Sacroiliac (SI) screw loosening may indicate persistent instability, non-union and contribute to pain. Yet, there is no reliable objective measurement technique to detect and monitor SI screw loosening. In 9 cadaveric pelvises one of two SI screw was turned back approximately 20 mm and subsequently assessed by optical measurement, fluoroscopy and a 3D scan using an image intensifier. CTs were segmented and a contour-based registration of the 3D models and the fluoroscopies was performed to measure SI backing out (X-ray module). Three independent observers performed measurements with three repetitions. Deviation of the measurement techniques to the 3D scan, intra- and interrater reliabilities and method equivalence to the 3D scan were assessed. The X-ray module and two fluoroscopic measurement techniques yielded a difference less than 5 mm compared to the 3D scan and equivalence to the 3D scan. Intrarater reliability was for two observers and almost all techniques very good. Three fluoroscopic measurement techniques and optical measurements displayed a very good interrater reliability. The 3D scan and X-ray module yielded the most precise values for SI screw loosening but only the fluoroscopic measurement of the inlet lateral loosening displayed a good reliability and equivalence to the 3D scan. In the elderly, this is most probably caused by a decreased bone stock in the alar region but also in the sacral body7. The incidence of SI screw loosening with a backing out of the SI screw was described with 2–20% and fractures of the vertical shear type were identified as risk factor9. Nonetheless, SI screw loosening did not generally lead to pain or indicate instability8. Especially if the SI screw loosening persists without progress, revision surgery is not required8. Though previous studies described SI screw loosening qualitatively7, quantitative measurement techniques were not mentioned bearing the risk to overlook SI screw loosening progression and leading to missing data regarding values indicating clinical relevant SI screw loosening.Sacroiliac (SI) screw loosening may cause pain and indicate missing fracture healing and persistent instability10. However, the reliability of radiological measurement methods has to be assessed before correlating functional and radiological outcome11.The assessment of the healing progress is not only based on the functional outcome, but also radiographic follow-up is of high interest10. Lefaivre et al.11 examined the interrater reliability of some promising radiographic techniques and observed poor to moderate results. Only the cross-measurement technique of Keshishyan et al.12 assessing pelvic instability yielded excellent results11. Especially for the posterior pelvic ring a poor reliability of radiographic measurements was observed13. In spine surgery, CT was proposed to assess implant loosening14. More sophisticated and expensive examinations such as single photon emission computed tomography (SPECT)/computed tomography (CT) are highly sensitive and specific detecting implant loosening15. However, the tracer uptake might be increased within the first year after surgery leading to false- positive results15. Taking the time of SI screw loosening into account (6–69 days) false-positive results might be determined1. There is lacking evidence describing techniques to assess specifically SI screw loosening with readily available conventional X-ray.Radiographic measurement techniques, previously introduced to determine pelvic fracture reduction, revealed a poor level of evidence, missing standardization, reliability assessment and a lack of instructions how to perform the measurementAs yet there are no reliable methods to quantify SI screw loosening, we aimed to develop and study different methods using cadavers. We investigated three-dimensional (3D) scan, optical measurements, a novel developed X-ray module and fluoroscopic measurement techniques to reveal an accurate and reliable measurement technique to detect and monitor SI screw loosening.Cadaver pelvises were obtained from body donors who gave their signed consent for the use of their bodies for educational and research purposes in medical school in accordance to the Saxonian Death and Funeral Act of 1994.et al.16 to test the stability of augmented vs. non-augmented SI screws. Therefore, both-sided alar fracture of the sacrum was set with an oscillating saw and the symphysis was cut16. In each pelvis, one side was fixed with a single non-augmented fenestrated SI screw in S1 and the other with a single bone cement (3 ml polymethylmethacrylat [PMMA]) augmented fenestrated SI screw in S1. For the present study, the non-augmented SI screw was retracted in each pelvis after biomechanical testing   and fluoroscopic images . This was defined as 1, an axial 3D scan with a voxel size of 0.38 × 0.38 × 1 mm was generated with the image intensifier Ziehm Vision FD Vario 3D© . The scan centre was set at the anterior-posterior centre of the SI joint of the loosened SI screw. For each pelvis a scan was carried out, exported in DICOM format and imported in Mimics . The distance of the SI screw turning back was measured in the slice of the maximum radiolucency . The turn back was measured from the tip of the screw to the end of the visible radiolucency created by the bone void of the manually turned back SI screw , each pelvis was placed on millimetre paper with the SI screw parallel to the millimetre paper. A K-wire in a drill sleeve was set parallel to the turned back SI screw with the tip in contact with the cortical bone. The drill sleeve was retracted up to the level of the screw head, placed on the millimetre paper and photographed  of Tics Fig. . The fol0). Subsequently, we duplicated the STL file of the SI screw. The duplicate was then set to the position found in the fluoroscopy  or line 5 (inlet) a line marking the superior lateral border or respectively the lateral border of sacral alae was set Fig. , line 3.The course of the SI screw was marked Fig. , line 4.To determine SI screw turn back, we used the following workflow after the import and registration of all fluoroscopic images of T1 − T0) of the values obtained before (T0) and after (T1) SI screw turn back in each pelvis in AP, inlet and outlet views. The medial loosening  and corresponding range independent of observer and measurement repetition using Graph Pad Prism 7 . The theoretical accuracy of each method was obtained from the pixel solution of the respective data.Data regardless of observer and repetition were merged and mean values for the 3D scan and other methods were generated for each pelvis specifically. The differences between the 3D scan and each method were calculated for each pelvis as absolute value. The here-obtained data were merged and are presented as mean ± SD including range.The raw data without generating a mean value were used to study the reliability for each measurement method using a MatLab script .20. Thus, measurements of each obtained value on the basis of the measurement method from a specific pelvis are interchangeable but the measurements of observers and the other pelvis are not. The grading of Landis and Koch22 were applied: ICC(1) 1 perfect, 0.81–1 very good, 0.61–0.80 good, 0.41–0.60 moderate, <0.4 poor. The 95% confidence interval (95% CI) was used to define upper  and lower limit . If the CI was <0, it was set to 0 related to the definition of the investigated range of ICC (0 to 1). Data are presented as mean and 95% CI.ICC (1) was chosen in accordance to McGraw and Wong 1996 to estimate the reproducibility of observer data in relation to the performed repetitions20. Hence, within the measurement methods the measurements for the pelvis might be changed but the observer data are not interchangeable. The criteria for ICC  were analysed regarding confidence interval and quality as done for the ICC (1). Data are presented as mean and 95% CI.Accordingly, to assess intrarater reliability the raw data for each measurement method, pelvis and the three observer data and their repetitions were used. Prior to analyses, a mean value of the three repetitions of each observer for each pelvis and measurement method was generated. The ICC  was chosen to compare the repeatability between observers14.The 3D scan was defined as “gold standard” as CT is the recommended radiological examination to detect implant related complications in spine surgeryA mean value of the three repetitions of each observer for each pelvis and measurement method was generated. The generated mean values of each measurement method were subtracted from the mean value of the 3D scan for each pelvis and observer. These difference values were averaged and presented. To verify the application of the 3D scan as “gold standard”, the data of the K-wire measured screw turn back  of the 3D scan. Values are presented