11) . Asn193 Strikingly, several residues propagating down L11 from Tyr152, and down α3 from Asn193, also adopt multiple conformations at higher temperatures . These rWe next discuss several similarities and a few differences between what we refer to as the 197 site and similar regions implicated by other recent studies of allostery in PTP1B. First, in addition to predicting the L16 site (see above), reciprocal chemical shift changes upon mutation suggested that several residues at both ends of the 197 site  are part of a region referred to as ‘Cluster I’ that is allosterically linked to the active site . HoweverWe also emphasize that the 197 site is structurally distinct from the two allosteric sites that have previously been targeted with small molecules to achieve inhibition , so any cat/KM the most, and the mutation farthest from the WPD loop (K197A) reduced kcat/KM the least  that were predicted to preserve the protein’s general structure, yet interfere with the conformational heterogeneity along the putative allosteric pathway lining the 197 site by removing interactions between alternative conformations. For Y152 we chose a mutation to glycine instead of alanine to more fully disengage residue 152 from the WPD loop, given that the Cβ and Hβ atoms of Y152 sterically engage with the WPD loop . All thrhe least . Our reshe least  and for he least ; small dOverall, we describe a large, collectively coupled allosteric network on one contiguous face of the protein . This neAlthough the results described above establish a conformationally coupled network within the structure of PTP1B, allosteric inhibition also requires binding sites for small molecules that can conformationally bias this network to modulate function. To identify potential allosteric ligand-binding sites in PTP1B, we mapped the small-molecule binding potential or ‘ligandability’ of the entire protein surface. Specifically, we used small-molecule fragments, which by virtue of their small size provide a relatively large sampling of drug-like chemical space . Astex POur PanDDA analysis of 1774 datasets revealed 381 putative binding events. We manually inspected each putative binding event, and were able to confidently model the fragment in atomic detail for 110 hits . OverallPanDDA initially identified >80 putative binding events in the active site. Many of these can be attributed to movements of the WPD loop , often iTo identify allosteric binders, we examined sites outside of the active site. Strikingly, we observed 24 bound fragments in the BB allosteric site . The posWe also examined fragments bound to the L16 site and the 197 site, which were suggested to be allosteric sites by our multitemperature analysis of apo PTP1B. Excitingly, both sites are fragment-binding hotspots: 17 fragments bind to the L16 site  and 30 fThe L16 site is between loop 16 (L16), the beginning of α1, and the end of α6. Most of the 17 fragments that bind here appear to ‘pry apart’ these elements  to creatThe 197 site is on the opposite side of the BB site, near α3 (including Lys197) and L11 (including Tyr152). Thirty fragments bind in the 197 site, with 14 in the primary subsite near Lys197, and 17 in a nearby but distinct subsite separated by a ‘ridge’ formed by the Gln157 and Glu170 sidechains  one fra. SimilarTo assess the effect of the bound fragments on the structure of PTP1B more globally, for each dataset we built an ensemble structure consisting of both the ground state and the bound state. Each dataset was modeled with an innovative PDB format as a multiconformer structure that represents both a heterogeneous apo state and a heterogeneous holo state. Due to limitations in the PDB model format and in the ability of conventional refinement programs to interpret and create reasonable restraints for this model type, either one conformation or four alternative conformations were used to describe each residue, often when only two were necessary. Due to this forced degeneracy, refinement of coordinates, occupancy, and B-factors must be highly restrained. We interpret the resulting occupancies as a good approximation of the fraction of unit cells that have a ligand present. Refining these ensemble structures using restraints that avoid overfitting allowed for some structural differences between the two states to emerge. In principle, these structural differences could give some prediction of the functional effects one might expect upon developing a higher affinity version of the molecule. The refined ensemble structures were of high quality . HoweverThe small-molecule fragments described above were identified by a naive screen and are not optimized for high-affinity binding to the 197 site or L16 site of PTP1B. Nevertheless, we selected 20 fragments that were deemed to bind in either site during early rounds of iterative PanDDA analysis   and testInstead, here we focus on an alternative strategy to validate the concept of allosteric inhibition at the 197 site: covalent binding to enhance ligand occupancy. Specifically, we used ‘Tethering’ , in whic121 space group, so we focused on K197C instead.For the allosteric 197 site in PTP1B, we chose to tether to a K197C mutant for several reasons. First, K197 is on the α3 helix, which is a key allosteric element in PTP1B . We pred1  . Michaelis-Menten kinetic analysis of K197C in the presence of 2 (50 µM) showed a statistically significant ~50% reduction in Vmax relative to DMSO treatment, but no significant effect on KM for the pNPP substrate  . Importad WT/WT* . In factubstrate . This suubstrate , once age DiFMUP . As withct on KM . These r2 acts specifically through the K197 site and to explore the mechanism of inhibition by 2, we solved a high-resolution  specific fragments to create potent new non-covalent allosteric inhibitors for WT PTP1B.Interestingly, several of the other noncovalent fragments bound to WT* overlay well with the aromatic rings of to K197C . This stOur analysis of PTP1B paints a portrait of an inherently allosteric system. Allostery is fundamentally tied to protein functions such as catalysis via the theme of conformational motions . Here, wen masse structural analysis, which let us reveal minor conformations and the shifts between them that allow a dynamic protein to function. We exploited families of structures in two different ways. First, we contrasted structures at several different temperatures  mutant, in the L192A (α3) mutant with an active-site inhibitor , and witOur work motivates specific future efforts to allosterically inhibit PTP1B activity for therapeutic purposes. However, the allosteric network illuminated here by probing PTP1B with non-biological perturbations  in vitro may also be relevant to how the enzyme is regulated in vivo, in at least three ways. First, in addition to directing subcellular localization , the quaFinally, it is interesting to note that different protein tyrosine phosphatases (PTPs) share a structurally conserved catalytic domain with PTP1B -- but have different variants of the α7 helix or even entirely different N- or C-terminal domains  that canFor all ‘wild-type’ PTP1B experiments here, we used what we refer to as the WT* construct: residues 1–321, with the C32S/C92V double mutation  to preveE. coli cells with plasmid, grew cells on LB + kanamycin plates overnight at 37°C, inoculated 5 mL starter cultures of LB + kanamycin with individual colonies, grew shaking overnight at 37°C, inoculated larger 1 L cultures of LB + kanamycin, grew shaking at 37°C until optical density at 600 nm was approximately 0.6–0.8, induced with 100 mM IPTG, and grew shaking either for 4 hr at 37°C or overnight at 18°C. Cell pellets (‘cellets’) were harvested by centrifugation and stored at −80°C in 50 mL conical tubes.Protein was expressed and purified as previously reported , with soFor purification, we first performed cation exchange with an SP FF 16/10 cation exchange column  in lysis buffer  with a multi-stage 0–1 M NaCl gradient ; PTP1B eluted around 200 mM NaCl. We then performed size exclusion with a Superdex 75 size exclusion column  in size exclusion buffer . PTP1B appeared highly pure in SDS-PAGE gels.3a, OTP = compound 12h). We were unable to solubilize OTP as used in  with at least a 200x volume ratio overnight at 4°C. We were unable to grow apo WT* PTP1B crystals initially, so we synthesized the active-site inhibitors OBA and OTP as in   and no glycerol. Drops were set at room temperature with 135 nL protein solution + 135 nL well solution + 30 nL seed stock, then trays were incubated at 4°C. Crystals appeared within a few days. The best seed stocks had been diluted 10-100x. These crystals were used for the BB3-soaking and fragment-soaking experiments.We crystallized apo K197C in the microbatch format with Al’s oil covering all wells. Protein was at 5–30 mg/mL in the same crystallization buffer as WT* but without DTT. The well solution was 0.3 M magnesium acetate, 0.1 M HEPES pH 7.5, 0.1% β-mercaptoethanol, 10–26% PEG 8000, 2% ethanol. Drops were set on ice with 1 μL protein solution + 1 μL well solution, then trays were incubated at 4°C. Crystals appeared within a few days. We also grew apo K197C crystals in a few other similar conditions.2 in the 96-well hanging-drop format. Protein was at 15 mg/mL in the same crystallization buffer as WT* but without DTT. The well solution was 0.2 M magnesium acetate tetrahydrate, 20% PEG 3350. Drops were set at room temperature with 100 nL protein solution + 100 nL well solution, then trays were incubated at 4°C. Crystals appeared within a few days.We crystallized K197C tethered to We used PDB ID 1sug for the apo WT 100 K dataset. The apo WT* 278 K dataset was collected at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12–2. All fragment-soaked datasets were collected at Diamond Light Source beamline I04-1. All other datasets were collected at Advanced Light Source (ALS) beamline 8.3.1.For apo WT* 180, 240, and 278 K, crystals had been grown in 2 μL drops with 10% glycerol in the mother liquor, then 1.5 μL of 50% glycerol was added several hours before data collection, resulting in a final concentration of ~27% glycerol. Some crystals were also dabbed into more 50% glycerol just before mounting.For BB3-complexed WT*, crystals were soaked with 125 nL of 10 mM BB3 (in DMSO). No glycerol was present in these crystals.For apo WT* and BB3-complexed WT*, crystals were looped and placed in a plastic capillary with ~70% mother liquor,~30% water to prevent dehydration during data collection, regardless of temperature; datasets were obtained at different temperatures simply by adjusting the cryojet temperature before placing the crystal on the goniometer. Helical data collection  was used to expose fresh regions with each shot, to minimize radiation damage.2-tethered K197C, crystals were simply looped and directly mounted on the goniometer in front of the cryojet. For apo K197C, a small