Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases. The transfer of phosphate groups onto proteins (protein phosphorylation) is one of the most important methods used to send signals inside cells. The enzymes that catalyze this process, called protein kinases, are themselves controlled by the phosphorylation of a flexible region called the activation loop.For many years it had been thought that the purpose of activation loop phosphorylation was to clamp the otherwise flexible activation loop in an active state that allows molecules that need to be phosphorylated to bind to the kinase. This assumption was based on static pictures of protein kinases obtained by X-ray crystallography, in which individual states are trapped and visualized in a crystal lattice. However, new methods and approaches now mean it is possible to visualize how the position of the activation loop changes as it moves in solution. By applying these techniques, Ruff et al. show that the static model is incorrect in a protein kinase called Aurora A. In this enzyme, the phosphorylated activation loop continues to switch back and forth between active and inactive states. Phosphorylation instead enhances the catalytic activity of the active state.Aurora A regulates several important steps in cell division, and plays important roles in several kinds of cancer. The discovery that activated forms of Aurora A can have different dynamic properties raises the possibility that inhibitor molecules could be designed to exploit these differences and block specific activities of Aurora A in cancer cells. To realize this goal we need to better understand how a kinase switching between active and inactive states affects the ability of inhibitors to interact with it. Stringent regulatory control of protein kinases is critically important for the integrity of cellular signal transduction. The catalytic activity of protein kinases is regulated by finely-tuned allosteric mechanisms that reversibly switch the kinase domain between active and inactive conformational states . DisruptPhosphorylation on a specific site in the activation loop of the kinase domain is the most widely conserved regulatory mechanism in kinases . X-ray sThe serine/threonine kinase Aurora A (AurA) is an essential mitotic protein that controls a variety of cellular processes including mitotic spindle assembly, centrosome maturation, and mitotic entry . These fWe recently showed that activation of AurA by Tpx2 is driven by a population shift from a DFG-Out to the DFG-In state . Since cWe set out to explain how phosphorylation of AurA on T288 leads to a ~100 fold increase in catalytic activity  Zorba e. We prev−1  , and werThe T288 phosphorylation site lies in the C-terminal segment of the activation loop, the correct positioning of which is essential for the binding of peptide substrates . In the We plotted the simulated L225-S284 distances as a function of the R255-T288 distance to assess how Tpx2 and phosphorylation affect the conformation of the activation loop . As expeAlthough unphosphorylated AurA is highly dynamic in the absence of Tpx2, the activation loop is not in fact disordered in the simulations. Instead, two discrete subpopulations are observed: one subpopulation corresponding to the active-like state with a long L225-S284 distance (~43 Å), and another with a much shorter distance (~38 Å), representing a DFG-In conformation in which the activation loop is not correctly positioned for catalytic function  in most An interesting feature of the autoinhibited DFG-In substate observed in the simulations is that the T288 residue, which immediately follows the helical segment in the protein sequence, is positioned close to the C-terminal end of the helix in almost all of the trajectories , with thWe wondered why the helical conformation of the activation loop has not been observed in X-ray structures of AurA. In fact, the activation loop adopts the active conformation in only a small subset of AurA structures, specifically those determined either in the presence of Tpx2  or otherOur MD simulations, which represent over a millisecond of simulation data, predict that phosphorylation has profound effects on the activation-loop conformation of AurA within the DFG-In state, both disrupting an autoinhibited substate and promoting an active substate that is primed for catalytic function. In an attempt to confirm the simulation result that the T288 phosphothreonine residue of phosphorylated AurA is correctly coordinated in the active state, we mutated its ion-pairing partner R180 to an alanine residue and measured the effect on kinase activity. The R180A mutant possessed 4-fold lower activity in the absence of Tpx2, whereas the activity in the presence of Tpx2 was only modestly affected . This isTo assess the effects of phosphorylation on the structure of the DFG-In state predicted by our simulations, we used double electron-electron resonance (DEER), a pulsed electron paramagnetic resonance (EPR) spectroscopy technique . DEER exWe first sought to confirm our above results that the phosphorylated kinase still samples both the DFG-Out and DFG-In states. DEER spectra (background-corrected dipolar evolution data) take the form of a damped oscillating signal in which the mean distance between the spin labels is encoded in the cube root of the oscillation period, and the width of the distance distribution is encoded in the degree of damping. DEER spectra acquired for the phosphorylated kinase bound to ADP or AMPPNP showed a rapidly decaying and heavily damped signal, consistent with the activation loop adopting multiple conformations  confirmeTo bolster these DEER experiments we performed molecular dynamics simulations of MTSL-labeled phosphorylated AurA in either the DFG-Out state (PDB ID: 5L8K) or the fully-active DFG-In state , totaling 75–110 microseconds of aggregate simulation data for each state. In these simulations, sampling of different spin label rotamers, combined with motion of the protein, gives rise to a range of predicted spin-spin distances. Although the simulated distributions for the DFG-In and DFG-Out states overlap, the DFG-In distribution is skewed to longer distances: distances beyond 45 angstroms are more populated in the DFG-In simulations, and distances beyond 50 angstroms are almost exclusively associated with the DFG-In state . PromineWe next turned to assessing how phosphorylation alters the structure of the DFG-In state. DEER experiments performed on unphosphorylated AurA bound to ADP gave qualitatively similar results to those obtained with the phosphorylated kinase bound to ADP, with a broad distribution of spin-spin distances consistent with similar populations of DFG-In and DFG-Out states  inset, aContrasting these results with those obtained with ADP  highlighInterestingly, the 4-angstrom increase in spin-spin distance observed in the DEER experiments performed with the L225/S284 labeling sites  inset isOur proposed model for the autoinhibited DFG-In substate of AurA provides an explanation for several puzzling observations. Firstly, the presence of a third state explains how phosphorylation can promote the active state without triggering a shift in the DFG equilibrium, the central result of this paper. By eliminating the autoinhibited DFG-In substate, phosphorylation redistributes the ensemble between the DFG-Out and active DFG-In substates . The facSecondly, our model accounts for the synergy between phosphorylation and Tpx2, observed in our fluorescence experiments, and reported previously . By unfoThe majority of eukaryotic protein kinases are activated by phosphorylation on the activation loop at a site equivalent to T288 in AurA . X-ray sBinding of Tpx2 to unphosphorylated AurA causes a pronounced population shift from the DFG-Out to the DFG-In state , in striIt is interesting to consider the dual activation mechanisms of AurA in the light of the closely-related AGC-family kinases. The AGC kinases possess a C-terminal hydrophobic motif that docks in cis onto a pocket above the αC-helix in a manner that closely resembles the interaction of Tpx2 with AurA . ActivatIn contrast, the highly dynamic nature of AurA activated only by phosphorylation may be a unique property of the Aurora kinases that reflects the loss of the hydrophobic motif in this lineage. These motions may facilitate further regulation of AurA by additional cellular factors, allowing for graded levels of catalytic activity. For instance, phosphorylated AurA has been shown to interact with Cep-192 , Bora M, and AjuIt is unclear whether activation of AurA by both phosphorylation and Tpx2 occurs in normal cells, where the Tpx2-bound spindle pool of AurA is thought to be predominantly unphosphorylated , and theThe DFG flip has long been considered one of the key regulatory mechanisms used by nature to control the catalytic activity of protein kinases , but theE.coli and purified as previously described  in FTIR buffer . Samples were concentrated to ~1 mM and loaded into a calcium fluoride sample cell mounted in a temperature-controlled housing (Biotools) for IR experiments. IR spectra were recorded on a Vertex 70 FTIR spectrometer (Bruker) equipped with a liquid nitrogen cooled indium antimonide detector with 2 cm−1 spectral resolution. Spectra were averaged across 256 scans, background subtracted using absorbance spectra of the buffer flow-through from sample concentration, and baselined using the polynomial method in the OPUS software (Bruker).Phosphorylated Q185C AurA protein samples  were prepared using a cysteine-light form of the kinase in which all endogenous solvent-accessible cysteines were removed by mutagenesis . After Nickel-affinity purification, repeated rounds of cation exchange chromatography were used to isolate the homogeneous singly phosphorylated species, with enrichment of the phosphorylated species tracked during purification by western blotting and activity assays . NitrileKinase activity was measured using the ADP Quest coupled kinase assay (DiscoverX) in a fluorescence plate reader (Tecan Infinite M1000 PRO) as described previously . Assays For FRET experiments, the AurA variant C290S/A C393S L225C S284C was expressed, purified and labeled with donor  and acceptor (Alexa 568) using cysteine-maleimide chemistry, as previously described . Labeled2, 1% DMSO and 0.1 mg/mL bovine-γ-globulins at AurA concentrations of 5–50 nM. Bulk FRET efficiency and inter-fluorophore distance were calculated from the ratios of the donor fluorescence in the presence and absence of acceptor, assuming a value of 62 angstroms for the Förster radius, as previously described (Ligand titration FRET experiments were performed in a Fluoromax 4 Spectrofluorometer (Horiba) at 22°C. Assays were performed in 15 mM HEPES pH 7.5, 20 mM NaCl, 1 mM EGTA, 0.02% Tween-20, 10 mM MgClescribed . The steescribed .D were determined using the spectra obtained with the D + A labeled sample. The ratio FD/FA (where FD is the donor fluorescence maximum and FA is the acceptor fluorescence maximum), a highly sensitive measure of ligand binding, was fit as a function of ligand concentration. For calculation of the KD for