as absolute deviation from “gold standard” (mean and 95% CI). The minimal detectable screw extrusion was determined based on the 95% CI from each method.A two one-sided test was used estimating equality/divergence of each method to the 3D scan. First, a mean value (1) for all measurement methods was generated for each observer based on the three repetitions for each pelvis. These mean values (1) were pooled for each measurement method and new mean values (2) including standard deviation were generated which were used for the analysisThe obtained data of SI screw loosening for each pelvis were merged and the mean ± SD as well as the range are indicated in Supplementary Tables The determined SI screw turn back in the 3D scan as well as the differences between the 3D scan and each method are summarized in Table None of the used method showed a difference of more than 10 mm compared to the 3D scan. The X-ray module, the medial outlet loosening and the inlet lateral loosening yielded differences less than 5 mm.The ICC (1) using 3D scan was very good for observer 1 and 2 and good for observer 3 (Table The following ICC (1) were determined for fluoroscopic measurements: AP medial loosening very good for observer 1 and 2 and good for observer 3, AP lateral loosening very good for all observers, outlet medial loosening was very good for observer 1 and 2 and good for observer 3, outlet lateral loosening very good for all observers, inlet medial loosening for observer 1 good, for observer 2 very good and for observer 3 poor, inlet lateral loosening very good for all observers.The ICC  was moderate for the 3D Scan  to construct its “equivalence interval”. Comparing the absolute differences of the measurement methods yielded values presented in Supplementary Table Based on the 95% CI of each method, the minimal measurable screw extrusion was calculated as followed: 3D scan 1.8 mm, optical measurement and X-ray module 2.3 mm, AP medial loosening 2.8 mm, AP lateral loosening 2.5 mm, outlet medial loosening 3.1 mm, outlet lateral loosening and inlet medial loosening 3 mm, inlet lateral loosening 2.5 mm.Compared to 3D scan, the most equivalent estimation of SI screw turn back was assessed by the application of the X-ray module and in fluoroscopy by measuring the inlet lateral loosening and the outlet medial loosening. These techniques yielded a deviation less than 5 mm compared to the 3D scan.28. A loosening rate of 2–20% for SI screws was reported8. Especially in the elderly population the loosening rate might rise up to 20%8. Radiography in AP, inlet and outlet projection are used to assess the instability of pelvic ring fractures31. Postoperatively, these radiographs are used to grade fracture reduction of the pelvic ring33. After mobilization, radiographs might reveal SI screw loosening indicating a possible persistent instability or non- union8. However, radiographic SI screw loosening was not always symptomatic, especially if there is no progression of the loosening8. Radiolucent lines, cup migration or gaps indicating cup loosening following hip arthroplasty35 or other criteria for implant loosening were previously not established on the pelvic ring possibly due to the impeded visualization of the posterior pelvic ring37.Percutaneous SI screw fixation is a common procedure for the fixation of unstable fractures of the pelvic ring8, therefore the definition of the critical SI screw loosening is not possible. To our best knowledge, no quantitative measurement techniques are published yet. Probably, the missing quantitative data are related to the impaired visualization of the posterior pelvic ring37 and the non- routinely performed CT scans.Currently, there are no quantitative data on the assessment of SI screw loosening available. Recent studies generally stated if the SI screw is loosened or not38. Though the registration of a 3D model to plain radiographs and the subsequent determination of the acetabular cup position yielded comparable results, the CT scan remains the “gold standard” for the determination of acetabular cup position39. The CT was also superior in the assessment of implant loosening following ankle replacement defined as osteolytic areas, when compared to radiographs40.Radiographs were found to be less accurate compared to CT determining the anteversion of the cup following hip arthroplasty45. This leads to a different projection of the posterior pelvic ring and individualized outlet and inlet projection angles were proposed47. However, these individualized angles are difficult to obtain in daily routine. In the present study, the very good ICC (1) and ICC  for the inlet lateral loosening were not affected by the missing adaption of angulation for the inlet fluoroscopic view. In contrast, the missing adaption of the angulation might explain the worse ICC  of the outlet medial loosening though it was less than 5 mm different from 3D scan and yielded equivalence to the 3D scan. Thus, the impaired visualization of the bony structures used for the measurements might led to the differing values between the observers48. The good results of the inlet lateral loosening might be resulted from the consistent upper SI joint projection in the inlet view irrespective to the pelvic tilt. Nonetheless, all these parameters are sensitive to patient positioning, i.e. when the patient lies oblique on the table , the distances might be distorted.Due to individual sacral and lumbar anatomy, the angulation for optimal visualization of pelvic anatomy in AP, inlet and outlet radiographs is variable37. Recently it was shown that the fluoroscopy after SI screw fixation is insufficient to exclude malpositioning and a postoperative CT scan is required49.Radiographic evaluation of the pelvic ring in the elderly is impaired and CT is recommended for fracture detection52. The improvement of multi-dimensional fluoroscopy by 3D image intensifiers allows appropriate evaluation of implant position and fracture reduction comparable to a CT scan53. We therefore considered the 3D scan as “gold standard” in the present study.In consideration of these data, also a possible impairment of SI screw loosening detection in radiographs might be discussed. Hence, in the present study radiographic measurements were compared to multi-dimensional fluoroscopy from a 3D image intensifier as “gold standard”. Indeed, 3D image intensifier based 3D scans are inferior compared to CT, but they still reveal satisfactory results regarding osteosynthesis position as reported for malpositioning rates of SI screws10. Intrarater reliability (ICC (1)) was for two observers very good using the majority of measurement techniques. Possibly, the data of the remaining observer led to the worse ICC  determined especially for the 3D scan and X-ray module. In addition, the worse ICC  for those methods might be caused by the higher individual observer’s choice reducing the reliability11. Here, the observer decides independently the slice for measurement in the 3D scan and for optimal sizing as well as accuracy of the contour-based registration in the X-ray module possibly affecting the ICC .Beside an accurate determination of the implant loosening, a good radiographic measurement method should also display appropriate ICC (1) and ICC 54.All fluoroscopic measurements irrespective to their projection  for the lateral loosening displayed a very good ICC , but only the X-ray module, outlet medial loosening and inlet lateral loosening yielded equivalence to the 3D scan. The AP view displayed at least good ICC (1) and ICC . However, no equivalence to the 3D scan and a higher absolute difference to 3D scan compared to other fluoroscopic measurements were detected. This might be caused by the disguised anatomical landmarks in the AP view, as previously discussed55.Beside the opportunity reducing the choices of the observer, computer-based registrations and their improvement may increase the reliability37. Thus, future studies are needed to support the presented findings in a clinical setup and prove their validity. The measurements of fluoroscopies following 3D model registration might also increase the reliability due to the correct scaling which might be absent in the clinical practice except by the introduction of a scaling reference56. In the present study, the reference was digitally implicated by the registration to the 3D model and the results should not be impaired. But for clinical studies a reference should be included in radiographs. Furthermore, the clinically relevant SI screw extrusion remains unclear. The optical measurement may be underestimated related to the “false” higher values measured digitally; however using the here used approach we were also able to investigate the reliability of this method and in future studies we also recommend to analyse the validity and reliability of calliper based