amount of ice was likely present on the crystal.For apo and For fragment-soaked PTP1B, WT* PTP1B crystals in MRC SwissCi 3-well sitting-drop trays were soaked with small-molecule fragments using acoustic droplet ejection technology and a database mapping individual fragments to individual crystals as described . PTP1B cMost crystals stuck to the bottoms of wells regardless of construct and tray format, but it was often possible to gently dislodge them, or to physically break them off then expose the unperturbed portion of the crystal to the X-ray beam. Each dataset in this study was collected from a single crystal.free flags for the 278 K WT* apo dataset, then transferred them to the MTZ file of every other dataset with the reflection file editor in PHENIX (free flags). We solved each structure by molecular replacement with Phaser  had the same number of indices; no observations were omitted in this step. We then phased each dataset with Phaser using a reference model that was created by interpreting a high-resolution DMSO-soaked apo dataset. Next, we refined each initial model from Phaser using phenix.refine with the following flags to prevent excessive coordinate drift: ‘reference_coordinate_restraints.enabled=True ‘reference_coordinate_restraints.sigma=0.1’. Structure factors were then dropped from MTZ files, leaving map coefficients as inputs to PanDDA. Filled map coefficients (from PHENIX) were used to avoid Fourier series truncation effects in PanDDA maps. The resulting models were used as input to PanDDA (see below).For fragment-soaked datasets, we used XDS and a custom script  to automatically determine resolution cutoffs for all datasets. The resolution cutoff was initialized at 1.4 Å and incremented until the following criteria were met for the highest-resolution bin: at least 1.0 I/σ(I), at least 50% CC1/2 , and at For For multitemperature WT/WT*, we refined the initial model using phenix.refine for 10 macrocycles with automated water picking turned off. Next, we inserted preliminary open and closed alternative conformations for the WPD loop, and refined for another 10 macrocycles with automated water picking turned on. Finally, we performed several rounds of manual rebuilding, including manual addition and deletion of protein, solvent, and glycerol conformations and refinement with automated water picking turned off. Anisotropic B-factors were not used in refinement. The α7 helix was modeled as alternative conformation A only, with the unmodeled B conformation presumed to correspond to the disordered state; this allowed the occupancy of the ordered state to be refined. It was necessary to provide explicit occupancy parameter files to phenix.refine in some cases. For many residues, conformations obtained from PDB ID 1sug or 1t49 or another of our datasets  were useful for ‘filling in’ missing density. Often the missing conformations would not have been obvious based on the map alone, but once inserted and refined they seemed to fit well. This cross-dataset conformational-sampling approach also had the effect of emphasizing differences between models from different temperatures while minimizing differences due to chance or arbitrary modeling choices. Nevertheless, we encourage future users of these datasets to compare across different temperatures based at least in part on the electron density, and not just our models. The building process was guided by all-atom structure validation with MolProbity . The 100Glycerol  was present in WT* crystals during each multitemperature data collection to maintain consistency with the 100 K WT structure (PDB ID 1sug), in which glycerol was used as a cryoprotectant. Several ordered glycerol molecules, including those in contact with the closed WPD loop and at the allosteric 197 site, were evident from electron density at all temperatures. However, in some cases, it was difficult to differentiate between ordered waters, glycerols, or simply noise in the map. For example, the electron density was uncertain at some of the elevated temperatures for some glycerols originally modeled in PDB ID 1sug. When glycerol was omitted from crystals, the WPD loop was entirely in the open conformation regardless of temperature, from cryogenic temperature to near room temperature (data not shown). Our interpretation is that ordered glycerols in the active site, which are evident from the electron density at all temperatures, make weak contacts with the WPD loop’s closed conformation, and thus shift the protein’s energy landscape to a regime in which the open vs. closed conformations are near enough to isoenergetic that temperature can modulate their populations. This interpretation is strengthened by the fact that these glycerol molecules align well with a bound mimic of the pTyr substrate (PDB ID 1pty), which causes the loop to close during the catalytic cycle.2-tethered K197C structure, we omitted 2 for many rounds of refinement, allowing the electron density for the missing molecule to become extremely convincing before we finally added it to the model. The distance between the sulfur atoms in K197C and the ligand was restrained to 2.15 Å with a σ of 0.1 Å for refinement.For K197C, we used a similar refinement procedure, including many manual tweaks of alternative conformations for protein and water atoms. For the For fragment-soaked datasets, we used the PanDDA approach  in a fewSome events for which we modeled bound fragments in the earlier PanDDA version 0.1 runs were not detected as events in the final PanDDA version 0.2 run. In these cases, we manually created event maps based on the 1-BDC background subtraction values from the earlier PanDDA runs. In most cases, visual inspection confirmed that these early events likely correspond to bound fragments.Many PanDDA ‘events’ in the active site corresponded not to ligand binding, but rather to protein conformational changes of the WPD loop, P-loop, and Y46 loop that are caused by oxidation of the catalytic Cys215, a natural PTP1B regulatory mechanism . Some otWe built a generic unbound-state model by interpreting both an average map for the highest-resolution bin and one of the best individual apo datasets. The WPD loop was modeled as open, Tyr152 was modeled with two alternative sidechain conformations on the loop 11 backbone that is compatible with the open WPD loop, and the N-terminus (start of α1) and C-terminus  were fit as well as possible. Ordered waters were also manually positioned. This generic unbound-state model was superposed onto each PanDDA input model in the correct reference frame, then refined, to create an unbound-state model for each dataset.For each fragment-bound state, we inspected the fragment binding site, plus the several interesting regions of the protein mentioned above, in detail interactively. Waters were copied over from the unbound-state model, then moved or deleted where they conflicted with the bound fragment and/or the PanDDA event map. For a small number of planar fragments, several copies of the fragment bind in a parallel stack bridging the 197 site and a symmetry-related copy of the WPD-loop residue Phe182 via a crystal-lattice contact. Some areas such as Tyr152 were modeled with alternative conformations in the bound state only when they were well justified in the event map; otherwise we generally adhered to the unbound-state model. The correct modeling choice for the termini was uncertain in some cases.To refine structures for the 110 datasets with one or more modeled fragments, first we created restraints files for the ligands with eLBOW . For a swork and Rfree. During this first PHENIX refinement stage to obtain water positions, occupancies were fixed to the original PanDDA BDC value for the ground state and 1-BDC for the bound state; occupancy was distributed evenly between substates when the ground state or the bound state had alternative conformations for some residues. We observed coordinate drift and unstable B-factors for the protein with PHENIX refinement. Therefore, we copied the water positions obtained from PHENIX into the initial ensemble models, and refined with Refmac  and unbound-state occupancies to 1–2*(1-BDC) , and generated new restraints files that included the water molecules by running the PanDDA utility giant.make_restraints with the same RMSD parameter as with giant.merge_conformations: ‘duplicates.rmsd_cutoff = 0.4’. Then, each ensemble model was refined with Refmac using giant.quick_refine with the ligand CIF and custom giant.make_restraints restraint parameter files using the protocol herein. First, each model was refined for the default 10 cycles, with the extra arguments to Refmac ‘MAKE HOUT Yes’, to preserve hydrogens, and ‘HOLD 0.001 100 100’ to restrain XYZ coordinates but still allow for some geometry regularization and encourage B-factor and occupancy convergence. Next, the output from that refinement was fed into a loop of Refmac refinement with the default 10 cycles per run, and the ‘HOLD 0.0001 100 100’ argument, essentially fixing the XYZ coordinates, while letting occupancies and B-factors refine. Our output was the result of the 4th round (1 round + 3 rounds) of refinement in Refmac. However, the occupancies refined with these refinements did not converge to the correct occupancy (as seen by huge difference peaks describing the ligand). We then refined these structures with PHENIX, fixing the XYZ coordinates and manually scanning across possible occupancies while refining B-factors with the following settings: refinement.refine.strategy = individual_adp, hydrogens.optimize_scattering_contribution = False, main.number_of_macro_cycles = 10, optimize_mask = True, optimize_adp_weight = True. While in principle one could interpret the difference density to pick an optimal refined occupancy, no other statistics calculated provided a clear choice of occupancy. We ultimately chose to deposit occupancies of the bound state at 2.2 times the event map occupancy (1-BDC). This occupancy choice was motivated by the trend previously found by For refinement of the ensembles representing multiconformer models of the apo and bound states, we first refined each ensemble structure with phenix.refine to obtain water positions. The first stage of PHENIX refinement was 10 macrocycles with no removal or addition of waters  to let the existing waters relax into local minima. The second stage of PHENIX refinement was another 10 macrocycles with automated removal and addition of waters (‘ordered_solvent = True’) to remove waters that were unable to reach local minima and add waters that were clearly missing. Adding and removing waters, when compared to only removing them, generally had negligible effect on MolProbity scores, but improved Rh Refmac . To do sCoot  was inst3)4 , and Na2CO3  in THF (8 mL) was stirred at 90 ˚C overnight. The reaction was allowed to cool, taken up in water, and then extracted 3x with EtoAC. Organic layers were combined, washed with brine, dried over Na2SO4, concentrated in vacuo and purified using flash chromatography (MeOH/DCM) to obtain 22.1 mg of product (10% yield). Calcd for C12H10F2NO (M+H+): 222.07; Found 222.8.A solution of 2,4-difluorophenyl boronic acid , 2-amino-4-bromophenol , Pd, and DIPEA  in THF (300 µL), EDCI·HCl  was added. The reaction was stirred at room temperature overnight. To this was added a solution of bis[2-ethyl]disulfide dihydrochloride  and TCEP  in THF/H2O . The reaction was stirred at room temperature overnight. The reaction was taken up in EtoAC/H2O, and extracted three times with EtOAc. Organic layers were combined, washed with brine, dried over Na2SO4, concentrated in vacuo and purified using flash chromatography (DCM/MeOH) followed by preparative HPLC (C18 column (50 × 19 mm), Methanol/Water–0.05% formic acid gradient: 10:90 to 100:0 over 12 min; 20 mL/min; 254 nm detection for 18 min.) to obtain 9.1 mg of 2 (23% yield). 