Tpx2 with saturating ADP bound, KD is near the concentration of fluorescent protein. Ligand depletion was accounted for by fitting the raw data to determine the plateau FD/FA value (representing ligand saturation), calculating the percent saturation for each total ligand concentration, and then back calculating the concentration of free ligand. FD/FA was then re-fit as a function of free ligand concentration.Dissociation constants Ko, with a GFP band pass filter in place (Semrock).The time-correlated single photon counting instrument used to collect time-resolved fluorescence data has been previously described . The ins2. Experiments were performed at 100–200 nM unphosphorylated or phosphorylated FRET-labeled AurA, in the presence and absence of 100–125 μM Tpx2 and 0.2–1 mM ADP or AMPPNP; one phosphorylated AurA experiment also contained 1 mM DTT. For both unphosphorylated and phosphorylated AurA, three independent experiments were performed and analyzed.Experimental buffer contained 15 mM HEPES pH 7.5, 20 mM NaCl, 1 mM EGTA, 0.02% Tween-20, 10 mM MgClData fitting was performed as previously described . Briefly2O in D2O. For DEER experiments, samples containing 30–60 μM MTSL-labeled AurA were prepared in the presence and absence of 100–200 μM Tpx2 and 300 μM ADP (8 mM MgCl2 was added to samples containing ADP). Final samples varied in v/v H2O concentration from 2–14%; however, no significant differences were observed in Tikhonov distributions derived from experiments performed in protonated and deuterated buffers. Samples were flash-frozen in an isopropanol dry ice bath followed by liquid nitrogen. Data shown are from one of two replicate experiments.DEER samples were prepared using the Cys-lite mutant construct of AurA C290S/A C393S C247A L225C S284C (or S283C), purified as described above. AurA was labeled with MTSL (Santa Cruz Biotechnology), purified by cation exchange chromatography, and concentrated. Labeling was verified using mass spectrometry, and MTSL-labeled samples retained close to full activity of unlabeled AurA in the presence and absence of Tpx2 . The proDEER spectra were detected at 65 K using an Elexsys E580 Q-Band spectrometer (Bruker Biospin) equipped with an ER 5107D2 resonator (Bruker Biospin) using the standard 4-pulse pulse sequence with π/2 and π pulses (including ELDOR) set to 16 and 32 ns respectively. The pump frequency was set to the central resonance position of the nitroxide echo-detected field swept spectrum while the observe position was set 24 G up-field to avoid excitation bandwidth overlap .Data were analyzed using custom software written in Mathematica which was based heavily on DeerAnalysis 2017 . The rawhttps://github.com/pandegroup/pdbfixer) version 1.2 was used to model in Tpx2 residues 23–29 (unresolved in 1OL5), add hydrogens belonging to standard dominant protein residue protonation states at pH 7.4, and remove phosphorylation from threonine residues 287 and 288 (WT AurA in complex with ADP was simulated with and without Tpx2. All simulations were started from the X-ray structure of WT AurA bound to Tpx2 and ADP in the presence of three magnesium ions (PDB ID: 1OL5 ). From t and 288 . Crystal and 288 . The pro and 288 .https://github.com/choderalab/AurA-materials), and subsequently converted into a hydrogen specification file for OpenMM’s Modeller by hand. The PDB file generated by PDBFixer was loaded into an OpenMM Modeller object where the hydrogens and bonds were added to the TPO residues using a Forcefield object instantiated with the AMBER99Bildn parameters as well as the custom TPO parameters described above. All crystallographic waters, ADP, sulfate ions and magnesium ions were handled as with the unphosphorylated WT AurA simulations.Simulations of phosphorylated WT AurA in complex with ADP were prepared as above, but the phosphothreonines (denoted TPO in the PDB file) at positions 287 and 288 were left in place and parameterized using the Sticht T1P AMBER parameters  retrieveAn OpenMM ForceField was instantiated using AMBER99SBildn force field parameters  for the −1. Nonbonded forces were modeled using the particle-mesh Ewald (PME) method with default parameters with a cutoff distance of 9.0 Å. All other settings remained at default values, except double precision was used throughout the minimization-and-equilibration process.Local energy minimization was performed in three separate steps in order to gradually introduce bond constraints. An OpenMM System was instantiated with no constraints on bonds or angles for the first minimization, which took place in vacuum  with no constraints on bonds or angles. After this minimization, a new System was instantiated with constraints on the lengths of all bonds involving a hydrogen atom, and minimization was repeated. The structure and positions of all atoms were then put into a new OpenMM Modeller object, where TIP3P waters were added to a cubic box extending 11 Å beyond the outermost protein atoms, along with neutralizing counterions and sufficient excess NaCl to achieve an effective salt concentration of 300 mM. Another System with constrained bonds to hydrogen was created from the solvated structure and minimized. To minimally relax the structure before deploying simulations to Folding@Home, 5000 steps of Langevin dynamics were run using a Langevin integrator with a time step of 2.0 fs, temperature of 300.0 K, and collision rate of 5.0 psThe resulting system, integrator, and state data from minimal equilibrations were serialized to XML format for simulation on Folding@Home using a simulation core based on OpenMM 6.3  for bothhttp://www.charmm-gui.org/?doc=input/solvator). In the first stage of this tool, all crystal waters and magnesiums in the structures where retained, while sulfates were deleted. Tpx2 was deleted at this stage for the non-Tpx2 conditions. Phosphorylation was either built in or deleted for the threonine at residue 288 in the second stage of the Solvator tool. Also in this stage, residues 225 and 284 were mutated to cysteines and the MTSL spin label (named CYR1) was added to those residues. C290 was mutated to serine in the unphosphorylated conditions and alanine in the phosphorylated conditions, to match the DEER experimental conditions. After the PDB file was generated, a rectangular solvent box was generated using 10 Å edge distance fit to the protein size, with 300 mM NaCl placed using the Monte-Carlo method.Because AMBER parameters for spin probes were not widely available, we used the equivalently modern CHARMM36 generation of forcefields for MTSL-labeled AurA, simulated in complex with ADP and with and without both TPX2 and phosphorylation, for a total of four possible combinations per starting structure. Simulations were started from two different starting configurations: DFG-in (PDB ID: 1OL5) and DFG-out (5L8K ). For thAn OpenMM ForceField was instantiated using CHARM36  force fi−1. Nonbonded forces were modeled using the particle-mesh Ewald (PME) method with a cutoff distance of 9.0 Å. All other settings remained at default values, except mixed precision was used throughout. After this, a second equilibration was run using 500000 steps of NPT dynamics using a Langevin integrator with a temperature of 300 Kelvin, collision rate of 90 ps−1, and timestep of 2.0 fs. A Monte Carlo Barostat was used with pressure of 1 atm and barostat update interval of 50 steps. To minimally relax this structure, 500000 steps of Langevin dynamics were run using a Langevin integrator with a two fs time step, 300 K temperature, and a collision rate of 5 ps−1.A local energy minimization was performed with no constraints on bonds or angles, which took place in the solvated water box output by CHARMM-GUI and loaded into an OpenMM object. After minimization, 5000 steps of NVT dynamics were run using a Langevin integrator with a time step of 1.0 fs, temperature of 50K and a collision rate of 90.0 psThe resulting system, integrator, and state data from the minimization and equilibration were serialized to XML format for simulation on Folding@Home using a simulation core based on OpenMM 6.3. This was done for all eight conditions, where each combination of starting structure, phosphorylation status and Tpx2 status was set up as a RUN. For each of the RUNs, 100 CLONEs with different initial random velocities and random seeds were simulated on Folding@home, where each clone ran for a maximum of 3 μs . In aggregate, each of the 12 configurations totaled between 75-110μs per starting configuration.https://doi.org/10.5281/zenodo.883859) distplot using the norm_hist parameter. The distance and dihedral contour plots were generated using the kdeplot function in Seaborn v0.8.1. All analysis scripts have been made publically available .Thank you for submitting your article "A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation" 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.The reviewers agree that this is a very detailed analysis of the dynamic features of the activation loop of an important protein kinase, and in particular of the DFG motif. It is more comprehensive than any previous study and also introduces new concepts for thinking about this important conserved feature of every protein kinase. The spectroscopic studies are well done and informative and some of the technologies are new. However, the overall organization of the paper is a weakness and the major concepts were not clearly articulated and in some cases appear contradictory. Most importantly, it was felt that the DFG motif cannot be considered independently of the other concerted and correlated motions that are mediated by the C-Helix and by the Tpx2 motif that docks directly onto the C-Helix. It is felt that key issues were lost in the interpretation of the results and in the discussion. To submit a revised manuscript there would need to be major revisions as outlined below. Given the potential importance of these findings we would be willing to consider a substantially revised manuscript.1) A major concern has to do with the relative roles of Tpx2 and phosphorylation in vivo. You note that Tpx2 operates at the mitotic spindle, where the kinase is kept unphosphorylated by the action of PP6, whereas at the centrosome activation is by phosphorylation at T288 on the activation loop, implying two distinct activation mechanisms. There was no discussion of what Tpx2 actually does in addition to recruiting a phosphatase. Your studies, as well as earlier literature, indicate that Tpx2 and phosphorylation can act synergistically in vitro, but it is not entirely clear whether phosphorylation alone or another factor that serves a role similar to Tpx2 might operate at the centrosome. A revised manuscript would require a clear mechanistic discussion concerning the role of activation loop phosphorylation and Tpx2 docking onto the C-Helix, which are correlated motions.2) The reviewers feel that the kinetic data in the last part of the paper are not particularly solid. For example, the stopped-flow experiments lead to the model that the DFG-Out configuration is needed for nucleotide release and could be an intermediate step in the catalytic cycle. However, these