measurements. Moreover, besides SI screw turning back, also a cutout of SI screws or a washer penetration occurs16. This implant failure mode should also be evaluated; however the SI screw turn back is the most common observed failure and the only one which might be clinically inconspicuous or indicate persistent instability underlining the need for a quantitative differentiation3.Our results are limited by the small sample size, especially for the fluoroscopic measurements (n = 5) leading to increased deviations compared to the remaining methods. The fluoroscopic measurement techniques presented better results due to the removal of internal organs and missing gas known to impair the visualization of the posterior pelvic ring49. In future studies the here presented measurement techniques should be validated in large clinical trials to reveal the clinically relevant SI screw loosening and correlate SI screw loosening to the functional outcome. Thus, the clinical consequence of SI screw loosening might be revealed. Here, we recommend measuring the lateral loosening in inlet radiographs.Although the X-ray module displayed the smallest deviation to the 3D scan, at least good ICC (1) and an equivalence to the 3D scan, the ICC  was poor. In contrast, determining the SI screw loosening as inlet lateral loosening showed beside a small deviation to the 3D scan, very good ICC (1), equivalence to the 3D scan also a very good ICC . Thus, using this method SI screw loosening can be monitored temporally and reduces the radiation for the patient by decreasing the need for CT scans. Moreover, the here presented results indicate the need for outlet and inlet views in the clinical follow- up after SI screw fixation. Furthermore, the X-ray module might be utilized to plan and control SI screw placement as well as to grade fracture reduction in radiographs after introducing reliable anatomical landmarks as known from hip arthroplasty for a faster registrationSupplementary Table S1-4_Clean


Protein kinases are crucial to coordinate cellular decisions and therefore their activities are strictly regulated. Previously we used ancestral reconstruction to determine how CMGC group kinase specificity evolved . In the present study, we reconstructed ancestral kinases to study the evolution of regulation, from the inferred ancestor of CDKs and MAPKs, to modern ERKs. Kinases switched from high to low autophosphorylation activity at the transition to the inferred ancestor of ERKs 1 and 2. Two synergistic amino acid changes were sufficient to induce this change: shortening of the β3-αC loop and mutation of the gatekeeper residue. Restoring these two mutations to their inferred ancestral state led to a loss of dependence of modern ERKs 1 and 2 on the upstream activating kinase MEK in human cells. Our results shed light on the evolutionary mechanisms that led to the tight regulation of a kinase that is central in development and disease. Eukaryotic biology, including cell growth, differentiation, proliferation and apoptosis. They are one of the largest protein families in eukaryotes  extracellular signal-regulated kinase 1 (ERK1) and ERK2, several structural elements are important for activity. These include the p+1 binding pocket that binds the substrate peptide proline, the regulatory αC helix, and the L16 loop. Upon phosphorylation of the TxY threonine and tyrosine residues, a number of changes occur. A network of electrostatic contacts with the phospho-threonine and phospho-tyrosine drives a conformation change around the p+1 substrate binding site, enabling substrate binding. The regulatory αC helix rotates inwards, allowing a glutamate on the αC helix (Glu-69) to form a salt bridge with Lysine (Lys-52) on the β3 strand that is crucial for catalytic activity. Extension of the C terminal L16 loop enables rotation of the N- and C-terminal domains, leading to ‘domain closure’, bringing Lys-52 closer to other catalytic residues, such as aspartates in the DFG and HRD motifs. Together, these conformational changes can lead to a 1000-fold increase in activity . Similartrans, where distinct molecules transfer phosphates to one another. Other kinases autophosphorylate in cis; that is the kinase catalyzes an intramolecular transfer of phosphates onto its own TxY. In the latter case, autophosphorylation of dual-specificity tyrosine regulated kinases (DYRK) and glycogen synthase kinase-3β(GSK3β) has been proposed to occur during intermediate stages of protein folding  for their activation  and a decrease in kcat  as a generic substrate. We found that all inferred ancestors prior to and including AncERK1-5 had relatively high activity, while AncERK1-2 had low activity, almost comparable to modern ERK1/2 . One altssed MEK , indicat in kcat . TherefoWe next aimed to determine the mechanism of the activity transition between AncERK1-5 and AncERK1-2. Almost all MAPKs have a conserved 'TxY' motif at the activation lip in their kinase domain, and dual phosphorylation of this motif drives kinase activation. Dual phosphorylation of the threonine and tyrosine residues of 'TxY' motif in ERK2 is catalyzed by MEK1/2. This dual phosphorylation causes the N- and C-terminal domains in ERK2 to rotate toward one another, thus organizing the catalytic sites . This coMultiple sequence alignment analysis showed that the ‘TxY’ motif was conserved among all inferred kinase ancestors . To testTo understand how this regulatory evolution occurred, we next sought to identify the specific mutations underlying the transition in kinase activity that occurred between AncERK1-5 and AncERK1-2. First, however, we wished to determine whether the activities of AncERK1-5 and AncERK1-2 were robust to uncertainties in ancestral sequence reconstruction. To address this issue, we synthesized alternative inferred ancestors combining all alternate residues with posterior probability >0.2 . The infth amino acid  and a transition from glutamine to methionine at the 89th amino acid . Indeed, reverting these amino acids to the inferred ancestral state in AncERK1-2 (referred to as the AncERK1-2-ins44R-Q89M mutant) was sufficient to confer high basal activity to this kinase  and determined this mutant to be just as active as the ERK1-74R allele. Indeed, we found that, in addition to arginine and asparagine, many other amino acid insertions also increased ERK1 activity . Thus, wBecause the ‘TxY’ motif was highly phosphorylated in inferred deep ancestral kinases, we hypothesized that increased ‘TxY’ phosphorylation would likely also be a mechanism of mutant ERK1 activation. Consistent with this idea, mutations in this motif to ‘AxY’, ‘TxF’ and ‘AxF’ all abolished the increased activity caused by the insertion mutation . FurtherE. coli bacterium that we use to express ERK1 and inferred ancestral kinases lacks endogenous kinases capable of phosphorylating the ‘TxY’ motif. Therefore, the increased phosphorylation of ‘TEY’ motif is likely caused by autophosphorylation  simulations. We focused on the unphosphorylated structure, which is only available for ERK2  of each mutant to WT. ERK2-ins55N showed increased flexibility at the glycine rich loop , ERK2-Q1We next investigated the dynamics of the catalytic residues. There are several residues critical for catalysis: K52/71, D147/166 and D165/184 (rat ERK2/human ERK1 numbering) . The salThe gatekeeper residue lies between two conserved non-consecutive structural ‘spines’ of hydrophobic amino acids, the R- and C-spines that connect the two lobes and reorganize upon kinase activation . We therPrevious MD simulations have shown changes in angles between the N- and C-terminal domains in gatekeeper mutants of ERK2. This domain closure is a key step in kinase activation . TherefoWe next took an unbiased approach to identify contacts that were gained or lost by determining a differential contact map between the mutant and wild type kinases for all residue pairs . In all To gain a deeper understanding of the dynamical impacts of mutations, we built Markov state models (MSMs) from a set of 125 highly informative inter-residue distances . MSMs help integrate information from multiple simulations to reveal the system’s kinetically distinct clusters of conformations, which are referred to as macrostates . We usedFinally, we compared our simulation data to a previously proposed mechanism  where gaWhile these MD simulations were not long enough to observe the full transition to an active kinase conformation , they paNext, we sought to determine whether the evolutionary insertion mutation led to higher ERK1 activity in vivo. We generated lentivirus encoding ERK1-WT or ERK1-ins74N and transduced NIH 3T3 cells with these two alleles. We used an ERK-KTR reporter , as our We also analyzed the phosphorylation level of the two ERK variants in vivo, by western blot using an antibody specific to phosphorylated