1H NMR  δ 9.55 , 8.17 , 8.06–8.04 , 7.54–7.45 , 7.22–7.19 , 7.12–7.00 , 3.94–3.78 , 3.15–2.99 , 2.54–2.52 . Calcd for C18H21F2N2O2S2 (M+H+): 399.1; Found 398.93.To a mixture of 3-amino-2',4'-difluoro-−4-ol , dithiodiglycolic acid , HOBt·HWe screened K197C against a previously synthesized library of 1600 disulfide fragments made available by the UCSF Small Molecule Discovery Center (SMDC) .50 values were calculated using nonlinear fitting in Prism 7 (Graphpad), n = 3.For the screen, tethering reactions were performed using the following conditions: 1x tethering buffer , with 500 nM of K197C, 1 mM β-mercaptoethanol, and 100 µM of fragment (0.2% DMSO), 1 hr at rt. Unless otherwise noted, tethering reactions for follow-up experiments and activity assays were performed using the following conditions: 1x tethering buffer, 1 µM of K197C, 0.1 mM β-mercaptoethanol, and 50 µM of fragment (2% DMSO), 1 hr at rt. For DiFMUP assays 100 µM of fragment (0.2% DMSO) was used during tethering. For crystallography, tethering reactions were performed using the following conditions: 1x tethering buffer, 0.76 mg/mL of K197C, 0.1 mM β-mercaptoethanol, 500 µM of TCS401, and 250 µM of fragment (2% DMSO), 2 hr at rt. A total reaction size of 3.5 mL was used for preparation of crystallography samples. Following labeling, the reaction was dialyzed into crystallization buffer overnight to remove TCS401 and unbound fragment. In all cases, the percent of tethering was measured using a Waters Xevo G2-XS Mass Spectrometer, and calculated by comparing the relative peak heights of the unmodified and modified protein. Tethering ECcat values were calculated using an pNPP extinction coefficient of 18,000 M−1 cm−1 and a path length of 0.29 cm. These parameters for WT* PTP1B were similar to those reported previously for WT . Protein was diluted to 200 nM in a variant of pNPP activity assay buffer . Enzyme activity assays were performed with 0.15 or 1 mM fragment in 2% DMSO  or with 2% DMSO without fragment as a control, with 5 mM pNPP. A no-enzyme well was also assayed. Absorbance at 405 nm for each reaction was monitored every 30 s for 5 min. The rate (mAU/s) of each reaction was calculated over the 5 min. These rates were compared with fragment vs. with DMSO.Fragment-RateNo Enzyme)/(RateDMSO-RateNo Enzyme))).For single-point assays of tethered K197C, completed tethering reactions (post 1 hr incubation) were diluted to a final concentration of 200 nM K197C with a variant of pNPP activity assay buffer  and pNPP . A no-enzyme well and a DMSO-only well were also assayed. Absorbance at 405 for each reaction was monitored every 30 s for 5 min using a Tecan Infinite M200 Pro. Percent inhibition was calculated using the following equation: 100, the same concentration of 2 as used during tethering, and pNPP . A no-enzyme well and a DMSO-only well were also assayed. Absorbance at 405 nm for each reaction was monitored every 30 s for 5 min using a Tecan Infinite M200 Pro. The rate (mAU/s) of each reaction was calculated over the 5 min. Percent inhibition was calculated using the following equation: 100(1- ((RateFragment-RateNo Enzyme)/(RateDMSO-RateNo Enzyme))). Ki values were calculated using nonlinear fitting in Prism 7 (Graphpad), n = 3.For 2 (50 µM), and 12 pNPP concentrations obtained by serial two-fold dilutions starting from 20 mM. Absorbance at 405 nm for each reaction was monitored every 30 s for 5 min using a Tecan Infinite M200 Pro. The rate (mAU/min) of each reaction was calculated over the 5 min. Data was plotted and fit using Prism 7 (Graphpad), n = 3. Vmax and KM values were calculated using the Michaelis-Menten nonlinear fit in Prism 7 (Graphpad). All p-values and significances were calculated using a FDR-Approach Multiple t-test in Prism 7 (Graphpad).For pNPP kinetics experiments with tethered complexes, completed K197C and WT* tethering reactions (post 1 hr incubation) were diluted to a final concentration of 200 nM K197C and WT* with a variant of pNPP activity assay buffer , Fragment-RateNo Enzyme)/(RateDMSO-RateNo Enzyme))), n = 3.For DiFMUP inhibition experiments with tethered complexes, completed K197C and WT* tethering reactions (post 1 hr incubation) were diluted to a final concentration of 2 nM K197C and WT* with a variant of DiFMUP activity assay buffer , and 9.4 µM of DiFMUP. Fluorescence at 450 nm with 358 nm excitation for each reaction was monitored every 30 s for 3 min using a Tecan Infinite M200 Pro. The rate (ΔF/min) of each reaction was calculated over the 3 min. Percent inhibition was calculated using the following equation: 100. All p-values and significances were calculated using a FDR-Approach Multiple t-test in Prism 7 (Graphpad).For DiFMUP kinetics experiments with tethered complexes, completed K197C tethering reactions (post 1 hr incubation) were diluted to a final concentration of 2 nM K197C with a variant of DiFMUP activity assay buffer , and 11 DiFMUP concentrations obtained by serial two-fold dilutions starting from 300 µM. Fluorescence at 450 nm with 358 nm excitation for each reaction was monitored every 30 s for 3 min using a Tecan Infinite M200 Pro. The rate (ΔF/min) of each reaction was calculated over the 3 min. Data was plotted and fit using Prism 7 (Graphpad), n = 3. VMulticonformer models and structure factors for the multitemperature WT and WT* , BB3-bound (6B8Z), K197C apo (6BAI) and tethered (6B95) datasets have been deposited in the Protein Data Bank .We have made publicly available several files that document our PanDDA analysis of all WT* fragment-soaked datasets. For each dataset, we provide a model of the unbound state, structure factors, an average map for the corresponding resolution bin, a PanDDA Z-map, and one or more PanDDA event map(s) as applicable. For fragment-bound datasets, we also provide the refined ground state model and the bound state model (before they were merged into an ensemble and refined) as separate PDB files, along with PHENIX, Refmac, and ligand restraint files used in the ensemble refinement. Additionally, we provide an overall PanDDA log file. These files are hosted at Zenodo at the following DOI: 10.5281/zenodo.1044103. Finally, using a new deposition procedure, refined ensemble structures for the 110 WT* fragment-bound datasets have been deposited to the PDB . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "New routes for PTP1B allosteric inhibition by multitemperature crystallography, fragment screening and covalent tethering" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.This manuscript investigates PTP1B through a combination of multiple-temperature X-ray crystallography and structure determination of hundreds of individual small-molecule fragments bound to PTP1B. The amount of work described is impressive and the experiments appear to be well executed. The authors noted that there are a number of allosteric inhibitors already described in the literature. The hypothesis was that the newly discovered allosteric site would enable them to design better PTP1B allosteric inhibitors. Unfortunately, none of the small molecule fragments bound to the allosteric site exhibited significant inhibitory activity against PTP1B. The authors then attempted tethering compounds to Cys197 and identified compound 2 as an inhibitor using this approach. However, the potency of compound 2 in the tethering assays was still weak, IC50 ca. 50 μm . Overall, while there was enthusiasm for the methodology and scholarship of the study, the lack of a clear pathway to a practical inhibitor for PTP1B reduced support for publication in Reviewer #1:This work consists of two main results. First, it identified an extensive allosteric network of PTP1B from the concerted conformational changes revealed by room-temperature X-ray crystallography. This represents an important methodology development besides the detailed structural elucidation of PTP1B regulatory mechanism it afforded. Secondly, the work identified several potential sites for allosteric small-molecule inhibition of PTP1B based on a large number of crystal structures from fragment soaking. The results are consistent with known PTP1B allosteric inhibition sites, and revealed a new so-called 197 site, which was further validated by a crystal structure of a small molecule covalently bound there when a Cysteine is introduced (the "tethering" technique) and by the allosteric inhibition of PTP1B confered by the binding. This work fell short of identifying a potent non-covalent small molecule inhibitor for wild-type PTP1B , which, of course, is much desired as PTP1B remains an undrugged yet therapeutically highly important target. Notwithstanding, the authors generated a substantial amount of data and new insights into PTP1B concerning its conformational changes and its patterns of interaction with small molecules.Reviewer #2:This manuscript reports the identification of a putative new allosteric site in PTP1B through a combination of multiple-temperature X-ray crystallography and structure determination of hundreds of individual small-molecule fragments bound to PTP1B. The amount of work described is impressive and the experiments appear to be well executed. The authors noted that there are a number of allosteric inhibitors already described in the literature. The hypothesis was that the newly discovered allosteric site would enable them to design better PTP1B allosteric inhibitors. Unfortunately, none of the small molecule fragments bound to the allosteric site exhibit any inhibitory activity against PTP1B. The authors then attempted tethering which has little impact to the story. The tethering is unlikely to lead to any drug that will have effect on a WT protein. Furthermore, they screened a new library for the tethering screen. Then what was the purpose of the fragment crystal soak screen? Developing a real inhibitor from the hits within the fragment crystal soak screen would have proved their point  better and been a real