experiments were performed in the absence of a peptide substrate. What happens under conditions where peptide is present? Overall, the arguments regarding a possible role of the "out" conformation in nucleotide release are not sufficiently substantiated, so we would ask that these portions be dropped from the manuscript unless they are fortified with more extensive measurements with peptide substrate.3) The activation segment clearly serves to activate kinases, but phosphorylation also provides the docking site for substrates. It is conceivable that binding of a specific substrate will stabilize the DFG-in conformation further and this effect could be dependent on a particular substrate. MYC for instance has been recently described as a substrate that interacts in a fashion that is dependent on the DFG conformation (DFG-out inhibitors displace it). Thus, it is likely that the dynamic properties will change in the presence of specific substrates.4) Another concern is that ADP and not ATP was used here; this is convenient as ATP will probably lead to additional autophosphorylation during the measurements. However, the γ phosphate, as well as the second metal ion, forms important interactions with the DFG and will almost certainly influence its dynamic properties, especially if the phosphatase is also nearby. ADP concentrations (50-100 uM) are significantly lower than ATP (1-5 mM) concentrations in cells. While AMP-PNP, a non-hydrolyzable ATP analog may not completely mimic ATP, it would be better that ADP. Why was it not used? These issues should be discussed to address the concern that the proposed model is more generally relevant than just to the narrow conditions studied here.5) The conclusion that the DFG-In population doesn't shift with phosphorylation is based in part on the nitrile-labeled Q185C data in 6) More explicit reference to earlier validation of the spectroscopic techniques, including effects (or lack thereof) on enzyme activity by the introduction of cysteines and probes, use of ADP vs. ATP as a cofactor, etc. should be provided. What new techniques and data are introduced here?7) To state that regulation of Aurora A by phosphorylation and by Tpx2 are normally mutually exclusive is a simplification that is not substantiated by your data. Without more data and information on the time scales and physiological partners of Aurora A under different conditions, this statement seems to be an over simplification.8) Another concern is with the final statement in the discussion regarding the evolution of kinases. The evolutionary pressure for kinases is probably not to optimize for catalytic efficiency (i.e. substrate binding and product release) but rather to optimize for regulation via a switch mechanism, which is what you are characterizing here.eLife. Your revised article has been favorably evaluated by Philip Cole (Senior Editor), a Reviewing editor, and two reviewers.Thank you for resubmitting your work entitled "A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The reviewers agree that your revised manuscript is substantially improved, including the addition of new data with AMP-PNP and the removal of the kinetic data, focusing on the dynamic features of the DFG motif and the synergy between the activation loop and the TPX2 docking motif. This is a comprehensive analysis of the dynamic features of these regions that converge at the active site and provides a foundation for others to think about this region. The use of multiple biophysical techniques is a great strength. Although each kinase is regulated in unique ways, all involve the critical coupling of these regions, and these motions are tightly regulated. The results are thus highly relevant to a large set of investigators in the signaling area.Having said this, the reviewers feel that interpretation of the results need further revision; these do not require additional experiments:1) The questions addressed in point 3 in the rebuttal letter highlight the peptide substrates and in fact talk about substrates likely diffusing in and out of the active site. These substrates bind weakly (Km 10 mM) so it is not likely that these are physiologically relevant. Indeed, experiments at high peptide concentration indicate no changes on the activation segment conformation. It is important to emphasize that in cells the substrates are not peptides, but proteins that are often tethered to a signaling complex by motifs that are not at the active site. Such substrates can have docking sites that increase their local concentration; if they are tethered by an additional site they are not limited by diffusion once the kinase is activated, even if it is activated in a very transient way. The authors should comment on this, as the whole system may be seen in a different way if one considers it as a tightly assembled signaling complex. In this scenario it is very likely that there could be an as yet unidentified functional equivalent of Tpx2 in the mitotic spindle.2) The authors note that MYC binds as a pseudosubstrate to AurA. It has, however, also been shown that type II inhibitors displace MYC, thus there is presumably tight coupling between the activation segment and substrate binding. It would be good to discuss this a bit more.3) The authors argue that the "…C-helix is likely also coupled to these elements, but we note that the scale of the conformational changes of the C-helix in AurA is small (as is the case in the entire AGC lineage due to the constraining influence of the B-helix in these proteins)". This is not evident from crystal structures that show flexibility of the alphaC helix. Admittedly, these structures lack the N-terminus but if the N-terminus constrains alphaC movement this would also pose a problem for the construct used in the spectroscopic data that uses a similar boundary. The authors should attempt to reconcile this issue in the discussion.4) The synergy between the two regions – the TPX2 docking site (referred to as the hydrophobic motif in other AGC kinases) and the activation loop – is a fascinating conclusion. While this synergy is critical in all kinases and highly regulated, it is not easily trackable in other kinases. Aurora A with its trans mechanism for docking to the hydrophobic motif is an exceptional model system to tease apart this synergy but the results are in general likely to be applicable to other kinases. The authors should at least pose the question of whether phosphorylation of the activation loop actually "drives" the assembly of the active kinase or "facilitates" the assembly of the active kinase.5) The authors should state clearly that the driving force for evolution of the protein kinases is likely to be regulation and not highest efficiency catalysis.6) With regard to correlated motions of the C-Helix and the Hydrophobic Spine, we think it is important to include this motif in your analyses as these all seem to be related motions, including the water molecules that you describe. Even though the C-Helix does not substantially move in the AGC kinases, as you point out, it is the alignment of the spine residues that is critical and in the case of the AGC kinases there has been much attention paid to the "capping" of the R-spine with the hydrophobic motif – a process that is highly regulated in unique ways for each AGC kinase. With Aurora A you have a very unique system which allows you to tease apart the allosteric features of the hydrophobic motif as it comes from a different protein. However, the concepts are likely to be similar. It would at least be useful to see one figure that compares the DFG motif and the water molecules, as well as the second Mg ion, with the R-spine in the DFG-In and DFG-out position – even if it is in the supplement.7) There is a question that relates to the C185 position, which is where you place one of the labels. This residue clearly is packed against the DFG phenylalanine in the DFG-out position. Cys itself is quite hydrophobic as is your probe. You demonstrate that the properties of the modified protein resemble those of the wt protein. However, do you know what happens if you replace this Cys with an Ala? Is this an activating mutation? It would seem that the unique position of this Cys might contribute to maintaining the DFG out inhibited state. Is this a possibility? The reviewers agree that this is a very detailed analysis of the dynamic features of the activation loop of an important protein kinase, and in particular of the DFG motif. It is more comprehensive than any previous study and also introduces new concepts for thinking about this important conserved feature of every protein kinase. The spectroscopic studies are well done and informative and some of the technologies are new. However, the overall organization of the paper is a weakness and the major concepts were not clearly articulated and in some cases appear contradictory. Most importantly, it was felt that the DFG motif cannot be considered independently of the other concerted and correlated motions that are mediated by the C-Helix and by the Tpx2 motif that docks directly onto the C-Helix.We never meant to suggest that the DFG motif can be considered independently of the motions of the activation loop and C-helix. Indeed, we take the striking similarity in the IR and FRET/EPR data – which all show similar nucleotide- and Tpx2-mediated conformational shifts – as a clear indication that conformational changes of different structural elements are tightly coupled across the kinase domain. We concur that the C-helix is likely also coupled to these elements, but we note that the scale of the conformational changes of the C-helix in AurA is small (as is the case in the entire AGC lineage due to the constraining influence of the B-helix in these proteins) and therefore challenging to measure directly, and we have therefore relied on the DFG motif and activation loop as readouts of the overall conformational state of the kinase.It is felt that key issues were lost in the interpretation of the results and in the discussion. To submit a revised manuscript there would need to be major revisions as outlined below. Given the potential importance of these findings we would be willing to consider a substantially revised manuscript.We regret that the original manuscript was confusing in places. We believe that the removal of the kinetics section as suggested by the reviewers, and our refocusing of the discussion on the key structural questions, particularly how phosphorylation triggers a structural rearrangement within the DFG-In state, have simplified the revised manuscript and improved the clarity and focus. We believe that the central result of the paper, that phosphorylation alters the structure of the DFG-In state to bring about activation, is now much more prominently and clearly articulated.1) A major concern has to do with the relative roles of Tpx2 and phosphorylation in vivo. You note that Tpx2 operates at the mitotic spindle, where the kinase is kept unphosphorylated by the action of PP6, whereas at the centrosome activation is by phosphorylation at T288 on the activation loop, implying two distinct activation