ERK. NIH 3T3 cells transduced with both wildtype and mutant ERK were treated with Trametinib. After treatment, proteins were extracted for western blot. The western blot result showed that the phosphorylation level of ‘TEY’ motif in wild type ERK1 was very low after treatment with Trametinib, while the cells expressing ERK1-ins74N exhibited robust phosphorylation . These rOver the past several decades biochemical, genetic and structural studies have provided deep insights into the regulation of kinases. However, the mechanisms by which this diverse array of regulatory mechanisms evolved from a common ancestor remain poorly understood. In this study, we analyzed the regulatory evolution of the ERK kinases that lie within the CMGC group. Ancestral reconstruction revealed that the ability of these kinases to autophosphorylate at their activation loop sharply decreased at the split between the ERK5 and ERK1-2 kinase subfamilies. While the basal activity of the ERK5 subfamily remained relatively high, the ERK1-2 kinase subfamily became strongly dependent on the upstream activating kinase MEK. We identified two mutations that together account for most of the difference between modern ERK1-2 and ERK5. Strikingly, reversal of these evolutionary mutations in modern ERK1 led to high kinase activity and autophosphorylation independent of the upstream activating kinase MEK .It has recently been discovered that changes in the β3-αC loop length of BRAF, EGFR, and HER2, none of which are in the CMGC group of kinases, also led to activation of these kinases . In thesIn previous studies on the evolution of specificity within the CMGC kinase group, we found that an inferred ancestral DFG +1 mutation was also sampled in cancer mutations throughout the kinome , therefoWhy, then, is ERK not a major oncogene? Oncogenic activation mutations in upstream kinases BRAF, MEK, EGFR and HER2 are frequently observed in cancer. Indeed, the Ras/RAF/MEK/ERK pathway is one of the most important signaling axes in oncogenesis. One possibility is that the upstream regulators have multiple targets in addition to ERK, and indeed this is certainly the case for Ras. Another intriguing possibility is that the dynamical properties of ERK signaling are crucial for its role in oncogenesis. ERK is not simply an ON/OFF switch, but rather oscillates or adapts in various contexts . It willWe found that the evolutionary changes that place ERKs 1 and 2 under stringent regulation synergize in their effects. One change, a polar substitution in the ‘gatekeeper’ residue of the active site has been previously described as an activating mutation . EvolutiThe second evolutionary change that we implicated in the decrease in autophosphorylation at the transition to AncERK1-2, a change in loop length between the β3 strand and αC helix, has not been reported before. The inferred deeper CMGC ancestors and the modern ERK kinases that have higher basal activity, ERK5 and ERK7, all had one extra amino acid in this loop . The revEach of these evolutionary changes confers an order of magnitude change in basal activity, and when combined, these two mutations increase the basal activity of ERKs 1 and 2 by more than two orders of magnitude. This additive, relatively independent effect of the two mutations may have provided evolutionary pathways that could have both conferred a selective advantage to intermediates and allowed for evolution towards a tightly controlled system without major fitness barriers.Our molecular dynamics simulations indicated that reversal of the inferred evolutionary mutations  had two clear effects on the rapid timescale (ns - μs) dynamics of ERK2. First, there was increased flexibility in the glycine rich loop and loop 16 . The reoEukaryotes.Previous ancestral reconstruction studies have highlighted the importance of epistasis in the diversification of function. For example, changes in the specificity of hormone-receptor ligand binding domains required stabilizing mutations  prior topET28b vectors were used for bacterial expression. Open Reading Frames (ORFs) of the Erk proteins were fused N terminally to the 6X histidine tag for purification. Glutathione S Transferase (GST) was inserted between the 6X His-tag and the Erk ORF in order to increase protein molecular weight. For site-directed and domain-swapping mutagenesis, primers containing the point mutation sites or domain junction sites for chimera protein were designed. The PCR fragments and the vectors were assembled according to the manufacturer’s instructions using Gibson Assembly Master Mix kit from New England Biolabs (NEB). All the constructs were verified via DNA sequencing.NIH3T3 ATCC CRL1658 Cell lines were verified using short tandem repeat DNA profiling (STR Profiling) by PCR amplification of 9 STR loci plus Amelogeninusing Promega GenePrint 10 System, Fragment Analysis with ABI 3730XL DNA Analyzer, comprehensive data analysis with ABI Genemapper software and final verification using supplier databases including ATCC and DSMZ. Cell lines were checked and tested negative for mycoplasma infection. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), penicillin, and streptomycin. The cells were incubated at 37°C and 5% CO2. Transfections of HEK293T cells were performed using TransIT-293 Transfection Reagent (Mirus), according to the manufacturer’s instructions. Viral supernatants were collected 48–72 hr following transfection of 293 T cells. After virus infection, NIH3T3 cells were selected with 1–2 μg/ml puromycin. For western blot, cells were first washed three times with PBS, then incubated in medium with no serum and treated with 2 µM Trametinib. After 12 hr, cells were lysed by addition of SDS loading buffer containing 20% glycerol, 4% SDS, 0.1 M Tris (pH 6.8), 0.2 M dithiothreitol (DTT), and phenol blue dye, followed by boiling at 100°C for 10 min.E. coli cells was performed as previously described  and transferred to a nitrocellulose membrane. Membranes were probed with anti-ERK  or anti-p-ERK .2, 50 mM NaCl, 0.75 µg/µl myelin basic protein (MBP), 100 µM ATP, and 0.05 µCi of [γ-32P]ATP. The kinase reactions proceeded for 30 min at room temperature and were terminated by addition of 6 µl 5x SDS loading buffer . All samples were separated on 4–12% Bis-Tris gels (ThermoFisher Scientific). The gel was dried and exposed to a phosphor screen. Phosphor screens were analyzed with a Typhoon 9500 scanner (GE) using ImageQuant software (GE). Michaelis–Menten curves were generated in a similar manner, except the buffer contained 300 µM ATP and varying concentration of MBP. Data were fit by nonlinear regression to the Michaelis–Menten model V0 = [ERK]* kcat *[S]/KM+[S] using Prism 8 (GraphPad software) and the fit was used to determine values for kcat and KM.0.05 µg purified recombinant 6x His-tagged Erk protein was used in a total volume of 10 µl (approximately 0.1 µM). Final reaction conditions were 20 mM Hepes, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 25 µM ATP, and 0.05 µCi of [γ-32P]ATP) without substrate. The autophosphorylation reactions proceeded for 30 min at room temperature and were terminated by addition of 6 µl 5x SDS loading buffer. All samples were separated on 4–12% Bis-Tris gel (ThermoFisher Scientific). The gel was dried and exposed to phosphor screen. Phosphor screens were analyzed with a Typhoon 9500 scanner. Autophosphorylation of ERK1 ins74N was measured at 5, 10, 15, and 20 min, as previously desctibed (0.1 µg purified recombinant 6x His-tagged Erk protein (approximately 0.2 µM) was incubated in kinase reaction buffer  for 1 hr at 55°C. The reduced cysteines were subsequently alkylated with iodoacetamide (2 μl of 0.5 M) for 45 min in the dark at room temperature. The protein samples were digested with trypsin (Promega) at a 100:1 (protein:enzyme) ratio overnight at room temperature. The pH of the digested protein lysates were lowered to pH < 3 using trifluoroacetic acid (TFA). The digested lysates were desalted using C18 solid-phase extraction . 