step forward in a novel allosteric inhibitor for PTP1B.Reviewer #3:This work advances our understanding of the subtle collective motions of a prototypical non-receptor protein tyrosine phosphatase, PTP1b. Previous work suggested that the rate of enzymatic catalysis of PTP1b is controlled by the motions of the conserved WPD loop, which has been trapped in different crystals at either open or closed conformations. The authors show the inherent mix of open and closed WPD loop in their reanalysis of the previously deposited PDB structure 1SUG and proceed to demonstrate via multi-temperature crystallography the gradual shift of population towards open WPD as a function of increasing temperature. The collective motion of WPD loop, residues in the alpha3, disorder of alpha7, and residues of L16, match the motions observed upon binding benzbromarone, suggesting the latter stabilizes a pre-existing conformation. The multi-temperature crystallography reveals temperature dependent changes of the 197 site, which might also couple to the WPD motions via Tyr152 and rotation of helix3, thus impacting catalysis. The authors perform a systematic crystallographic fragment screen and identify fragments binding to the benzbromarone site, to L16, and the 197 site. They expand one fragment hit to build a tethered fragment binding to the K197C mutant, for which they show biochemical evidence of inhibition due to tethering and also obtain a high-resolution crystal structure. The crystallography and fragment screening work is solid, and the proposal of a new allosteric site, within reach of a previous site may prove to be important. I'd love to see follow up work with non-tethered inhibitors binding to the same site, but the current work is interesting in its own right.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors and the evaluation has been overseen by Philip Cole as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript reports on an approach to identify new allosteric networks and allosteric binding pockets in PTP1B phosphatase based on room-temperature X-ray crystallography and crystallographic fragment screening. An extensive allosteric network was identified in agreement with previous reports and, while the work fell short of identifying a potent allosteric PTP1B inhibitor, at least one of the binding pockets is confirmed by covalent "tethering".Essential revisions:The reviewers identified a number of concerns in several aspects:1) Overstatements and over-claims. The manuscript will benefit if the authors focus on the large amount of data  they produced and remove and temper their claims.a) The claim that new pockets are identified by RT crystallography needs qualification. The claim that L16 newly identified is not accurate as it was previously reported by the Loria laboratory. The K197 pocket was also previously reported by Loria lab and Peti lab with a different name. These previous works need to be adequately discussed in this context. The authors need to explain if and how these cryptic binding sites can be revealed from the RT crystallography data without the knowledge a priori.b) The allosteric network identification is interesting, but a substantial part of the network has been reported in 4 publications (2 by the Loria laboratory and 2 by the Peti laboratory). These results need to be properly acknowledged and discussed. Again, would the network be obvious without the prior knowledge?c) Some of the added speculative discussion can be misleading and needs to be tempered. For example, the discussion of the BB inhibitors and the corresponding site might be misleading to readers that are not well versed in mechanistic studies or drug discovery. The word "validates" is too strong , since the referenced paper was largely based on unsubstantiated or weak claims. Even though the present work provides support for some of these speculative ideas it does not validate them.d) The discussion of flat SAR is misleading: "On the other hand, they may also accommodate different small-molecule variants equally well, such that it is difficult to improve upon inhibition - that is, the structure-activity relationship (SAR) is flat." This argument is questionable as it would be an extreme coincidence to perfectly compensate the binding energetics with corresponding structural rearrangements. The possibility cannot be excluded that the flat SAR is due to an inhibition mechanism that is unrelated to that particular binding site, which is not uncommon occurrence in PTP1b drug discovery efforts.2) Methodology rigora) How observations are derived from the RT crystallography data in a rigorous way is not well explained throughout the text. Alternative conformations are fit in weak density in published and new crystal structures – however, would it be possible to just place waters in such incomplete density and the statistical analysis of the data improves? What is the cut off for rigor and statistics to identify biologically relevant alternative conformations etc., and, further, that it can be interpreted for the identification of allostery. This issue is crucial to the general applicability of the approach and specifically to the identification of both the allosteric network and the cryptic sites in PTP1B without using prior knowledge.b) In the fragment screening, many fragments were seen to bind with PTP1B in many locations. How would one know which of these locations are sites for allosteric inhibition if they have not been independently identified previously. It seems in such cases one can only make educated guesses based on druggability estimation of a fragment's binding site and its relation to the structural-functional mechanism of the target's regulation. Or there might be more rigorous ways to approach this issue. This is worth an adequate clarification and discussion.3) Presentation issuesa) The figures lack continuity and clarity. It seems that in each figure PTP1B is shown from a different orientation, without relaying the orientation to a general overview, which makes it difficult for the reader.b) The wording in many statements in the manuscript is exaggerated or unnecessarily strong, which needs revision.c) There are many typos throughout the manuscript.4) One interesting observation is that the tethering of the compound leads to change in Km? What is the mechanism for this change? A change in the active site? This should be repeated with a set of substrates . Such data may shed new light to the mechanism of allosteric inhibition of PTP1B. [Editors’ note: the author responses to the first round of peer review follow.]eLife.All reviewers are complimentary about the technical execution of the work. From that discussion, you identified a need to make the findings about the allosteric network more compelling to appeal to the more critical reviewer. We agree, and the revised manuscript emphasizes the major goal of long-range allosteric control of protein function  and discusses the more limited ramifications for new actionable therapeutic molecules (drug discovery) on this specific protein. Our change to a new title, “An expanded allosteric network in the dynamic enzyme PTP1B revealed by multitemperature crystallography, fragment screening, and covalent tethering”, instead of “New routes for PTP1B allosteric inhibition by multitemperature crystallography, fragment screening and covalent tethering”, summarizes this shift in focus. The revised manuscript better places the allosteric mechanism discovered by multi-temperature crystallography in the context of mutagenesis, recently published NMR work, post-translational modifications, and the partial antagonist effect of the tethered molecule. Moreover, our retrospective evaluation reveals that multi-temperature crystallography plus protein:fragment structures predict the known BB allosteric inhibitor for PTP1B, which demonstrates the potential value of our approach for other targets without any known allosteric inhibitors. By emphasizing the parts of the work that the reviewers were uniformly positive about and placing the parts they were less enthusiastic about in a more self-critical context, we hope that the revised manuscript will be suitable for further consideration at Reviewer #1:This work consists of two main results. First, it identified an extensive allosteric network of PTP1B from the concerted conformational changes revealed by room-temperature X-ray crystallography. This represents an important methodology development besides the detailed structural elucidation of PTP1B regulatory mechanism it afforded. Secondly, the work identified several potential sites for allosteric small-molecule inhibition of PTP1B based on a large number of crystal structures from fragment soaking. The results are consistent with known PTP1B allosteric inhibition sites, and revealed a new so-called 197 site, which was further validated by a crystal structure of a small molecule covalently bound there when a Cysteine is introduced (the "tethering" technique) and by the allosteric inhibition of PTP1B confered by the binding.Thank you for this excellent summary of our work.This work fell short of identifying a potent non-covalent small molecule inhibitor for wild-type PTP1B , which, of course, is much desired as PTP1B remains an undrugged yet therapeutically highly important target. Notwithstanding, the authors generated a substantial amount of data and new insights into PTP1B concerning its conformational changes and its patterns of interaction with small molecules.As discussed below for reviewer #2, we agree that a potent non-covalent inhibitor for wildtype PTP1B is desirable, but beyond the scope of this manuscript. As reviewer #1 notes, our contribution here is to illuminate the intrinsic allosteric network of the enzyme, map the ligandability of the entire enzyme, and confirm the existence of a functional allosteric linkage from one remote site to the active site. It is unlikely that this site can bind a potent enough molecule without additional interactions, for example with the disordered C-terminal tail, being formed to “cap” the site.Reviewer #2:This manuscript reports the identification of a putative new allosteric site in PTP1B through a combination of multiple-temperature X-ray crystallography and structure determination of hundreds of individual small-molecule fragments bound to PTP1B. The amount of work described is impressive and the experiments appear to be well executed.We thank the reviewer for these kind words about our work.The authors noted that there are a number of allosteric inhibitors already described in the literature.There are only two allosteric inhibitors for PTP1B, each with specific limitations. The first type, the BB series, has an IC50 in the μM range and did not reach the clinical stage. The second type, exemplified by MSI-1436, targets the disordered C-terminus – but nonspecifically, at multiple binding