mechanisms. There was no discussion of what Tpx2 actually does in addition to recruiting a phosphatase. Your studies, as well as earlier literature, indicate that Tpx2 and phosphorylation can act synergistically in vitro, but it is not entirely clear whether phosphorylation alone or another factor that serves a role similar to Tpx2 might operate at the centrosome. A revised manuscript would require a clear mechanistic discussion concerning the role of activation loop phosphorylation and Tpx2 docking onto the C-Helix, which are correlated motions.The reviewers raise a good point. Our assertion that the roles of Tpx2 and phosphorylation were largely independent in vivo was based on considerable previous work by other labs. For instance, the work of Toya et al., 2011 showed that spindle-associated AurA is not phosphorylated on T288, and that a T288A mutant of AurA does not block the spindle functions. Further, work by Zeng et al., 2010 has confirmed that the major population of AurA on the spindle is not phosphorylated on T288 due to the action of the PP6 phosphatase. Similarly, studies of Tpx2 localization and knockdowns  showed that Tpx2 is not present at the centrosome, does not interact with centrosomal AurA and is not required for its centrosomal localization and function. These in vivo studies formed the basis for our assertion that the two activation mechanisms are independent. We have edited the Introduction section to more clearly describe these points.We concur with the reviewers that our biochemical data showing striking synergy between Tpx2 and phosphorylation could be considered puzzling in light of the in vivo work, and might potentially indicate the existence of a small but functionally-important cellular pool of AurA that is activated by both Tpx2 and phosphorylation. In light of this we have toned down the assertion of the independence of the Tpx2 and phosphorylation mechanisms in the revised Discussion section. However, we note that the presence of both synergy and independence in the actions of Tpx2 and phosphorylation was previously reported by Dodson and Bayliss, 2012 (“Activation of Aurora-A kinase by protein partner binding and phosphorylation are independent and synergistic”). We now include a new section at the end of the Results that describes how our model for a novel autoinhibited DFG-In substate provides an explanation for the synergy: disruption of the autoinhibited state by phosphorylation promotes the assembly of the Tpx2 binding site.Finally, we have further amended the Discussion section of the manuscript to make the following important connection with the related AGC family kinases and our previous work, which provides some context for considering the relative synergism versus independence of phosphorylation and Tpx2. Our previous work  showed that the ability of AurA to be strongly activated by either phosphorylation alone or by Tpx2 alone (at least in vitro) was due to an enhanced regulatory spine in the Aurora kinase lineage involving a unique polar residue that coordinates a water-mediated hydrogen bond network in the active site. We showed that removal of this feature by mutagenesis, essentially converting AurA into an AGC-like kinase, prevented individual activation of AurA by phosphorylation or Tpx2 alone, such that both were required simultaneously for activation. This is analogous to the situation found in the AGC kinases where hydrophobic motif engagement and phosphorylation occur as a concerted activation process. We believe it likely that the emergence of the unique hydrogen bond network in AurA was necessary to allow for independent activation of spindle- and centrosome-associated AurA pools. We have now included a discussion of this point in the revised manuscript.We certainly concur with the reviewers that the existence of a Tpx2-like element that further modulates AurA activity at the centrosome is possible; while identification of this is outside of the scope of this work, we have mentioned the possibility of further modulation of centrosomal AurA by protein-protein interactions in the revised Discussion section.2) The reviewers feel that the kinetic data in the last part of the paper are not particularly solid. For example, the stopped-flow experiments lead to the model that the DFG-Out configuration is needed for nucleotide release and could be an intermediate step in the catalytic cycle. However, these experiments were performed in the absence of a peptide substrate. What happens under conditions where peptide is present? Overall, the arguments regarding a possible role of the "out" conformation in nucleotide release are not sufficiently substantiated, so we would ask that these portions be dropped from the manuscript unless they are fortified with more extensive measurements with peptide substrate.We agree with the reviewers that the kinetic data were a weakness of the original paper. Although we maintain that the question of the functional role of the large DFG-Out subpopulation of phosphorylated AurA is an important and interesting question, we concur that this is outside the scope of the current work, and that the inclusion of the kinetic data unnecessarily complicated the manuscript and led to confusion. We have therefore decided to remove the kinetic section and refocus the manuscript on the key structural effects of phosphorylation.3) The activation segment clearly serves to activate kinases, but phosphorylation also provides the docking site for substrates. It is conceivable that binding of a specific substrate will stabilize the DFG-in conformation further and this effect could be dependent on a particular substrate. MYC for instance has been recently described as a substrate that interacts in a fashion that is dependent on the DFG conformation (DFG-out inhibitors displace it). Thus, it is likely that the dynamic properties will change in the presence of specific substrates.The reviewers bring up an excellent point about the potential role of substrates in modulating AurA conformation. We performed several experiments in an attempt to dissect the effects of substrate binding on the conformational dynamics of AurA. Unfortunately, the available consensus substrate peptides for AurA are relatively poor, with measured Km values in the millimolar range. We in fact performed IR, FRET and EPR experiments in the presence of 10 mM of the substrate peptide kemptide, which conforms to the consensus sequence determined for AurA by Ferrari et al., 2005, and found no effect on the conformation of the kinase using any of these methods. We have included the EPR data in the revised paper . We thinWe note that MYCN actually binds as a pseudosubstrate to AurA, and although it binds tightly and does indeed trap the kinase in the active state, as shown by the work of Richards et al., 2016, it is important to appreciate that this binding is a non-catalytic scaffolding function of AurA, and does not reflect a normal kinase:substrate interaction.4) Another concern is that ADP and not ATP was used here; this is convenient as ATP will probably lead to additional autophosphorylation during the measurements. However, the γ phosphate, as well as the second metal ion, forms important interactions with the DFG and will almost certainly influence its dynamic properties, especially if the phosphatase is also nearby. ADP concentrations (50-100 uM) are significantly lower than ATP (1-5 mM) concentrations in cells. While AMP-PNP, a non-hydrolyzable ATP analog may not completely mimic ATP, it would be better that ADP. Why was it not used? These issues should be discussed to address the concern that the proposed model is more generally relevant than just to the narrow conditions studied here.We did include steady-state FRET experiments with AMPPNP in the original manuscript, and observed very similar results to those with ADP. However, given the importance of this question we agreed with the reviewers that we needed to repeat the other experiments with AMPPNP as well. We have now repeated both the time-resolved FRET and DEER experiments with AMPPNP and observe very similar results to those obtained with ADP . In partAlthough it might seem surprising that coordination of Mg-ATP by the DFG Aspartate does not trap the kinase in the DFG-In state, it arguably makes sense from the standpoint of using the DFG equilibrium for regulatory control, as Tpx2 does. If ATP binding was tightly coupled to the DFG equilibrium, then the high concentration of ATP in the cell would force AurA into the DFG-In state, overriding any ability of Tpx2 or other factors to influence the DFG equilibrium. Instead, nature has ensured that nucleotide is only weakly coupled to the DFG equilibrium, so that binding and release can occur without overriding the regulatory status of the enzyme.5) The conclusion that the DFG-In population doesn't shift with phosphorylation is based in part on the nitrile-labeled Q185C data in The reviewers are absolutely correct that the kinase activity of the nitrile-modified enzyme is minimal without Tpx2, and that this could in principle be a sign of a deleterious effect of the labeling on the conformational equilibrium. Indeed, we would not seek to conclude solely from the IR data that the DFG equilibrium is not shifted by phosphorylation. However, the conformation of the DFG motif is tightly coupled to that of the activation loop, as is established from numerous x-ray structures of kinases as well as our results with Tpx2, and the TR-FRET and DEER data do both support the interpretation that phosphorylation has minimal effect on the conformational equilibrium of the activation loop. Indeed, the slight difference between the DEER distributions of AurA:ADP with and without phosphorylation mentioned by the reviewers see : the DFGWe agree that the inclusion of the kinetic data led to confusion and have removed this section from the revised manuscript and refocused the discussion on how phosphorylation alters the structure of the DFG-in state of AurA to activate the kinase, the central conclusion of this work.We did attempt to fit the distance distributions to Gaussian functions as suggested by the reviewers to estimate the degree to which the DFG-Out and DFG-In states are populated in each sample. However, because of the complexity of the Tikhonov-derived distance distributions, with three to four distinct peaks contributed by each structural state due to alternative rotamers of the spin probes, this fitting procedure is problematic and we did not find the results to be very reliable or informative. We decided to focus instead on more qualitative descriptions of the distributions which nonetheless serve to illustrate the central points that a) the conformational ensemble of phosphorylated AurA is heterogeneous in the absence of Tpx2 and b) phosphorylation alters the structure of the DFG-In state as reflected in the contribution of a 52-Å distance to the distribution.We feel that in the revised manuscript the apparent inconsistencies between the different methods have been resolved. Specifically, all three experimental methods strongly support the key observations that:A) Unphosphorylated and