40% acetonitrile (ACN) in 0.5% acetic acid followed by 80% acetonitrile (ACN) in 0.5% acetic acid was used to elute the desalted peptides. The peptide eluate was concentrated in the SpeedVac and stored at −80 C.1 µg of each sample was loaded onto a trap column  connected to an analytical column  using the autosampler of an Easy nLC 1000 (Thermo Scientific) with solvent A consisting of 2% acetonitrile in 0.5% acetic acid and solvent B consisting of 80% acetonitrile in 0.5% acetic acid. The peptides were eluted into either an Orbitrap QExactive (Thermo Fisher Scientific) or Orbitrap QExactive HF-X mass spectrometer (Thermo Fisher Scientific) respectively, using the following gradient: 5–35% in 60 min, 36–45% in 10 min, followed by 45–100% in 10 min. Protein samples for Erk1 and Erk1-ins74N were acquired on an Orbitrap QExactive mass spectrometer, with full MS spectra recorded at a resolution of 70,000, an AGC target of 1e6, with a maximum ion time of 120 ms, and a scan range from 400 to 1500 m/z. Following each full MS scan, twenty data-dependent MS/MS spectra were acquired. The MS/MS spectra were collected with a resolution of 17,500 an AGC target of 5e4, maximum ion time of 120 ms, one microscan, 2 m/z isolation window, fixed first mass of 150 m/z, dynamic exclusion of 30 s, and normalized collision energy (NCE) of 27. Protein samples for AncCDK.MAPK, AncMAPK, AncJPE, AncERK, AncERK1-5 and AncERK1-2 were acquired on the QExactive HF-X using data dependent acquisition mode (DDA) or parallel reaction monitoring mode (PRM). In both cases, high resolution full MS spectra were recorded with a resolution of 45,000, an AGC target of 3e6, a maximum ion time of 45 ms, and a scan range from 400 to 1500/z. For samples acquired using DDA mode, MS/MS spectra were collected with a resolution of 15,000 an AGC target of 1E5, maximum ion time of 120 ms, one microscan, 2 m/z isolation window, fixed first mass of 150 m/z, dynamic exclusion of 30 s, and normalized collision energy (NCE) of 27. For samples acquired using PRM mode, PRM scans were acquired for the peptides of interest following each full MS scan. The MS/MS spectra were collected with a resolution of 15,000 an AGC target of 2E5, maximum ion time of 200 ms, one microscan, 2 m/z isolation window, fixed first mass of 150 m/z, dynamic exclusion of 30 s, and normalized collision energy (NCE) of 27.For all samples, the MS/MS spectra were searched against a given protein sequence of interest using Byonic . The masTo assess the activity of Erk in mammalian cells, ERK-KTR was expressed from the CMV promoter in vector pLentiCMV (Addgene #59150). ERK kinase was expressed along with a dsRed reporter expressed from an IRES. Cells were seeded on six well plates one day before imaging. At 30% confluency, media was changed to media without FBS and containing Trametinib  2 hr prior to imaging. Cells were imaged with a Nikon Eclipse Ti fluorescence microscope. All imaging was carried out at constant conditions of 37°C, 5% CO2, and constant humidity. The subcellular distribution of the KTR reporter was analyzed with Image J. Cells expressing both Kinase and KTR reporter were used for analysis.To perform MD simulations of ERK2-ins55N, ERK2-Q103M (gatekeeper mutation), and ERK2-ins55N + Q103M (double mutant) mutants, we used the unphosphorylated ERK2 (PDB ID: 1ERK). We introduced the insertion to ERK2 (55N) using Discovery Studio 4.5 software , and the−1 collision rate. We controlled the pressure by a Monte Carlo molecular-scaling barostat with an update interval of 50 steps. We treated non-bonded interactions with the Particle Mesh Ewald  (-)   and Smit+) (-)  parameteing@home  for eachTo study the flexibility of the kinase, we calculated root mean square fluctuations (RMSFs) for each trajectory using NumPy . These cTo study the motion between N and C domains we calculated the domain closure as the angle between the helices C and E. We did a least squares fit of a vector ('helix axis') for each helix to the positions of the carbonyl C and O atoms, and then recorded the angle between those vectors . To alloTo understand the mutation-induced changes in the interactions between residues that are important for activity and/or along the regulatory and catalytic spines, we calculated the closest heavy atom residue-residue distances using all frames using MDTraj  for eachWe calculated the closest heavy atom residue-residue distances for all pairs of residues for each trajectory using MDTraj , convertWe trained the XGBoost  gradienthttps://osf.io/dp4cb/.The molecular dynamics simulation data is available via the Open Science Framework at: https://github.com/choderalab/ERK-ancestral-materials .The code used to analyze the data is available at We identified orthologs of human ERK1, ERK2, ERK5 and ERK7 using a protein BLAST  search aAll primary data can be accessed at the following urls:InputFasta File:http://webdoc.nyumc.org/phylobot/Sang2019_Input_fasta_ERK_resurrection.fastaOutput Fasta File:http://webdoc.nyumc.org/phylobot/Sang2019_Output_fasta_ERK_ancestors.fastaSQL Database containing all resurrection data:http://webdoc.nyumc.org/phylobot/Sang2019_ERK_ASR.dbThe full reconstruction can be found at:http://phylobot.com/350478581/msaprobs.PROTCATLG/ancestors In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been favorably reviewed by three peer reviewers, and the evaluation has been overseen by John Kuriyan as the Senior Editor and the Reviewing Editor. The following individual involved in review of your submission has agreed to reveal her identity: Dorothee Kern (Reviewer #3).Thank you for submitting your article "Ancestral resurrection reveals mechanisms of kinase regulatory evolution" 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.In this paper, Sang et al. use ancestral reconstruction to study the evolution of MAP kinase regulation. The authors analyzed the specificity and activity of several kinases that represent common ancestors of major kinase families within the CMGC group. Interestingly, they found that the dependence on upstream MAPKK phosphorylation occurred at the transition between the common ancestor of the ERK 1/2/5 kinases (AncERK1-5) and the common ancestor of ERK 1/2 (AncERK1-2). Specifically, the authors present convincing data that a two amino acid change late in evolution between the ancestor of ERK5 and ERK1 and 2 toward ERK1-2 caused a large decrease in autophosphorylation rate, and autophosphorylation was replaced by MEK phosphorylation. This is a beautiful result!Overall, the study is well designed and presented, and the results will be of high interest to investigators studying kinase regulation.eLife paper, which described the divergence of substrate specificity in related kinases using similar approaches, and thus warrants publication as an eLife Research Advance after revision.The manuscript builds upon the authors' previous Major issues that must be addressed, with additional experiments as necessary:Although this is broken down into separate points, 1 to 6 below, all of these issues are related to putting the activities of the relevant kinases on a more rigorous footing. We ask that a substantial response be prepared for these questions, but the authors should judge whether particular experiments can be done in a realistic timeframe or not. Note that these questions are important, so a simple response that none of the experiments can be done in a timely fashion will not be deemed a satisfactory response.1) The authors use MBP phosphorylation as readout for intrinsic activity, but as in their previous paper, they show that each ancestral kinase has a slightly different substrate specificity. Is it possible that the differences observed for MBP phosphorylation are due to difference in sequence specificity, rather than intrinsic activity?2) In this assay, does MEK have any significant activity toward MBP? This should be measured as a control.cat, KM, or a combination.3) The activity measurements done in the paper are endpoint assays of myelin basic protein phosphorylation. For at least the key proteins , the authors should carry out kinetic experiments. This would allow them to determine whether the effects are driven by changes in k4) What are the phosphorylation states of each protein prior to the MBP phosphorylation reactions? It would be important to compare the activities of all proteins for the same phosphorylation states (nonphosphorylated versus doubly phosphorylated proteins). The activity data presented in Figure 1B represent the results of different mixtures of non- and phosphorylated kinases according to Figure 2B and C.For this, the quantification of phosphorylation for all samples need to be measured first, and then activity data collected for clean species . Particularly AncERC1-5and AncRC1-2 are missing in respect to analysis of differential phosphorylation rates, the crucial proteins where the switch seem to happen in evolution.5) A distinction should be made throughout the manuscript between "intrinsic activity" and regulation by activation loop auto-phosphorylation. Differences in "intrinsic activities" would ideally be assessed by comparing the enzymatic activities of all kinases in the same phosphorylation states. In other words, how active are each of the unphosphorylated kinases, and how active are those same kinases after activation loop phosphorylation? What the authors are measuring in the beginning of the Results section, and in Figure 1B, appears to be the consequence of a difference in the ability to auto-phosphorylate and thus auto-activate, not necessarily a difference in intrinsic activity.6) Related to these points, it would be informative to see raw data (phosphorimages) from the MBP phosphorylation