sites, and in a pose (or poses) that were not structurally resolved, all of which limit the ability to rationally improve allosteric inhibition. Our work identifies new sites capable of binding diverse small molecules, and we discuss how only some of these sites can likely be made “allosteric”. Our retrospective analysis of the BB binding site and new analysis of the tethering to the 197 site show how linking the knowledge of binding (by fragment screening) and motion  could be used to generate candidates for development of new, potent allosteric inhibitors that overcome the limitations of the BB and MSI-1436 series in the future. We have rewritten the corresponding paragraph in the Introduction to better clarify these points, as follows:“… an allosteric inhibitor that binds to a less-conserved and less-polar surface site could bypass the limitations of active-site inhibitors. Two classes of compounds have been identified that allosterically inhibit PTP1B, although each has limitations. The first class of compounds are based on a benzbromarone (BB) scaffold and inhibit allosterically by binding to the space normally occupied by the regulatory C-terminal α7 helix [10]. Recent work combining mutagenesis, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations revealed how rotations of the α3 helix and a discrete switch of the catalytic WPD loop are impacted by these BB allosteric inhibitors [11]. Unfortunately, the BB molecules were not successfully translated to the clinic. The second class are natural products, including a molecule called MSI-1436, that bind to multiple sites that primarily involve the disordered C-terminus [4]. However, the binding poses were not structurally resolved, limiting our ability to understand the molecules’ allosteric mechanism and rationally improve their potency. For example, a variant of MSI-1436 had improved inhibition but a distinct response to mutations at the putative binding sites, suggesting an unknown change in mechanism [12]. MSI-1436 passed Phase I clinical trials but was not advanced to Phase II [13]. A new approach to revealing the intrinsic allosteric circuitry of proteins would reveal new opportunities to develop allosteric inhibitors for PTP1B that could overcome the limitations of these existing molecules. Such an approach would additionally set the stage for efforts to dissect allosteric regulatory strategies in other biologically important phospho-signaling proteins.”The hypothesis was that the newly discovered allosteric site would enable them to design better PTP1B allosteric inhibitors.Our hypothesis was that the new structural biology methods we apply would reveal sites which can bind small molecules and are functionally coupled to the active site. Indeed, our multitemperature X-ray crystallography to map allosteric connectivity identifies a small subset of binding sites identified by fragment screening, which maps the relative ligandability of the entire protein surface. Remarkably, the methods do converge, in that the sites with the highest number of molecules bound are also the ones identified by multitemperature crystallography. The key result here is that orthogonal methods for mapping how well a site might influence the conformation/activity of the active site, and for mapping the capability of small molecules to bind at that site, converge to the same places. We have added the following text to the Introduction to emphasize this result and its implications:“To prioritize putative allosteric sites rather than benign binding sites, we focused on the subset of fragment-binding sites that were also conformationally coupled to the active site based on multitemperature crystallography of apo PTP1B. Notably, the sites chosen in this way bound more fragments than did any other sites -- suggesting that conformational heterogeneity may be important for both allostery and ligand binding.”Importantly, the approach described above is additionally (retrospectively) validated by successfully identifying fragment poses that predict the previously reported BB allosteric inhibitor scaffold , discovered using independent methods. We have now more strongly emphasized this key point with a new figure, Figure 7—figure supplement 1, which not only details how fragments predict central elements of the BB scaffold, but also provides evidence for potentially fruitful new chemical extensions to that scaffold. We added the following text to the Results section to emphasize this point:Strikingly, we observed 24 bound fragments in the BB allosteric site . The poses of many of these fragments overlap portions of the BB scaffold . However, many of them also contain chemical groups that suggestively protrude in new directions from the BB scaffold . This retrospective result validates the idea that fragment screening identifies binding sites, and specific fragment poses in those sites, that can be fruitfully exploited for allosteric inhibition.Unfortunately, none of the small molecule fragments bound to the allosteric site exhibit any inhibitory activity against PTP1B.The small-molecule fragments have very low affinity due to their small size and lack of any optimization for binding to specific sites in PTP1B, so it was not at all surprising that we did not see any inhibition from them. Indeed, our goal in using fragments was instead to map the bindable surface of the protein. However, the tethering experiments serve a complementary purpose by ultimately confirming a functional link from the allosteric site to the active site. We have clarified these points at several points in the text, including the following in subsection “Validating a functional allosteric linkage with covalent tethering”:“The small-molecule fragments described above were identified by a naive screen and are not optimized for high-affinity binding to the 197 site or L16 site of PTP1B. Nevertheless, we selected 20 fragments that were deemed to bind in either site during early rounds of iterative PanDDA analysis   and tested whether they have allosteric effects using enzyme activity assays. Unsurprisingly, we did not observe inhibition of enzyme activity by the fragments up to the maximum concentrations we were able to assay due to solubility of the fragments. It is important to note that this is not surprising given the fragments’ relatively simple chemical structures and low affinities . However, looking ahead, the dozens of cocrystal structures with small-molecule fragments bound at promising new allosteric sites we have reported here offer a foothold for future medicinal chemistry efforts to design allosteric inhibitors for PTP1B.Instead, here we focus on an alternative strategy to validate the concept of allosteric inhibition at the 197 site: covalent binding to enhance ligand occupancy. Specifically, we used “Tethering”…”The authors then attempted tethering which has little impact to the story. The tethering is unlikely to lead to any drug that will have effect on a WT protein.We agree with the reviewer that converting a covalent tethered compound for a mutant protein to a non-covalent drug for the wildtype protein remains a challenge beyond the scope of this work. However, we disagree that the tethering has little impact: the main goal of the tethering experiment here was to confirm that the allosteric 197 site is functionally coupled to the active site – i.e., to establish the concept of long-range control of protein function from this site. Indeed, our experiments confirm that a local perturbation (the tethered compound) at the 197 site, which is distal from the active site, significantly modulates the activity of the enzyme. This observation is critical for revealing that the 197 site can be thought of as a true allosteric site, rather than a benign, non-functional binding site. Thus, the tethering experiments ultimately confirm the prediction of allosteric coupling between the 197 site and active site that was made based on our multitemperature X-ray analysis of the apo enzyme.Furthermore, they screened a new library for the tethering screen. Then what was the purpose of the fragment crystal soak screen?The goal of the fragment screen was to identify places on the protein surface that bind molecules. The tethering screen followed up on the fragment screen on a single site – chosen because it had the highest number of molecules bound . The fragment and tethering libraries are different due to the requirement for a disulfide linkage in the tethering experiment. Thus, the fragments were free to contain essentially any chemical groups, whereas the tethering molecules all were required to feature a disulfide group, and were all screened against a specific mutant (K197C) of the protein. Converting fragment hits from our initial screen to disulfide-containing fragments would have been difficult and slow. It was simpler to rescreen the identified site against the existing disulfide fragment library.Overall, the fragment screen mapped the ligandability of the entire surface , and the tethering screen identified a compound that tethers well (indicating it binds appreciably) to the 197 site and is functionally linked to activity. Essentially the two approaches (fragments vs. tethering) involve a tradeoff between structural resolution and affinity. By combining both approaches, we have learned that the most bindable site in PTP1B has an inherent allosteric connection to activity.We have added the following text to the Discussion section to clarify the inherent value of both the fragment and tethering screens:First, we used a new high-throughput method with small-molecule fragments to map the ligandability of the entire surface of PTP1B with high structural resolution. Although individually these fragments have low affinity, collectively the 110 protein:fragment structures we report reveal overlapping poses with a multitude of precise binding interactions at specific sites in PTP1B . Second, we used covalent tethering to probe the functional effects of a high-occupancy ligand at one promising allosteric site. Our work takes advantage of a synergy between fragments and tethering. Fragments bind weakly but allow for the visualization of hundreds of chemical entities bound to distinct sites in a protein. By contrast, tethering does not provide high-throughput structural information, but allows for targeted perturbations to functionally probe a specific site, and additionally can provide lower-throughput structural information about chemical matter that allosterically inhibits a protein variant.Developing a real inhibitor from the hits within the fragment crystal soak screen would have proved their point  better and been a real step forward in a novel allosteric inhibitor for PTP1B.We agree with the reviewer that a “real” non-covalent inhibitor for PTP1B is desirable, as it has been a highly sought-after therapeutic target for many years, but that it is also an extremely