phosphorylated AurA reside in a similar conformational equilibrium between DFG-Out and DFG-In states.B) Tpx2 binding is required for both unphosphorylated and phosphorylated AurA to homogeneously adopt the DFG-In state.6) More explicit reference to earlier validation of the spectroscopic techniques, including effects (or lack thereof) on enzyme activity by the introduction of cysteines and probes, use of ADP vs. ATP as a cofactor, etc. should be provided. What new techniques and data are introduced here?E. coli, and the crystal structure of the Cys-lite construct determined by Burgess et al., 2015 which was essentially indistinguishable from the WT. We have now cited these advances in the Materials and methods section.We have edited the manuscript to ensure that the validation data are clearly presented and previous work appropriately cited throughout. We have now included additional information on the preparation of the phosphorylated Q185CN form of AurA for IR in 7) To state that regulation of Aurora A by phosphorylation and by Tpx2 are normally mutually exclusive is a simplification that is not substantiated by your data. Without more data and information on the time scales and physiological partners of Aurora A under different conditions, this statement seems to be an over simplification.As discussed above in response to point 1, we concur that we may have overstated this point, which was based on the in vivo work of other groups and not on our own data, which indeed could be seen to potentially support a physiological role for Tpx2 and phosphorylation acting together. We have edited the manuscript to tone down this assertion and removed the “mutually exclusive” phrase. We have also included in the revised Discussion section a comparison between the Tpx2/phosphorylation mechanisms of AurA and the activation mechanisms of the related AGC family kinases. We feel this discussion helps put our work in context and more accurately describes the dual activation mechanisms of AurA as a variation on a theme rather than an entirely unique property.8) Another concern is with the final statement in the discussion regarding the evolution of kinases. The evolutionary pressure for kinases is probably not to optimize for catalytic efficiency (i.e. substrate binding and product release) but rather to optimize for regulation via a switch mechanism, which is what you are characterizing here.We agree with the reviewers that the primary selective pressure operating on kinases is regulatory, and in the light of the removal of the kinetics section and the refocusing of the manuscript on the structural story, we have amended this last section of the Discussion. We do think that the potential function of the DFG-Out subpopulation is an intriguing question, but agree that this should be left for a future analysis.1) The questions addressed in point 3 in the rebuttal letter highlight the peptide substrates and in fact talk about substrates likely diffusing in and out of the active site. These substrates bind weakly (Km 10 mM) so it is not likely that these are physiologically relevant. Indeed, experiments at high peptide concentration indicate no changes on the activation segment conformation. It is important to emphasize that in cells the substrates are not peptides, but proteins that are often tethered to a signaling complex by motifs that are not at the active site. Such substrates can have docking sites that increase their local concentration; if they are tethered by an additional site they are not limited by diffusion once the kinase is activated, even if it is activated in a very transient way. The authors should comment on this, as the whole system may be seen in a different way if one considers it as a tightly assembled signaling complex. In this scenario it is very likely that there could be an as yet unidentified functional equivalent of Tpx2 in the mitotic spindle.This is an excellent point and we have amended the Discussion section to include a sentence pointing out how complex formation and substrate docking in vivo may further modulate the dynamics of the phosphorylated enzyme.2) The authors note that MYC binds as a pseudosubstrate to AurA. It has, however, also been shown that type II inhibitors displace MYC, thus there is presumably tight coupling between the activation segment and substrate binding. It would be good to discuss this a bit more.We have included a mention of the NMYC pseudo substrate interaction and how it modulates the kinase dynamics in the revised Discussion section, in the same section that deals with the broader issue of substrate docking raised in point 1.3) The authors argue that the "…C-helix is likely also coupled to these elements, but we note that the scale of the conformational changes of the C-helix in AurA is small (as is the case in the entire AGC lineage due to the constraining influence of the B-helix in these proteins)". This is not evident from crystal structures that show flexibility of the alphaC helix. Admittedly, these structures lack the N-terminus but if the N-terminus constrains alphaC movement this would also pose a problem for the construct used in the spectroscopic data that uses a similar boundary. The authors should attempt to reconcile this issue in the discussion.We have amended the Discussion section of the manuscript to further discuss the role of the aC-helix and how it is coupled to phosphorylation and Tpx2 binding.To be clear we did not intend to suggest above that the aC-helix doesn’t move at all in AurA, but rather to draw a clear distinction between the dynamics in AurA/AGC kinases versus in the classes of kinases like the CDKs and Src family in which the aC-helix undergoes a large-scale movement into an alternative, stable, and heavily-populated autoinhibitory state. The latter mode of motion is inhibited by the B-helix in AurA/AGC kinases. The reviewers are completely correct that the aC-helix may still be “dynamic” in AurA/AGC kinases, as evidenced by disorder in crystal structures. Indeed we detected these dynamics in our earlier molecular dynamics work  and found that they were suppressed by Tpx2. The smaller magnitude of the motions characteristic of these latter “dynamics” simply make it difficult to study them experimentally by e.g. DEER spectroscopy, which is why we have not measured these dynamics experimentally. This would be an interesting topic for a future work but is clearly outside the scope of the current study.4) The synergy between the two regions – the TPX2 docking site (referred to as the hydrophobic motif in other AGC kinases) and the activation loop – is a fascinating conclusion. While this synergy is critical in all kinases and highly regulated, it is not easily trackable in other kinases. Aurora A with its trans mechanism for docking to the hydrophobic motif is an exceptional model system to tease apart this synergy but the results are in general likely to be applicable to other kinases. The authors should at least pose the question of whether phosphorylation of the activation loop actually "drives" the assembly of the active kinase or "facilitates" the assembly of the active kinase.We believe that our data, particularly the EPR results, show that phosphorylation really drives the assembly of the active state, albeit without stabilizing the active state relative to the DFG-Out state, which is in and of itself remarkable. The way we prefer to think about it is that the data argue that phosphorylation acts by altering the detailed shape of the conformational energy landscape for the DFG-In state, rather than by changing the depth of the free energy well (with respect to that for the DFG-Out state). We have amended the wording in the Discussion to clarify this point.5) The authors should state clearly that the driving force for evolution of the protein kinases is likely to be regulation and not highest efficiency catalysis.We have amended the Discussion to make this point explicit.6) With regard to correlated motions of the C-Helix and the Hydrophobic Spine, we think it is important to include this motif in your analyses as these all seem to be related motions, including the water molecules that you describe. Even though the C-Helix does not substantially move in the AGC kinases, as you point out, it is the alignment of the spine residues that is critical and in the case of the AGC kinases there has been much attention paid to the "capping" of the R-spine with the hydrophobic motif – a process that is highly regulated in unique ways for each AGC kinase. With Aurora A you have a very unique system which allows you to tease apart the allosteric features of the hydrophobic motif as it comes from a different protein. However, the concepts are likely to be similar. It would at least be useful to see one figure that compares the DFG motif and the water molecules, as well as the second Mg ion, with the R-spine in the DFG-In and DFG-out position – even if it is in the supplement.We have added a new figure to the supplement  showing 7) There is a question that relates to the C185 position, which is where you place one of the labels. This residue clearly is packed against the DFG phenylalanine in the DFG-out position. Cys itself is quite hydrophobic as is your probe. You demonstrate that the properties of the modified protein resemble those of the wt protein. However, do you know what happens if you replace this Cys with an Ala? Is this an activating mutation? It would seem that the unique position of this Cys might contribute to maintaining the DFG out inhibited state. Is this a possibility?In our earlier work  we replaced the Q185 residue with several different amino acids found at that position in different ePKs. In total we tested Q185C, Q185N, Q185L, Q185M and Q185H. We found that all mutations were deleterious to the activity of the phosphorylated enzyme, but that the activity of most of the mutants could be largely or completely rescued by activating with both phosphorylation and Tpx2. Interestingly, Q185N was the most deleterious mutation, and could only be partially rescued. We did not test the Q185A mutation, as alanine is not commonly found at this position in ePKs. It is certainly possible that some of the effects of the substitutions on kinase activity are due to differentially stabilizing the DFG-Out and DFG-In states, but as described in our Nat Chem Biol paper, we attributed most of the effects to the disruption of the water-mediated hydrogen bond network nucleated by Q185. Given the generally good agreement between the IR data, obtained with the unnatural Q185CN probe, and the FRET and EPR data, obtained with a native Q185 residue in the active site, it seems likely that any perturbation of the DFG equilibrium by the probe is relatively minor, although we can’t rule out some effect. We have added a sentence to the IR section pointing out this caveat.