reactions in Figure 1B as a figure supplement, rather than just a normalized quantification of endpoint intensities.7) Finally, we ask that the amino acid sequences of all proteins (modern and resurrected) be included in the supplementary materials, and it would be of value to give the number of substitutions in the text. The full tree used for ancestral sequence reconstruction (ASR), including the species, needs to be included for reproducibility for the field. A supplementary figure that shows the probabilities of the nodes, and information of the posterior probabilities for the full sequences is essential to evaluate ASR.Other important issues that require response:1) The authors should avoid language that implies that the ancestral protein sequence is actually known. A sophisticated reader will understand what is meant, but we are concerned that the way that much of the paper is written a strong impression is given that the ancestral sequence is actually synthesized. This is not just a semantic point, because it may turn out that errors in the sequence reconstruction have led to misleading results. We do not consider this point to invalidate the conclusions of this study, but we ask that the language used to describe the "ancestral" sequences be changed throughout the manuscript. A few comments, given below, should help explain what we mean.The method of ancestral sequence reconstruction is a powerful approach to dissect molecular differences between close-related biomolecules. However, the use of the dramatic term "resurrection" should be avoided throughout this manuscript, as it implies that we have explicit knowledge of the true protein sequences found in ancestral organisms, which is then "resurrected". The more common term, "reconstruction", is more appropriate, as it indicates that a sequence model of a plausible ancestral protein is being generated using the best available evidence. An example of a statement that implies that the ancestor is truly known, and that the authors should avoid is the following:"As part of this study, we observed that a deep ancestor of this group had relatively high intrinsic activity even in the absence of any activating kinase of or allosteric activators."In the beginning of the main section of the paper, the authors use the following phraseology, which correctly captures the inherent uncertainty in the "resurrected" sequences: "We synthesized the coding sequences for these inferred ancestors".JNK, p38. Therefore the Introduction, Abstract and title should be modified to reflect the data from this manuscript .2) The manuscript is focused on the specific late evolution between ERK5 and ERK 1 and 2, and there is no information about the evolution of regulation towards CDKs, MAPKs, 3) To enlighten the reader about the evolutionary trajectory, key information to add would be how modern ERK5, ERK 7 that branch off the ancestors used in this study are behaving in respect to activities of their nonphosphorylated and phosphorylated activation loop states towards substrates, and their ability to autophosphorylate.4) What is the explanation of a much higher activity of AncERK1-2 with the two mutations in the presence of MEK relative to AncERK1-2 in the presence of MEK? Could the authors also include the data for the reverse experiment, AncERK1-5 M89Q 44Rdel, both in the absence and presence of MEK, in Figure 3?5) 300 ns trajectories are very short MD simulations, and it is quite unlikely that refolding of the L16 loop coupled to domain closure is on that time scale. NMR data for ERK has suggested that these processes occur on the millisecond time-scale. Thus, the MD simulations are not convincing due to the discrepancy in time scale between the suggested conformational changes and the length of the simulations, and rather weaken the manuscript than strengthen it. The authors should consider carefully what information is contributed by the MD simulations, and then describe that with appropriate cautionary notes. Alternatively, remove the MD simulations from the manuscript.Should the MD simulations be retained, the authors should clarify what atoms are being used to measure the distances between residues in the analyses of MD simulations. In particular, for salt bridges, as in the one analyzed in Figures 5C, F, it would be most informative to look at distances between terminal or near-terminal atoms of the side chains .6) The figures could be better arranged and assembled, it is hard to jump back and forth between gels and activity figures. Could all data be presented in a quantitative format (activity towards MBP and autophosphorylation)?7) The authors should include an LC/MS quantification of TEY motif phosphorylation for AncERK1-5 and AncERK1-2 .8) Figure 4E does not conclusively establish the mechanism of autophosphorylation; His6-ERK1 ins74N could still be undergoing intermolecular (trans) autophosphorylation. This point would need to be established by kinetic experiments, e.g. by measuring the concentration dependence of autophosphorylation.9) Figure 6D: the authors should present a quantification of the Western blots .10) The activation loops of the ancestral kinases differ in length and sequence. Notably, the ancestral CDK.MAPK kinase has a -3 Arg residue, for which this kinase still has some residual preference. If canonical sequence-specificity is not relevant because of a cis-autophosphorylation mechanism, it is still worth noting that the sequence of the activation loop could affect activation loop dynamics. The authors should discuss how the composition of activation loop itself might impact their findings. This is somewhat addressed by the chimera experiments but would still be worth mentioning.eLife. Your revised article has been favorably evaluated by John Kuriyan (Senior Editor and Reviewing Editor), and three reviewers. The manuscript is now, in principle, suitable for publication in eLife, but the reviewers have identified some remaining issues that need to be addressed before acceptance, as outlined below. Please use your best judgement in revising the manuscript with reference to these points, and we will make a final decision as quickly as possible. When submitting the revised manuscript, please provide a point-by-point response to the review comments. Also, please add figure numbers to each individual figure in the submitted manuscript, and include page numbering on each page of the manuscript – the lack of such numbering introduced an element of confusion in the review of the previous manuscript.Thank you for resubmitting your work entitled "Ancestral reconstruction reveals mechanisms of ERK regulatory evolution" for further consideration at The premise of this study, that ancestral sequence reconstruction can be used to identify molecular mechanisms by which kinase regulation evolved, is quite intriguing. The authors do a nice job of exploring this premise within the MAP kinase family. The results are novel and of broad interest to the kinase field. Furthermore, the authors have adequately addressed most of the concerns raised after the original submission, although a few remaining points require attention before acceptance.JNK, p38 is not discussed either in the manuscript. Without the proper analysis on those ancestors the authors may wish to remove them and to keep the focus on the ERK ancestors. The latter is the story of this manuscript, and this part is interesting and convincing.1) A major issue is that for AncERK, AncJPE, AncMAPK, AncCDK.MAPK (which are evolutionary further from the modern proteins), still no posterior probabilities are provided. They need to be shown, as previously asked by the review. In addition, no experiments were performed to test for their phosphorylation status, and no experiments were done on their modern counterparts. Only activity was measured for these ancestors , but their relation in respect to modern CDK, MAPK, 2) The manuscript still does not correctly describe the quantification of the percentages of non-phosphorylated, single and double phosphorylated (TxY motif) proteins used in the activity studies. Figure 2—figure supplement 1B is provided, but one cannot follow what is exactly shown, and how these data can be used for quantification of phosphorylation. In the text it is described as "more" or "less" phosphorylation. Since differences in autophosphorylation is the center of the findings of this manuscript, this needs to be quantified accurately. Are these full-length proteins, or peptides shown in this figure, and the results of what kind of experiment are shown? Even ERK 1 appear to have some phosphorylation. What does "modified TxY " mean?3) The part of the manuscript pertaining to Point No. 4 in the "Other Important Points" in the original review, still needs further clarification in the manuscript. If this result is puzzling and