high bar for publication.We have refocused several aspects of the manuscript around what we do think is the major step forward here: revealing an expanded allosteric network in PTP1B via multitemperature crystallography, mapping the ligandability of the entire surface to find potential allosteric sites with high-throughput small-molecule fragment screening and structure determination, and confirming a functional linkage from a new allosteric site to the active site. Although the tethered molecule we report at the allosteric 197 site achieves only modest inhibition, the partial allosteric inhibition we observe is strong evidence that the 197 site represents an important new foothold for allosteric inhibitor development for PTP1B. As described above, we have added text to the Discussion section that comments on the mechanistic basis of this partial antagonism.toward the goal of developing potent non-covalent inhibitors for PTP1B, our work takes on additional value by being highly generalizable to other systems.Overall, our work introduces a new approach for establishing long-range control of conformation and function. This is a general approach that we have applied to PTP1B. In addition to the novel result of the 197 site, our approach successfully identifies the core scaffold and conformational hypothesis of the previously characterized BB allosteric inhibitors . Moreover, the unprecedented number of small-molecule:PTP1B cocrystal structures we report offers invaluable opportunities for specific medicinal chemistry follow-up experiments using fragment-based drug design. While we have moved the field Reviewer #3:This work advances our understanding of the subtle collective motions of a prototypical non-receptor protein tyrosine phosphatase, PTP1b. Previous work suggested that the rate of enzymatic catalysis of PTP1b is controlled by the motions of the conserved WPD loop, which has been trapped in different crystals at either open or closed conformations. The authors show the inherent mix of open and closed WPD loop in their reanalysis of the previously deposited PDB structure 1SUG and proceed to demonstrate via multi-temperature crystallography the gradual shift of population towards open WPD as a function of increasing temperature. The collective motion of WPD loop, residues in the alpha3, disorder of alpha7, and residues of L16, match the motions observed upon binding benzbromarone, suggesting the latter stabilizes a pre-existing conformation. The multi-temperature crystallography reveals temperature dependent changes of the 197 site, which might also couple to the WPD motions via Tyr152 and rotation of helix3, thus impacting catalysis. The authors perform a systematic crystallographic fragment screen and identify fragments binding to the benzbromarone site, to L16, and the 197 site. They expand one fragment hit to build a tethered fragment binding to the K197C mutant, for which they show biochemical evidence of inhibition due to tethering and also obtain a high-resolution crystal structure.This is a nice summary of our work from the reviewer.The crystallography and fragment screening work is solid, and the proposal of a new allosteric site, within reach of a previous site may prove to be important. I'd love to see follow up work with non-tethered inhibitors binding to the same site, but the current work is interesting in its own right.As mentioned above, we concur that development of a potent non-covalent inhibitor is desirable and is an excellent topic for future work - but we agree with the reviewer here that our current report has quite broad scope and will be of interest to others.[Editors' note: the author responses to the re-review follow.]Summary:This manuscript reports on an approach to identify new allosteric networks and allosteric binding pockets in PTP1B phosphatase based on room-temperature X-ray crystallography and crystallographic fragment screening. An extensive allosteric network was identified in agreement with previous reports and, while the work fell short of identifying a potent allosteric PTP1B inhibitor, at least one of the binding pockets is confirmed by covalent "tethering".We agree with this summary from the reviewers.Essential revisions:The reviewers identified a number of concerns in several aspects:1) Overstatements and over-claims. The manuscript will benefit if the authors focus on the large amount of data  they produced and remove and temper their claims.We agree with the reviewers that the novel crystallographic data are exciting. To focus on these data and avoid overstating any claims, we have made several changes to the text. For example, we changed this sentence in the Abstract:“Our results converge on new allosteric sites that are conformationally coupled to the active-site WPD loop, hotspots for fragment binding, and distinct from other recently reported allosteric sites in PTP1B.”… to the following:“Our results converge on allosteric sites in PTP1B that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding.”We also changed this paragraph in the Introduction:“Overall, by pinpointing promising functionally linked, ligandable allosteric sites plus structurally resolving dozens of small-molecule binders at those sites, our work opens doors for future development of potent non-covalent small-molecule allosteric inhibitors for PTP1B. More broadly, we illustrate a new approach to characterizing and rationally exploiting coupled conformational heterogeneity to enable long-range control of protein function that is highly generalizable to other systems.”… to the following:“Overall, by highlighting promising allosteric sites and ligands that bind to them, our work may aid future development of potent non-covalent small-molecule allosteric inhibitors for PTP1B. More broadly, we illustrate a generalizable approach to characterizing and exploiting coupled conformational heterogeneity to enable long-range control of protein function.”Other examples of simplifying our claims are scattered throughout the manuscript, some of which are included in responses to other comments below.a) The claim that new pockets are identified by RT crystallography needs qualification. The claim that L16 newly identified is not accurate as it was previously reported by the Loria laboratory. The K197 pocket was also previously reported by Loria lab and Peti lab with a different name. These previous works need to be adequately discussed in this context. The authors need to explain if and how these cryptic binding sites can be revealed from the RT crystallography data without the knowledge a priori.We agree with the reviewers that previous work has used NMR, simulation and mutagenesis to identify sites that closely correspond to the 197 site and L16 site that we have identified here. We have therefore removed the word “new” throughout the manuscript when describing these sites. Overall, we feel that our contributions complement these excellent studies by the Peti and Loria groups in a number of ways by revealing atomic details, implicating distinct residues, and identifying small molecules that bind at these sites (as elaborated below).First, our work using multitemperature crystallography independently identifies similar allosteric sites as did Peti and Loria et al. by other methods including NMR, MD, mutagenesis, and crystallography. We note that, although publication dates may initially suggest otherwise, our work was in fact performed over several years in parallel with (and without knowledge of) those studies; thus, it is highly gratifying to see several studies converge to some similar conclusions. Moreover, our use of multitemperature crystallography affords direct experimental resolution of relevant conformational states of PTP1B throughout the allosteric network in atomic detail. To emphasize this aspect, we have added the following text to the Introduction:“Our findings provide support for the previously characterized allosteric network in PTP1B that responds to BB inhibitors [11]. Moreover, they reveal extensions of this network, including additional allosteric binding sites that are distinct from the BB site . Similar regions of PTP1B have been implicated as allosteric sites based on mutagenesis coupled with traditional cryogenic X-ray crystallography, molecular dynamics simulations, and NMR spectroscopy [11][15]; here we complement those studies by using multitemperature crystallography to reveal in atomic detail the conformational heterogeneity that allosterically links these sites to the active site.”We also added this sentence to Results section:“Our findings thus shed additional light on the mechanism by which loop 11 allosterically communicates with the active site, thus complementing other recent studies using mutagenesis, MD, and NMR to map allostery in PTP1B [11][15].”Second, there are some minor differences between the residues implicated in allosteric sites between these studies. In the L16 site, we identify residues that the Loria group did not . In the 197 site, we implicate some residues that are not implicated by the Loria group and/or Peti group. These small differences may in part arise from our use of a different technique, multitemperature multiconformer crystallography, which enables us to directly visualize the conformational ensemble for every residue in the protein. To explain this aspect, we have added the following text for the L16 site in the Results section:“The L16 site was not identified as part of the allosteric network in PTP1B based on a study using mutagenesis, NMR, and MD [11]. However, in a more recent study, several residues lining what we call the L16 site  were included in a region called “Cluster II”, which was suggested to be a previously unidentified allosteric site based on reciprocal NMR chemical shift perturbations upon mutation of this site or the WPD loop [15]. Our work here using multitemperature crystallography complements these findings by independently identifying this allosteric site using a new methodology, and by revealing in atomic detail how multiple conformational states at the L16 site may aid communication with the active site. Interestingly, a separate approach combining molecular dynamics and machine learning also recently pointed to this area as a potential “cryptic” binding site [31]. Therefore, the L16 site may be not only energetically coupled to the active site, but also capable of forming an under-appreciated small-molecule binding pocket via the conformational heterogeneity we observe.”We also added the following text for the 197 site in the Results section:“We next discuss several similarities and a few differences between what we refer to as the 197 site and similar regions implicated by other recent studies of allostery in PTP1B. First, in addition to predicting the L16 site (see above), reciprocal chemical shift changes upon mutation suggested that several residues at both