Drug hypersensitivity reactions (HSRs) are increasing in the 21st Century with the ever expanding availability of new therapeutic agents. Patients with cancer, chronic inflammatory diseases, cystic fibrosis, or diabetes can become allergic to their first line therapy after repeated exposures or through cross reactivity with environmental allergens. Avoidance of the offending allergenic drug may impact disease management, quality of life, and life expectancy. Precision medicine provides new tools for the understanding and management of hypersensitivity reactions (HSRs), as well as a personalized treatment approach for IgE (Immunoglobuline E) and non-IgE mediated HSRs with drug desensitization (DS). DS induces a temporary hyporesponsive state by incremental escalation of sub-optimal doses of the offending drug. In vitro models have shown evidence that IgE desensitization is an antigen-specific process which blocks calcium flux, impacts antigen/IgE/FcεRI complex internalization and prevents the acute and late phase reactions as well as mast cell mediator release. Through a “bench to bedside” approach, in vitro desensitization models help elucidate the molecular pathways involved in DS, providing new insights to improved desensitization protocols for all patients. The aim of this review is to summarize up to date information on the drug HSRs, the IgE mediated mechanisms of desensitization, and their clinical applications. Drug hypersensitivity reactions (HSRs) are adverse effects of drugs ,2. AmongThe classification of HSRs relies on the clinical presentation of typical symptoms and their timing ,7, and wCutaneous symptoms , as well as respiratory and gastrointestinal symptoms are the most common mast cell activation related clinical presentations. More severe reactions with vital sign changes, throat tightness, or swelling can also appear ,2,7,15. Reactions which occur during or within 1–6 h after the drug administration are classified as immediate reactions ,8. This Type IV reactions, classically known as delayed reactions, have a more heterogeneous presentation and typically appear several days or weeks after the exposure from the start date of drug administration ,2,8. TheThese phenotypes and endotypes can be further assessed through biomarkers such as skin testing (ST), and specific IgE and basophil activation tests (BAT), which help identify mast cell involvement in the HSRs as well as cross reactivity between drugs ,27,28,29Drug desensitization (DS) was developed due to the pressing need to reintroduce drugs in a safe fashion in patients who had developed IgE/non IgE type I HSRs to critical antibiotics and/or other drugs. The first reported case was in 1942 in an English soldier in urgent need of penicillin at a time when no alternatives existed; further advances were made in the 1980s when the first oral and intravenous protocols for penicillin were created. The safety and efficacy of penicillin desensitization was widely described without reports of deaths or anaphylaxis to such a degree that it was even used in high risk populations such as pregnant women who had prick positive penicillin allergy and required treatment for syphilis a 35,36],3635,36]Desensitization today is indicated when patients have HSRs mediated by mast cell activation to their first line therapy without comparable alternatives ,30,38,39Understanding the molecular mechanisms of the HSR, patient’s comorbidities, skin testing, and genetic markers are critical to determine whether desensitization is indicated as well as the potential risk of reaction during the procedure.DS has been established to be safe and effective for IgE mediated drug HSRs. Patients with immediate reactions to taxanes and other chemotherapies in which the IgE mechanism cannot be demonstrated have also been successfully desensitized ,6. HIV pAfter risk stratification, a flexible protocol with 4 to 16 steps  and escalating the dose 2 to 2.5 times every 15 min is applied. Usually the starting concentration of the solution in a 4 bags/16 steps protocol is 1/1000 to 1/100 in a 3 bags/12 steps protocol, reaching the target dose at the end of the procedure b 3,5,6,,36,42,44Recently, algorithms to manage HSRs through DS have been described for antibiotics and chemotherapies ,12,25,27While empiric desensitization protocols were established clinically to treat patients in need, the mechanisms of desensitization were evaluated in vitro to understand the cellular and moelcular players. The first in vitro studies occurred in animals; later, human basophils were used for proof of concept in vitro experiments by performing BAT (basophil activation test). Currently both in vivo and in vitro studies are used to understand the cellular and molecular pathways of mast cell and basophils involved in HSRs and anaphylaxis. A major step in improving clinical desensitization protocols was insight from in vitro studies on how dosing and timing during the desensitization protocol inhibited cell degranulation and cytokine production.Mast Cells (MCs) are key effectors in many immune responses including IgE and non-IgE mediated HSRs and are believed to be the primary target cell in DS .During HSRs, MCs can be activated through IgE or non-IgE dependent pathways. Recent studies have shown that small molecule drugs may induce systemic non-IgE mediated reactions through the activation of human MC G-protein coupled receptor (MrgprX2) ,22. The 2. For allergen recognition, the α chain of the receptor binds to IgE through the two extracellular Ig-like domains on the Fc region of the antibody. The membrane-tetraspanning β chain and the two disulfide-linked identical γ chains contain a single immune-receptor tyrosine-based activation motif (ITAM) which is responsible for signal transduction. Phosphorylation of γ-subunit ITAMs is important in initiating and inducing downstream propagation of the intracellular signaling. On the other hand, phosphorylation of β-subunit ITAM has been suggested to have an amplifier and/or a suppressor function for the γ chain mediated signaling events. The Lyn tyrosine kinase is constitutively associated with the cytoplasmic tail of the FcεRI β chain .,53.45,53Furthermore, challenging with the same antigen after being desensitized does not induce activation , howeverFurthermore, the treatment of desensitized cells with calcium ionophore A23187 or non-desenstizing antigen resulted in a high level of b-hexosaminidase release and calcium flux; indicating that desensitized cell mediators were not depleted and the non-IgE–mediated activation pathway was intact after DS . In summInitial studies indicated that MC unresponsiveness after desensitization was due to internalization of FcεRI through progressive cross-linking at low antigen concentration, indicating that cell surface receptors were depleted before the next dose . More reIn IgE mediated MC activation, the antigen valency and dose have been demonstrated to be key factors directly affecting FceRI behavior. Low doses of multivalent-antigen can create small clusters of Ag/IgE/FcεRI complexs which keep the receptors mobile on the cell surface. However, high doses or valency, result in larger cluster aggregates of receptors which induces their immobilization and subsequence internalization ,64. Whetgp49B1 (LILRB4) receptors belong to the immunoglobulin (Ig) superfamily; which has two extracellular Ig-like domains and two ITIMs in its cytoplasmic domain. Colligation of gp49B1 (LILRB4) to crosslinked FcεRI will counter-regulate IgE-dependent MC activation in an ITIM dependent manner which has been evident in both in vitro and in vivo models. Mutation or deletion of gp49B1 (LILRB4) ITIM prevents the inhibition of MC activation. ITIM phosphorylation is required to recruit protein tyrosine phosphatases SH2-containing tyrosine phosphatase SHP 1, needed to dephosphorylate key signaling molecules, and consequently dampen the cellular response ,66. FcγRPrevious studies have shown that the inositol phosphatase SHIP can acts as a “gatekeeper” and negatively regulates MC degranulation for all antigen doses . In addiSubthreshold antigen doses induce a small amount of intracellular calcium mobilization in MCs ,53,54. DRecent studies have shown the participation of actin cytoskeleton reorganization in calcium mobilization in many cells including MCs. Challenges with the same antigen after desensitization does not induce β-hexosaminidase release, calcium mobilization, or rearrangement of actin filaments ,53. The The classical description of drug HSRs from Gell and Coombs is now complemented by the new understanding of phenotypes, endotypes, and corresponding biomarkers. This allows an expanded reaction classification, such as “cytokine storm-like reactions” to be recognized in HSRs to moAbs, oxaliplatin, and taxanes. New biomarkers from mast cells and other immune cell mediators , Phospholipase A(PFA)) will be added in the future to allow for better categorization .Desensitization is a revolutionary approach for the safe reintroduction of immunogenic drugs. Mast cells and basophils have long been known to be the cellular targets involved in desensitization; however the inhibitory mechanisms of desensitization are still being elucidated. DS takes advantage of inhibitory mechanisms which prevent activated mast cell signal transduction and pro-inflammatory mediator release.2+ channels, and signaling molecules. This compartment is specific to the antigen and excludes non-desensitized receptors (Impairment of Ag/IgE/FεcRI complex internalization during desensitization can lead to these complexes remaining mobile on the cell surface. Low antigen doses administered incrementally along with actin remodeling could lead to an aggregation of these mobile complexes forming a secluded compartment of desensitized antigen specific receptors, Caeceptors .Successful human DS protocols are based on in vitro IgE mast cell desensitization models and provide outstanding safety for all patients with severe allergic reactions in need of first line therapies. Future research will uncover the molecular pathway of in vitro desensitization which will permit more effective and safer human protocols.