currently cannot be explained unambiguously with a model, it is important to articulate this for the reader.4) The MD simulations are done on ERK2 , but all the experiments in the manuscript are performed on ERK1 . Why is this?There is difficulty in seeing how the MD figures provided now help explain why the mutants of ERK1 autophosphorylate the TxY motive in cis much more efficiently than wtERK1. In Figure 5 all proteins have a bimodal distribution for the plotted distances . If one of these two substates is interpreted to be the active conformation, one would not expect the experimental result of a big difference in phosphorylation state between these proteins. The authors correctly note that there is no difference I domain closure either. These points should be clarified. Major issues that must be addressed, with additional experiments as necessary:Although this is broken down into separate points, 1 to 6 below, all of these issues are related to putting the activities of the relevant kinases on a more rigorous footing. We ask that a substantial response be prepared for these questions, but the authors should judge whether particular experiments can be done in a realistic timeframe or not. Note that these questions are important, so a simple response that none of the experiments can be done in a timely fashion will not be deemed a satisfactory response.1) The authors use MBP phosphorylation as readout for intrinsic activity, but as in their previous paper, they show that each ancestral kinase has a slightly different substrate specificity. Is it possible that the differences observed for MBP phosphorylation are due to difference in sequence specificity, rather than intrinsic activity?This is a good point. To check this possibility more carefully, we added two experiments. First, we expanded our specificity arrays to include AncERK1-5 . There is not much change in specificity between AncERK1-5 and ERK2. We were unable to get good specificity array data for AncERK1-2 due to low activity. However, we believe it unlikely that a major specificity change would occur from AncERK1-5 to AncERK1-2 and then back again from AncERK1-2 to ERK2. Nevertheless, we also checked the relative activities of AncERK1-5 and AncERK1-2 with a second substrate, ELK1: see the new Figure 1—figure supplements 4A and 4B. We see the same drop in activity from AncERK1-5 to AncERK1-2 with this second substrate. Taken together, we are confident that our results indicate changes in activity without a major contribution from specificity changes.2) In this assay, does MEK have any significant activity toward MBP? This should be measured as a control.We tested this possibility and found that MEK has negligible toward MBP . MEK is known to be highly specific for ERK, so we were happy to see this result. To further confirm that MEK can activate AncERK1-2, we co-expressed MEK and AncERK1-2, purified the ancestor away from MEK and found that this approach also led to increased AncERK1-2 activity . Together, these experiments reinforce the conclusion that MEK can activate AncERK1-2.cat, KM, or a combination.3) The activity measurements done in the paper are endpoint assays of myelin basic protein phosphorylation. For at least the key proteins , the authors should carry out kinetic experiments. This would allow them to determine whether the effects are driven by changes in kM and decreased kcat. Interestingly, the values for AncERK1-2 look very similar to reported values of unphosphorylated ERK2 and the values for AncERK1-5 look very similar to phosphorylated ERK2 . We have added these data to a new Figure 1D.We undertook the suggested characterization and found that the decrease in activity is due to a combination of increased KProwse, C.N., and Lew, J. (2001). Mechanism of Activation of ERK2 by Dual Phosphorylation. J Biol Chem 276, 99–103.4) What are the phosphorylation states of each protein prior to the MBP phosphorylation reactions? It would be important to compare the activities of all proteins for the same phosphorylation states (nonphosphorylated versus doubly phosphorylated proteins). The activity data presented in Figure 1B represent the results of different mixtures of non- and phosphorylated kinases according to Figure 2B and C.For this, the quantification of phosphorylation for all samples need to be measured first, and then activity data collected for clean species . Particularly AncERC1-5and AncRC1-2 are missing in respect to analysis of differential phosphorylation rates, the crucial proteins where the switch seem to happen in evolution.M and kcat data reported above).We characterized the phosphorylation state of the TEY motif in AncERK1-5 and AncERK1-2. We added these data to Figure 2D. AncERK1-2 is almost completely unphosphorylated and AncERK1-5 is highly phosphorylated (consistent with the K5) A distinction should be made throughout the manuscript between "intrinsic activity" and regulation by activation loop auto-phosphorylation. Differences in "intrinsic activities" would ideally be assessed by comparing the enzymatic activities of all kinases in the same phosphorylation states. In other words, how active are each of the unphosphorylated kinases, and how active are those same kinases after activation loop phosphorylation? What the authors are measuring in the beginning of the Results section, and in Figure 1B, appears to be the consequence of a difference in the ability to auto-phosphorylate and thus auto-activate, not necessarily a difference in intrinsic activity.Thank you for pointing this out. We believe we are really looking at differences in phosphorylation state due to differential autophosphorylation (see above experiments). We have updated this language and interpretation in the text.6) Related to these points, it would be informative to see raw data (phosphorimages) from the MBP phosphorylation reactions in Figure 1B as a figure supplement, rather than just a normalized quantification of endpoint intensities.We have added examples of these raw data to the Figure 1—figure supplement 2A.7) Finally, we ask that the amino acid sequences of all proteins (modern and resurrected) be included in the supplementary materials, and it would be of value to give the number of substitutions in the text. The full tree used for ancestral sequence reconstruction (ASR), including the species, needs to be included for reproducibility for the field. A supplementary figure that shows the probabilities of the nodes, and information of the posterior probabilities for the full sequences is essential to evaluate ASR.http://phylobot.com/350478581/msaprobs.PROTCATLG/ancestorsWe have added a supplementary figure with the posterior probabilities of the reconstructions for each of the kinases studied . We have also provided a link to all reconstruction data including the full trees, sequences for every ancestor and all probabilities: Other important issues that require response:1) The authors should avoid language that implies that the ancestral protein sequence is actually known. A sophisticated reader will understand what is meant, but we are concerned that the way that much of the paper is written a strong impression is given that the ancestral sequence is actually synthesized. This is not just a semantic point, because it may turn out that errors in the sequence reconstruction have led to misleading results. We do not consider this point to invalidate the conclusions of this study, but we ask that the language used to describe the "ancestral" sequences be changed throughout the manuscript. A few comments, given below, should help explain what we mean.The method of ancestral sequence reconstruction is a powerful approach to dissect molecular differences between closely-related biomolecules. However, the use of the dramatic term "resurrection" should be avoided throughout this manuscript, as it implies that we have explicit knowledge of the true protein sequences found in ancestral organisms, which is then "resurrected". The more common term, "reconstruction", is more appropriate, as it indicates that a sequence model of a plausible ancestral protein is being generated using the best available evidence. An example of a statement that implies that the ancestor is truly known, and that the authors should avoid is the following:"As part of this study, we observed that a deep ancestor of this group had relatively high intrinsic activity even in the absence of any activating kinase of or allosteric activators."In the beginning of the main section of the paper, the authors use the following phraseology, which correctly captures the inherent uncertainty in the "resurrected" sequences: "We synthesized the coding sequences for these inferred ancestors".We have reworded the manuscript to better reflect these uncertainties and removed the term “resurrection”.2) The manuscript is focused on