ends of the 197 site  are part of a region referred to as “Cluster I” that is allosterically linked to the active site [15]. However, that study did not implicate additional key residues on the α3 helix, e.g. Asn193 and Lys197. Second, mutagenesis, NMR, and cryogenic crystallography implicated several elements of our 197 site as being part of the larger allosteric network in PTP1B: loop 11 (including Tyr152 and Tyr153), the α3 helix , and the α7 helix [11]. Chemical-shift-restrained molecular dynamics simulations further suggested that Tyr152 on loop 11 and Asn193 on the α3 helix have mutually coupled alternative conformations [11]. However, here we highlight additional residues (e.g. Asp148 and Glu157 on the β strands on either end of loop 11) as being conformationally coupled to each other and to the rest of the allosteric network and the active site, and which may collectively form a binding pocket. Therefore, our work accomplishes two things with regards to these existing studies. First, we add support to their findings by reaching similar conclusions using orthogonal methods. Second, we complement the other studies by revealing additional amino acid residues that may play roles in binding and allosteric communication at the 197 site.”Finally, in contrast to the Peti and Loria work, our study identifies dozens of structures with ligands bound to PTP1B, including dozens in the two key allosteric sites, and one with a molecule that was experimentally demonstrated to inhibit enzyme activity. This is an important, unique contribution relative to previous work. To emphasize this point, we have added text in several places, e.g. the following in the Introduction:“Our work builds on previous studies of these sites in PTP1B [11][15], which did not report chemical matter that binds to them.”b) The allosteric network identification is interesting, but a substantial part of the network has been reported in 4 publications (2 by the Loria laboratory and 2 by the Peti laboratory). These results need to be properly acknowledged and discussed. Again, would the network be obvious without the prior knowledge?Indeed, the allosteric network is obvious from our multitemperature X-ray analysis. We feel it is a credit to all of these research groups that our results agree with each other, despite using very different methods . See above for further discussion on this point. Moreover, our multitemperature X-ray experiments provide atomistic detail that complements NMR measurements . Finally, we add value by providing many structures of PTP1B in complex with small-molecule fragments, which both validate that the featured putative allosteric sites are ligandable and provide starting poses for future drug discovery efforts by others.c) Some of the added speculative discussion can be misleading and needs to be tempered. For example, the discussion of the BB inhibitors and the corresponding site might be misleading to readers that are not well versed in mechanistic studies or drug discovery. The word "validates" is too strong , since the referenced paper was largely based on unsubstantiated or weak claims. Even though the present work provides support for some of these speculative ideas it does not validate them.underscores for emphasis):To avoid misleading the reader by speculating, we have made several changes throughout the manuscript. For example, we have changed this sentence in the Introduction  The discussion of flat SAR is misleading: "On the other hand, they may also accommodate different small-molecule variants equally well, such that it is difficult to improve upon inhibition -- i.e., the structure-activity relationship (SAR) is flat. " This argument is questionable as it would be an extreme coincidence to perfectly compensate the binding energetics with corresponding structural rearrangements. The possibility cannot be excluded that the flat SAR is due to an inhibition mechanism that is unrelated to that particular binding site, which is not uncommon occurrence in PTP1b drug discovery efforts.We agree with the reviewers that other mechanisms which would also explain the flat SAR should not be excluded. To indicate this, we have added the following to the Discussion section:“Notably, it is possible that the flat SAR observed at the BB site is due to an inhibition mechanism that is unrelated to the particular binding site, which is not uncommon in PTP1B drug discovery efforts – if this is the case, targeting the 197 site may face similar hurdles.”2) Methodology rigora) How observations are derived from the RT crystallography data in a rigorous way is not well explained throughout the text. Alternative conformations are fit in weak density in published and new crystal structures – however, would it be possible to just place waters in such incomplete density and the statistical analysis of the data improves? What is the cut off for rigor and statistics to identify biologically relevant alternative conformations etc., and, further, that it can be interpreted for the identification of allostery. This issue is crucial to the general applicability of the approach and specifically to the identification of both the allosteric network and the cryptic sites in PTP1B without using prior knowledge.We agree that it is important to ensure that our models are not fitting to noise . Moreover, significant past research has validated the concept of modeling multiple protein states as “alternative conformations”. We have added the following text to the Results section when multiconformer models across temperatures are first introduced to clarify this point:“Such models are equally good and usually better explanations of the experimental X-ray data , and have been used to understand many biologically relevant phenomena at protein:water interfaces [26], dynamic enzyme active sites , and allosteric networks perturbed by mutations [28].”To further ensure that our multiconformer models for room-temperature and other elevated-temperature datasets in this paper are well-justified, we have now performed an additional analysis in which we removed the alternative conformations, re-refined the new single-conformer models, and compared the resulting R-factors to the multiconformer models’ R-factors. The result is that the multiconformer models’ R-factors are better (lower) in each case. To emphasize this point, we have added this new text to Results section:“Removing the alternative conformations and re-refining the resulting single-conformer models, either with or without automated solvent placement, yields deteriorated statistics (Table 1—source data 1), which confirms that the multiconformer models are appropriate explanations of the experimental data at each temperature.”… and a new supplementary table containing these data:Finally, the shift across temperatures provides an additional validation that protein conformational shifts, rather than water, is responsible for the density. We are working on joint refinement procedures that use these related datasets to increase the data:parameter ratio as a future direction.b) In the fragment screening, many fragments were seen to bind with PTP1B in many locations. How would one know which of these locations are sites for allosteric inhibition if they have not been independently identified previously. It seems in such cases one can only make educated guesses based on druggability estimation of a fragment's binding site and its relation to the structural-functional mechanism of the target's regulation. Or there might be more rigorous ways to approach this issue. This is worth an adequate clarification and discussion.Indeed, it is a general challenge in biophysics to predict what sites on a protein surface are good candidates for allosteric inhibition. Even when specific binding sites are known, it is difficult to predict whether small-molecule binding at them will allosterically inhibit protein activity. This is why our multitemperature X-ray analysis coupled to multiconformer modeling for apo PTP1B was so important: it provides a mechanistic way to predict which parts of the protein are capable of transmitting signals to the active site via an allosteric network of coupled conformational motions. Binding sites that abut this allosteric network are more likely to be good sites for allosteric inhibition using small molecules. We have added the following text to the Discussion section to emphasize this key point:“Many of the 11 binding sites outside the active site are likely to be benign. Importantly, our multitemperature X-ray analysis of the apo protein provides a way to predict which binding sites are instead likely to be allosterically coupled to function. Based on the result that the same small number of sites are both (a) implicated as allosteric by multitemperature X-ray analysis of conformational changes in apo PTP1B and (b) the most ligandable sites from the fragment screen across the entire surface, we conclude that conformational changes may be important for allosteric ligand binding in this protein.”3) Presentation issuesa) The figures lack continuity and clarity. It seems that in each figure PTP1B is shown from a different orientation, without relaying the orientation to a general overview, which makes it difficult for the reader.Due to their allosteric nature, the sites we describe are in various areas of PTP1B, which unfortunately does require different viewing orientations in order to see them without obstruction. We apologize if this was confusing. We do note that in the first revision, we included an additional overview figure  which specifies all the structural sites that are illustrated in subsequent figures, as requested by the original reviewers. We also note that movie versions of several key figures are available, which provide a better 3-dimensional sense. However, to further assist the reader in this regard, we have added text to each structure-based figure that better orients the reader with respect to Figure 1 and/or other relevant overview figures. Because almost all figures are in one of a small number of orientations (“front side” vs. “back side”), but are zoomed in to different regions from these orientations, we feel this is a helpful strategy for guiding the reader. For example, the caption for Figure 4 now includes the following text:“The viewing orientation in A-D is as in Figure 1B (“back side” of PTP1B), except zoomed in on the loop 16 site .”b) The wording in many statements in the manuscript is exaggerated or unnecessarily strong, which needs revision.We have adjusted the tone of the manuscript to be more subdued in many instances. For example, we removed the subjective word “beautifully” from this sentence in Results:“Remarkably, this L16 alternative conformation sampled by apo PTP1B [beautifully] matches the L16 conformation when PTP1B is allosterically inhibited by BB2…”As another example, we removed the word “exciting” from this sentence in the Discussion:“Our work reveals [exciting] new opportunities for long-range control of PTP1B function by impinging upon tendrils of this expanded allosteric network with small molecules.”c) There are many typos throughout the manuscript.We thank the reviewers for their attention to detail. We have fixed the typos that we observed while carefully revising the manuscript.4) One interesting observation is that the tethering of the compound leads to change in Km? What is the mechanism for this change? A change in the active site? This should be repeated with a set of substrates . Such data may shed new light to the mechanism of allosteric inhibition of PTP1B.Although there is a small change in KM, it is not statistically significant and therefore our original manuscript treated it as no change. To clarify this point – and to contrast it with the significant change in Vmax – we have added this Vmax and KM data to the manuscript as panels E and F in Figure 8—figure supplement 2 along with appropriate captions:We have also added the following text to the body of the manuscript referencing these new panels:2 (50 µM) showed a statistically significant ~50% reduction in Vmax relative to DMSO treatment, but no significant effect on KM for the pNPP substrate .”