The occurrence of late-glacial/deglacial sea ice maxima are consistent with sea ice/land ice hysteresis and land−glacier retreat via the temperature−precipitation feedback. We also identify interactions of sea ice with phytoplankton growth and ocean circulation patterns, which have important implications for glacial North Pacific Intermediate Water formation and potentially North Pacific abyssal carbon storage.Sea ice and associated feedback mechanisms play an important role for both long- and short-term climate change. Our ability to predict future sea ice extent, however, hinges on a greater understanding of past sea ice dynamics. Here we investigate sea ice changes in the eastern Bering Sea prior to, across, and after the Mid-Pleistocene transition (MPT). The sea ice record, based on the Arctic sea ice biomarker IP Modelling studies propose sea ice to be one of the underlying mechanisms for the Mid-Pleistocene transition. Here, the authors show Mid-Pleistocene subarctic North Pacific sea ice dynamics based on biomarkers and biogenic opal accumulation rates, supporting the importance of sea ice for climate change. However, it is only through the identification of long-term sea ice dynamics that our understanding of the role of sea ice for climate change can improve and hence our ability to predict future sea ice extent.Sea ice plays a key role in both long-term4, reaching its maximum extent approximately at the shelf edge  formation as a result of brine rejection during sea ice freezing. Today, NPIW is formed in the Sea of Okhotsk, whereas glacial NPIW (GNPIW) was at least partly formed in the Bering Sea11. Increased ventilation and extent of GNPIW during cold phases12 demonstrates the importance of the Bering Sea for glacial oceanic circulation patterns in the North Pacific realm and beyond12. Investigating the interactions of sea ice dynamics with ocean circulation and productivity patterns and identifying the role of sea ice for major climate transitions is critical for our understanding of Arctic and sub-Arctic climate. One such climate transition during the Quaternary Period is the Mid-Pleistocene transition .In modern times, sea ice in the Bering Sea forms in the Chukchi Sea and in polynyas along the southward facing coastlines on the eastern Bering Sea shelf. Thereafter, sea ice is advected south-westwarddge Fig. . Nutrien16, yet the change in G/IG frequency occurs without any attributable change in orbital forcing. Thus, the MPT marks a shift in the response of the climate system to orbital forcing, likely caused by internal climate mechanisms. Conceptual modelling has identified potential key feedback mechanisms involving sea ice, such as the so-called ‘sea ice-switch’ hypothesis (SIS)17, which suggests that sea ice can modify the climate state, switching it between a growing and a retreating land glacier mode, via a temperature−precipitation feedback. This hypothesis makes two critical predictions. First, the SIS invokes a gradual deep ocean cooling and change in ocean vertical mixing as the underlying cause for increased high-latitude sea ice extent during the MPT17. Second, the SIS proposes a land versus sea ice hysteresis, with large sea ice extent across early deglaciations2. Recent modelling studies also suggest that the periodicity of G/IG cycles is linked to changes in the interhemispheric pattern of sea ice growth18. However, while modelling studies clearly suggest the likely importance of sea ice for controlling climate change across the MPT18, complementary high-resolution proxy-based reconstructions of sea ice dynamics are yet to be reported.The MPT marks a fundamental shift in frequency and amplitude of northern hemisphere glaciations from 41 ka glacial/interglacial (G/IG) cycles to quasi-periodic glaciations at 100 ka25 (Ice Proxy with 25 carbon atoms)19, a proxy for seasonal Arctic sea ice, together with those indicative of open water conditions20 and in combination with proxies for primary productivity21, such as the mass accumulation rate of biogenic opal , enable high-resolution reconstructions of past sea ice dynamics. The IP25 biomarker is a mono-unsaturated highly branched isoprenoid (HBI) lipid produced by certain sea ice diatoms during spring23, providing proxy evidence for past seasonal sea ice. To date, IP25 has been readily identified within sediments dating back to the Plio-Pleistocene boundary24 and has even been detected in sediments of Miocene age25, albeit from different locations. Sedimentary IP25 abundance has been found to reliably reflect variations in seasonal sea ice extent, while absent/low IP25 is normally considered to reflect ice-free or extended sea ice cover regimes32. Consistent with these interpretations, IP25 is present in surface sediments from sites in the sub-polar North Pacific that experience seasonal sea ice cover during modern times, but is absent from year-round ice-free locations32. Further, elevated concentrations of a tri-unsaturated HBI biomarker (HBI III), shown recently to be produced by certain diatoms in polar environments33, reflect the spring ice-edge bloom within the open waters of the marginal ice zone (MIZ), at least within the Barents Sea20, which has a similar annual sea ice cycle to the Bering Sea. In contrast, lower abundances of HBI III20 and variable abundances of other phytoplankton biomarkers, including brassicasterol34, are found in year-round ice-free settings of the Barents Sea and Norwegian Sea. However, lower abundances of phytoplankton biomarkers also generally occur under perennial sea ice conditions, similar to IP25. Measurement of MARopal provides an alternative means of distinguishing these two extremes in sea ice cover, especially as siliceous phytoplankton are the most important primary producers in the Bering Sea today35. In the subarctic North Pacific, MARopal represents first-order changes in primary productivity36, such that extended sea ice cover leads to decreased productivity in the region36. Previously, Méheust et al.21 used the sedimentary biogenic opal content in the western Bering Sea across the Last Glacial Maximum to Holocene transition (Termination I), in order to distinguish between different sea ice states.Recent advances in the development of source-specific biomarkers for paleoenvironmental reconstructions, including IP25 and HBI III can also provide insight into seasonal sea ice dynamics, although these relationships are not yet fully understood. Thus, observational surface sediment calibration studies37 and downcore records38 have shown that a weak or inverse relationship between IP25 and HBI III is associated with a strong seasonal sea ice cycle, whereas a positive in-phase relationship likely reflects a fluctuating sea ice margin, with reduced seasonality, and smaller changes in the position of the winter and summer sea ice edge. Other biomarkers, including brassicasterol, are also indicative of open water settings29, although the complication of other potential sources , somewhat limits their use beyond a qualitative indication of general phytoplankton production. However, the relationship between IP25 and brassicasterol can provide context with respect to phytoplankton production in the high-productivity region of the eastern Bering Sea slope6.Additionally, beyond looking at the absolute biomarker concentrations, the nature of the correlation between IP40 , precludes a robust evaluation of sea ice dynamics on G/IG timescales. Here, we address this gap by presenting a high-resolution sea ice reconstruction for the MPT, between 1.22 and 0.8 Ma, from IODP Site U1343. We further examine our MPT findings by comparisons with data obtained from the same core (Site U1343) corresponding to the pre-MPT 41 ka (~1.53−1.36 Ma) and late Pleistocene 100 ka (0.50−0.34 Ma) G/IG cycles to test previous hypotheses of a strong causal link between sea ice and the changing nature of G/IG cycles. Our reconstruction is based on IP25 from Site U1343, together with HBI III and MARopal36. Building on the approach of Méheust et al.21, we utilise MARopal with threshold concentrations of IP25 and HBI III (see Methods) in order to provide a classification of the sea ice states recorded at Site U1343. We identify a substantial increase in sea ice in the Bering Sea and the appearance of transient late-glacial/deglacial sea ice maxima across the MPT, in support of land glacier retreat via a temperature−precipitation feedback mechanism. Together with existing regional and global climate records, we additionally propose that sea ice extent in the eastern Bering Sea plays an important role for GNPIW formation and potentially North Pacific carbon storage.At present, the only reconstruction of sea ice variability from the subarctic North Pacific spanning the MPT is based on sea ice diatoms from International Ocean Discovery Program (IODP) Site U134318Ob) record from Site U1343 is of low temporal resolution between 2.4 and 1.2 Ma (~10 ka). Wavelet analysis of the U1343 δ18Ob42, however, shows significant 41-ka periodicity from 1.5 Ma in accordance with the global δ18Ob (LR04) stack16. Although the relative timing of sea ice changes in comparison to G/IG cycles is uncertain, resulting from the low resolution of the U1343 δ18Ob record prior to 1.2 Ma  51 to 44 represent the time interval from 1.53 to 1.36 Ma and thus precede the onset of the MPT at around 1.2 Ma. The benthic foraminifera oxygen isotope  and perennial sea ice  conditions  is indicative of a fluctuating sea ice margin, with smaller changes in the position of the summer and winter sea ice edge.IP18Ob indicates dominant 100-ka periodicity of G/IG cycles from 0.7 Ma42.Our most detailed interval spans MIS 36 to 20 (1.22−0.8 Ma), thus covering the onset and the majority of the MPT (1.2−0.7 Ma). Wavelet analysis of Site U1343 δ25 was identified in 71 out of the 78 samples analysed, demonstrating the presence of seasonal sea ice in the eastern Bering Sea throughout most of the MPT, consistent with the observations from MIS 51 to 44. IP25 concentration exhibits distinct variability on G/IG timescales, with increased values throughout most glacial intervals ). The reasons for this are unclear, but potentially indicate the influence of occasional MIZ sedimentation.IP25 values is observed consistently across all glacial periods studied post MIS 28 (~1.0 Ma) concomitant with a shift in the timing of the glacial IP25 maximum from the mid-glacial to the late-glacial/deglacial , indicating a more fluctuating sea ice margin with predominantly MIZ conditions. This is framed by intervals of no apparent correlation between these two biomarkers from 1.22 to 1.0 Ma, MIS 36 to 29  and 0.95−0.8 Ma, MIS 24 to 20   represents two G/IG cycles during the post-MPT quasi-100 ka climate variability.25 was quantified in 26 out of 32 samples analysed for biomarkers. A broad gradual increase of IP25 and HBI III, accompanied by a high MARopal, occurs during cooling phases of the interglacial intervals, indicative of seasonal sea ice of increasing duration, with predominantly MIZ conditions , yet low IP25 content  consistent with the late MPT interval (0.95–0.8 Ma).IPons Fig. . Biomarkons Fig. , Table 2ns Fig. − sed, yetns Fig. − sed, yetns Fig. − sed, yetns Fig. − sed, yet25) and phytoplankton (HBI III) biomarkers, together with previously reported MARopal from Site U1343, demonstrate major changes in sea ice dynamics in the eastern Bering Sea during the past 1.5 Ma, especially across the interval covering the MPT  IPled Fig. , indicat2−1.0 Ma 25 and HBI III, together with a shift to a statistically significant in-phase relationship between these two biomarkers   Fig. . The gra 28 Fig. . Determi25 and HBI III we measured a third biomarker, brassicasterol , which has a variety of sources, including marine and lacustrine phytoplankton and potentially even sea ice algae39. As such, we use brassicasterol to investigate the importance of sea ice for phytoplankton growth in the eastern Bering Sea and, indeed, we find a weak positive relationship  between IP25 and brassicasterol   into thIII Fig. , suggest25, low HBI III and high MARopal. Temporal IP25 variability across G/IG cycles is similar to that of the interim state with higher IP25 concentration indicative of increased sea ice cover during the late-glacial/deglacial. Exceptionally, MIS 22 is characterised by lower IP25 values compared to the three preceding glacial periods , beginning at MIS 25, is