“Michaelis-Menten kinetic analysis of K197C in the presence of In addition, we have now performed new experiments with DiFMUP (as suggested by the reviewer) as an alternative substrate. As with pNPP, we observe no statistically significant change in KM , but a reduction in Vmax . The results across both substrates are therefore consistent with a non-competitive allosteric mechanism. We have commented on this result as follows in the “Validating a functional allosteric linkage with covalent tethering” section:To further profile the inhibitory effect of tethering of 2 on K197C, we assayed the ability of the tethered complex to dephosphorylate the alternative substrate DiFMUP [45]. As with pNPP, the tethered complex was inhibited, with kinetic analysis showing a dramatic reduction in Vmax, but no significant effect on KM . These results once again support a partial noncompetitive allosteric mechanism of inhibition.We have also added a new figure  detailing the Vmax and KM data as well as inhibition data with DiFMUP:In addition to these clarifications and new data, as requested by the reviewer, we have added the following summary of our finding of a non-competitive allosteric mechanism :We note that the non-competitive allosteric mechanism observed suggests that tethering 2 to K197C may shift the protein’s energy landscape in such a way as to alter the kinetics of WPD loop motions. Future work to explore this issue would nicely complement the crystallographic and functional analysis we provide here.


Companion animals are also affected by IgE‐mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles.CAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic.Custom‐designed allergen microarray was produced on the basis of the ImmunoHorses showed individual IgE‐binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S‐albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet.In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen‐specific preventive and therapeutic concepts. BSAbovine serum albuminCAPcaponizedCy3cyanine 3 fluorophoreHDMhouse dust miteHhourHRPhorse radish peroxidaseISAC131ISAC, custom‐designed with 131 allergen moleculesISACimmuno‐solid‐phase allergen chipISU‐EISAC standardized units for IgEISUISAC standardized unitsMinminuteRAOrecurrent airway obstructionrFag e 2recombinant Fag e 2RTroom temperatureSDS‐PAGEsodium dodecyl sulphate–polyacrylamide gel electrophoresisTBSTTris‐buffered saline with Tween 201Allergies also affect domestic animals, such as dogs, cats and horses.Aspergillus, in vitro IgE diagnosis and intradermal IgE levels do not support IgE‐mediated mechanisms.In horses, allergen hypersensitivityThe phenomenon of insect bite hypersensitivity (IBH) which especially occurs in Icelandic ponies and leads to “summer eczema” with severe pruritus, alopecia and crustingInterestingly, environmental allergen sources such as house dust mites (HDM), mouldsIn addition, several food allergen sources have been described being potentially relevant for horses, including oats, wheat or corn.Diagnosis of allergic conditions and desensitization therapy in domestic animals is today performed with allergen extracts.In recent years, innovative methods for reliable allergy diagnosis have been developed. In contrast to crude allergen extracts, molecular allergy diagnosis is based on single natural or recombinant allergen molecules. Molecular allergy diagnosis has entered clinical practice in humans,In particular, the immuno‐solid‐phase allergen chip (ISAC) has revolutionized human allergy diagnosis. The first ISAC microarray has enabled serum IgE testing on 94 allergensMalassezia associated with a number of different skin disorders such as atopic eczema,To this aim, we expanded an allergen chip for IgE serology in animals adding 19 more molecules of potential relevance in veterinary allergy diagnosis Table . We consIn a pilot study, using sera from clinically well‐characterized allergic horses with various symptoms and horses without clinical allergy Table , we esta22.1Sera from 41 horses suffering from diverse allergic symptoms and 10 horses without apparent clinical symptoms that were included , median age 12 years (range 2‐26 years), from Austria, Switzerland, Germany, Iceland and Japan were analysed (Table Sera were used retrospectively from other studies which were approved by the Ethics Committee of the University of Veterinary Medicine Vienna (GZ 68.205/0236‐II/3b/2011 und 68.205/0151‐II/3b/2013), by the Animal Experimental Committee of the Canton of Berne, Switzerland (No. BE 51/13) and by the Animal Care and Use Committee of Japan Racing Association (#2016‐9).2.2In total, 131 allergen molecules , additionally 19 allergen molecules from 9 different allergen sources) were spotted using the ImmunoCAP ISAC technology as previously described). ISU values above 0.3 ISU were considered positive.All detection steps were carried out according to the manufacturer's instruction, with slight modifications: Thirty μL undiluted horse serum was incubated on ISAC131 for 2 hours, and glass slides were then washed with ThermoFisher washing fluid. IgE detection (1 hour at RT) was performed 1 hour with 30 μL monoclonal mouse anti‐horse IgE antibody clone 3H10  self‐labelled with Cy3 , diluted 1:100 in IgE dilution buffer (Thermo Fisher). After final washing, fluorescence was measured in an ISAC microchip‐reader  with excitation at 532 nm (green laser). As the human IgE standard designed for IgE detection in human sera on ISAC112 could not be used, a work‐around was developed with the team of ThermoFisher, using an alternative positive signal with horse IgE on a spotted allergen as reference and comparing all values hereto in a semiquantitative manner. Microarray image data were analysed by MIA , and the ISAC standardized units (ISU) were calculated by comparing with a calibration curve  Figure B. ISU va33.1A representative sample of 51 equine patients from different geographic areas, Iceland, central Europe  and Japan, was assembled and horses grouped according to their clinical history ,Culicoides nubeculosus) responsible for summer eczema,Serum samples of all horses were tested on ISAC131 multiplex array Figure A, B for M. sympodialis Mal s 10 and Mal s 12 allergens. IgE‐binding intensities were highest in the 5 horse sera reacting to bee Api m 1 (mean 4.9 ISU).Representative results of equine IgE binding on ISAC131 Figure , into peIn group B with human and animal antigens Figure B, 12 horAmong pollen allergens Figure C, 18 horOverall, the most abundant IgE sensitizations were seen to Fag e 2 > Cyn d 1 > Aln g 1, but we were not able to find correlations between sensitization patterns in the different clinical cohorts  Cyn d 1, nPhl p 4, nApi g 5, nCup a 1 and MUXF3 on ISAC131, expresses cross‐reactive carbohydrate determinants (CCDs) which principally could lead to nonspecific IgE binding.In addition to the animal allergens on commercial ISAC112, we added animal and human antigens on ISAC131, however, without revealing any significant sensitization in the investigated horse cohorts. The most frequent sensitizer was Can f 3, serum albumin from dog, whereas in only one horse IgE to Can f 5 was found, suggesting exposure to a male dog.House dust mites (HDM) are abundant in households, transported by furry animals and involved in initiation and perpetuation of atopic eczema and asthma. Their enzymatic potency is involved in inflammation, while indispensable for specific sensitization.Alternaria allergen Alt a 1 in 6 horses, and a single horse reacted with IgE to Aspergillus allergen Asp f 1. Hay dust may be contaminated with moulds, to which specific IgE (together with specific IgG) can be formed which previously were correlated with equine bronchitis.There were a few indications of environmental exposure to German cockroach by IgE to Bla g 1 and 5, and little evidence for fungal exposure via IgE to M. sympodialis a lipid‐dependent yeast, part of our normal skin microbial flora, but also associated with several common skin disorders such as atopic eczema/dermatitis,Only sporadic sensitizations to Mala s 1, Mal s 6, 10 and 12Culicoides allergens Cul n 1 and Cul n 2,We failed to establish the IgE‐microarray testing for https://commons.wikimedia.org/w/index.php?curid=25570098), exactly from where our horse patients originate. The buckwheat plant (Fagopyrum esculentum) belongs to the mustard family and may elicit phototoxic reactions.The most significant finding, however, was the high prevalence of IgE to Fag e 2 from buckwheat in 37 of 51 tested horses from all groups, including eczema, respiratory symptoms, urticaria and nonsymptomatic. Considering that sensitization usually correlates with exposure, the high sensitization rate is plausible. Buckwheat predominantly grows in Middle Europe and Asia , Biomay AG, Vienna, Austria, and Viravaxx, Vienna, Austria, and serves as a consultant for the latter two companies. EJJ is shareholder of AllergyCare ‐ Allergy Diagnosis and Study Center, Vienna, Austria.LE performed ISAC tests and immunoblots and drafted the manuscript; GH helped in buckwheat extraction and immunoblots; IM, RV, SV, CR, AS and RS characterized and provided allergens for the custom‐designed ISAC131 microchip; SB, EKH, IF, KK, EM, HM, HS and AT clinically characterized allergic horses and provided equine sera and corresponding ethical approvals; IPS designed and helped in mouse immunizations; EJJ designed the study, supervised experiments and finalized the paper. All authors approved the final version of the manuscript. Click here for additional data file. Click here for additional data file.