characterised by an ice-free eastern Bering Sea  recorded at Site U1343  records exist across the MPT, one from Deep Sea Drilling Project (DSDP) Site 607 in the North Atlantic49 and another from Ocean Drilling Program (ODP) Site 1123 in the South Pacific50  to the surface and promoting vertical mixing. Eddy formation is correlated to the strength of the Bering Slope Current7  and sea ice (1.0 Ma). Additionally, the North Atlantic BWT remains low during the late Pleistocene, which could account for the observed increase in sea ice extent during glacial intervals from 0.50 to 0.34 Ma. Yet, more regional, orbitally resolved records of Pleistocene BWT are needed to confirm a long-term deep ocean cooling of the North Pacific and to investigate leads and lags of BWT versus sea ice change in the marginal seas of the Arctic Ocean.With regard to the SIS hypothesis, we also consider the possible influence of long-term deep ocean cooling on sea ice dynamics51 and from regional glacier advances52. The increase in sea ice extent in the Bering Sea is consistent with a long-term decrease of North Pacific SSTs   from ~1.1 Ma would support homogenous cooling and increased salinity stratification during glacial periods promoting sea ice formation as a result of decreased heat capacity of the surface ocean. An increase in North Pacific SSTs between 0.95 and 0.85 Ma has been attributed to a northward movement of the North Pacific Polar Front54. However, MIS 12 and 10 show increased sea ice extent  recorded at ODP Site 84956  and IODP Site U1342 , Δδ13C(849-U1342), is used as a proxy for Bering Sea GNPIW formation/ventilation as proposed by Knudson et al.10  values56, subtraction of site-specific δ13Cb values enables local influences to be determined10. Site U1342 is located in the southern Bering Sea on the Bowers Ridge  at 818 m water depth. Currently, Site U1342 is bathed in NPDW with very low δ13CDIC10. During glacial periods, however, GNPIW was formed in the Bering Sea and reached down to the seafloor (at U1342), transporting high δ13CDIC surface waters to greater depth10. Thus high (low) Δδ13C(849-U1342) represents reduced (enhanced) GNPIW influence at Site U1342  are inversely correlated  the late Pleistocene59. Future work should focus on reconstructing the depth of GNPIW across the late Pleistocene and its ability for carbon sequestration, as this would likely have been influenced by the amount of brine formation and thus sea ice dynamics. Additionally, future multi-proxy reconstructions of past sea ice variability in the marginal seas of the North Pacific should be coupled with investigations of GNPIW ventilation and carbon biogeochemistry as such studies could help unravel the role of sea ice for deep ocean carbon storage.Increased sea ice cover during glacial periods also has important implications for ocean-atmosphere gas exchange. Presently, deep reaching eddies bring high-CO17. This study is an important step forward in understanding the role of sea ice for the MPT, but will need to be supplemented by further high-resolution (and continuous) orbitally resolved sea ice records over the past 1.5 Ma to determine the exact timing of sea ice build-up across G/IG cycles. Additionally, sea ice records from other Arctic marginal seas across the MPT should help confirm the occurrence of deglacial sea ice maxima in late-to-post MPT glacial periods and to further understand their implications. Finally, we note that additional studies of Bering Sea surface ocean chemistry are needed to determine the role of sea ice for CO2 sequestration and ultimately deep ocean carbon storage via GNPIW formation.In summary, our sea ice reconstruction from Site U1343 in the Bering Sea shows a twofold change across the MPT Fig.  with imp41. At present day it is bathed in NPDW that enters the Bering Sea through several deep passes in the Aleutian Island Arc60. The surface circulation in the Bering Sea forms a cyclonic gyre  flows northward and leaves the Bering Sea through the 50 m deep Bering Strait—the gateway between the Pacific and Arctic Ocean. Mesoscale eddies developing in the eastern Aleutian basin bring nutrient-rich waters to the surface and maintain high primary productivity7 in the eastern Bering Sea. Other shelf edge processes such as tidal mixing and transverse circulation further enhance the horizontal exchange of nitrate-rich basin waters and iron-rich shelf waters6 sustaining high primary productivity  along the continental margin, also called the ‘Green Belt’. The eastern Bering Sea shelf is characterised by an intensive spring phytoplankton bloom, as a result of nutrient release during sea ice melting62. In recent decades the Bering Sea has experienced substantial retreat in the winter sea ice margin and earlier sea ice melting in spring with important implications for the marine ecosystem63, demonstrating the sensitivity of sea ice to climate change. The combination of Site U1343 being close to the present day winter sea ice margin  was retrieved off the eastern Bering Sea continental margin , and normalised to E. batialis, the most abundant species at Site U134342. Due to sample resolution this approach yields a highly refined age model for the last 1.2 Ma and a refined age model between 1.2 and 2.4 Ma42. The age model based on oxygen isotope stratigraphy is in good agreement with datum events based on bio- and magnetostratigraphy42.In total five holes were drilled at Site U1343 (A−E) out of which three  were used to construct a composite depth scale (splice) based on the physical properties of the cores from 0 to 269.92 m CCSF-A. Below the splice, U1343E cores with unknown gaps are appended from ~270 to 779 m CCSF-A64. Additionally, removal of elemental sulphur was performed65 and sterol fractions were collected. Prior to analysis samples were kept in cold storage at 7 °C. Samples were freeze dried at −45 °C and 0.2 mbar for 48 h using a Thermo Savant Modulyo D freeze drier and an Edwards K4 Modulyo freeze drier at Plymouth and Cardiff University, respectively. After freeze drying, samples were homogenised using a dichloromethane (DCM)-cleaned agate pestle and mortar and 3 g of sediment was weighed into 7 ml glass vials with aluminium-lined polypropylene screw caps. In addition to the sediment samples, a blank and two samples of standard sediments with known biomarker concentrations were added to each extraction batch. Standard sediments are from the Canadian Arctic Archipelago and 0.5 g of sediment were weighed in per sample. Ten microlitres of 0.01 mg mL−1 9-octylheptadec-8-ene (9-OHD) and 5 α-androstan-3βol solution were added to each sediment vial and procedural blank as internal standards for HBI and sterol quantification, respectively. The samples were extracted three times using a mixture of DCM (high performance liquid chromatography (HPLC) grade) and methanol  2:1 (v/v). Gas chromatography of the first extraction batch showed high concentrations of elemental sulphur in Site U1343 sediment samples that interfere with IP25 analysis. Therefore sulphur removal65 using tetrabutylammonium sulphite reagent was performed for all samples prior to silica column chromatographic purification of the total organic extracts (TOE)64. During silica chromatography, non-polar components  were eluted with hexane and collected in new pre-labelled 7 mL glass vials (TOE-2), whereas more polar hydrocarbon fractions (sterols) were eluted using hexane/methyl acetate  and collected in separate pre-labelled 7 mL glass vials (TOE-3). TOE-2 was dried under N2 flow at 25 °C and re-dissolved in 150 μL hexane before being transferred to 300 μL GC glass vials, concentrated to 20 μL under N2 flow at 25 °C, and capped with aluminium crimp-top caps and Teflon septa . Due to low abundance of HBI lipids and high concentrations of n-alkanes HBI fractions from the oldest time interval (1.53−1.36 Ma) were additionally purified using silver-ion chromatography (5:95 AgNO3:SiO2) to remove n-alkanes. TOE-3 was dried under N2 flow at 25 °C and derivatised using N,O-Bis(trimethylsilyl)trifluoroacetamide . All samples were analysed by gas chromatography-mass spectrometry (Agilent 7890A GC coupled to a 5975 series mass selective detector fitted with an Agilent HP-5ms column) at Plymouth University using the operating conditions specified in Belt et al.64. The identification of individual lipids was based on their characteristic retention times and mass spectra and quantification was achieved by integrating the peak area of selected ions (m/z 350 (IP25); 346 (HBI III); 470 (brassicasterol)) in comparison to the peak area of the internal standards added to each sample64. Quantification of individual lipids also considers an instrumental response factor obtained from known concentrations of biomarker lipids in the standard sediments64.HBI lipids were extracted from 3 g of freeze dried homogenised sediments as described in Belt et al.25 , HBI III (most prominent in the MIZ), and the mass accumulation rate of biogenic opal 36, measured in IODP Site U1343 in the eastern Bering Sea. IP25 and U1343 MARopal are weakly anti-correlated , consistent with the interpretation, that an increased sea ice cover leads to decreased primary productivity as a result of light limitation in the surface ocean.In order to reconstruct different sea ice states, we use a combinatory approach of IP21, who used threshold values for IP25 and sedimentary biogenic opal content in the western Bering Sea to identify sea ice regime shifts over the past 20 ka BP. Site U1343 is located on a topographic high off the eastern Bering slope, which reduces the influence of downslope transport. However, to avoid potential siliciclastic dilution effects, we used the MARopal36, rather than the sedimentary biogenic opal content (wt. %). Here we developed a multi-proxy classification, where threshold values of IP25, HBI III and the MARopal are used to identify four different sea ice states  and are summarised in Fig. opal is based on the past 20 ka. Sea ice reconstructions using IP25 and sea ice diatoms from the western Bering Sea21 and the Umnak Plateau44, respectively, indicate a change from a more extended sea ice cover to seasonal sea ice/ice-free conditions around 15 ka BP across Termination I, suggesting that this is a common feature of sea ice dynamics in the Bering Sea. The MARopal value in Site U1343 at 15 ka is 4 g cm−2 ka−1  are characterised by variable MARopal values. This could be a result of the dynamic high productivity region overlying the core Site of U1343, indicating that sea ice concentration, even though of major importance, might not be the only influence on primary productivity along the eastern Bering slope. Only three data points do not fit with our overall sea ice state classification  is small to begin with, confidence intervals are quite large. Ninety-five per cent confidence intervals are estimated using equi-tailed bootstrapping and are of the type Student’s t.For calculating Pearson’s All data generated during this study supporting its findings are supplied via the NERC Polar Data Centre (UK-PDC), doi:10.5285/9caf74c4-7054-4539-81b8-d4f942afc358.Supplementary InformationPeer Review File