the specific late evolution between ERK5 and ERK 1 and 2, and there is no information about the evolution of regulation towards CDKs, MAPKs, JNK, p38. Therefore the Introduction, Abstract and title should be modified to reflect the data from this manuscript .We have changed the title to reflect the focus on ERK.3) To enlighten the reader about the evolutionary trajectory, key information to add would be how modern ERK5, ERK 7 that branch off the ancestors used in this study are behaving in respect to activities of their nonphosphorylated and phosphorylated activation loop states towards substrates, and their ability to autophosphorylate.We have added language to indicate the high basal activity of ERK5 and ERK7 to the text.4) What is the explanation of a much higher activity of AncERK1-2 with the two mutations in the presence of MEK relative to AncERK1-2 in the presence of MEK? Could the authors also include the data for the reverse experiment, AncERK1-5 M89Q 44Rdel, both in the absence and presence of MEK, in Figure 3?This is a good question. We have added experiments to address this issue. Adding MEK to the AncERK1-2 Q89M ins44R doesn’t increase the kinase activity much. Adding MEK to AncERK1-2 increases activity ~6 fold, but this leads to < 10% of the activity of the active mutant. We believe that this inefficiency is due to the known high specificity of MEK for ERK1 and 2. We speculate that MEK doesn’t recognize AncERK1-2 as efficiently, accounting for the lower efficiency of activation of AncERK1-2.We tried experiments adding MEK to AncERK1-5  and saw only a slight potentiation. We also added MEK to AncERK1-5 M89Q del44R and again saw only an ~2-fold increase in activity. It is likely that AncERK1-5 is an even worse substrate for MEK than AncERK1-2. We have added these experiments to Figure 3—figure supplement 5.5) 300 ns trajectories are very short MD simulations, and it is quite unlikely that refolding of the L16 loop coupled to domain closure is on that time scale. NMR data for ERK has suggested that these processes occur on the millisecond time-scale. Thus, the MD simulations are not convincing due to the discrepancy in time scale between the suggested conformational changes and the length of the simulations, and rather weaken the manuscript than strengthen it. The authors should consider carefully what information is contributed by the MD simulations, and then describe that with appropriate cautionary notes. Alternatively, remove the MD simulations from the manuscript.We have greatly expanded our MD analysis using roughly three orders of magnitude more data. We performed extensive analyses of multiple  long MD simulations per sequence, generating ~500 microseconds aggregate simulation data for each sequence, and compared the WT and mutants. In total, we generated and analyzed a total of ~ 2 ms of aggregate simulation data.We did not observe a sufficient number of very slow conformational rearrangements  to be able to construct a Markov state model of the complete conformational dynamics. Therefore, we have taken care not to make such claims in the manuscript. However, this new far larger simulation dataset (> 3 orders of magnitude more than the previous experiment) is consistent with the leading edge of what has been done for biomolecular simulations of kinases, e.g., compare to the Scientific Reports paper from Sultan and Pande where they have 1.7 ms aggregate from the simulation of two kinase sequences (we have ~ 2 ms for 4 sequences):7, 15604.Sultan, M.M., Denny, R.A., Unwalla, R., Lovering, F., and Pande, V.S. (2017). Millisecond dynamics of BTK reveal kinome-wide conformational plasticity within the apo kinase domain. Sci Rep We are happy to report that the more active configuration of catalytic residues is maintained in these much more extensive data . We can also clearly see synergy between the two mutations. Furthermore, each mutation gives increased flexibility at two points in the kinase . While we can’t comment on domain closure and have therefore removed this language, we do see increased variance in domain angles . These last data are presented with strong caveats. We believe these data provide starting points for future biochemical and simulation work and therefore are valuable to the study.These new data were obtained in collaboration with John Chodera and Rafal Wiewiora at MSKCC, therefore they are now included as co-authors on the study. They have seen and approved the manuscript and are in agreement with their placement in the author list. They collaborated with Myvizhi Selvan, a new postdoc in the lab of Zeynep Gumus, therefore she is now added as an author. The work of Selvan Vatansever was removed from the manuscript following earlier criticisms and as a result she did not ultimately contribute to the data in the projects, therefore she has been removed as an author. All of these changes have been approved by the laboratories involved.Should the MD simulations be retained, the authors should clarify what atoms are being used to measure the distances between residues in the analyses of MD simulations. In particular, for salt bridges, as in the one analyzed in Figures 5C, F, it would be most informative to look at distances between terminal or near-terminal atoms of the side chains .We are indeed using the nearest neighbor heavy (non-hydrogen) atoms. We have added this language to the text.6) The figures could be better arranged and assembled, it is hard to jump back and forth between gels and activity figures. Could all data be presented in a quantitative format (activity towards MBP and autophosphorylation)?We have added the data in more quantitative format where possible.7) The authors should include an LC/MS quantification of TEY motif phosphorylation for AncERK1-5 and AncERK1-2 .We have added these data in Figure 2D.8) Figure 4E does not conclusively establish the mechanism of autophosphorylation; His6-ERK1 ins74N could still be undergoing intermolecular (trans) autophosphorylation. This point would need to be established by kinetic experiments, e.g. by measuring the concentration dependence of autophosphorylation.We have added these data in Figure 4—figure supplement 4. The concentration dependence of autophosphorylation is consistent with intramolecular autophosphorylation.9) Figure 6D: the authors should present a quantification of the Western blots .We have added this quantification.10) The activation loops of the ancestral kinases differ in length and sequence. Notably, the ancestral CDK.MAPK kinase has a -3 Arg residue, for which this kinase still has some residual preference. If canonical sequence-specificity is not relevant because of a cis-autophosphorylation mechanism, it is still worth noting that the sequence of the activation loop could affect activation loop dynamics. The authors should discuss how the composition of activation loop itself might impact their findings. This is somewhat addressed by the chimera experiments but would still be worth mentioning.We have added text that discusses this possibility in the section on chimeric kinases.[…] 1) A major issue is that for AncERK, AncJPE, AncMAPK, AncCDK.MAPK (which are evolutionary further from the modern proteins), still no posterior probabilities are provided. They need to be shown, as previously asked by the review. In addition, no experiments were performed to test for their phosphorylation status, and no experiments were done on their modern counterparts. Only activity was measured for these ancestors , but their relation in respect to modern CDK, MAPK, JNK, p38 is not discussed either in the manuscript.We have added this discussion (subsection “Reconstruction of the lineage leading to ERK kinases”).Without the proper analysis on those ancestors the authors may wish to remove them and to keep the focus on the ERK ancestors. The latter is the story of this manuscript, and this part is interesting and convincing.We discuss the regulation of p38, ERK1-2 and CDKs in the Introduction as well as the outgroups DYRK and GSK groups of kinases.We have added the posterior probabilities for all of these ancestors . We have also performed extensive additional mass spectrometry and have analyzed the phosphorylation status and added these results . Therefore, we have kept the deeper ancestors in the paper .2) The manuscript still does not correctly describe the quantification of the percentages of non-phosphorylated, single and double phosphorylated (TxY motif) proteins used in the activity studies. Figure 2—figure supplement 1B is provided, but one cannot follow what is exactly shown, and how these data can be used for quantification of phosphorylation. In the text it is described as "more" or "less" phosphorylation. Since differences in autophosphorylation is the center of the findings of this manuscript, this needs to be quantified accurately. Are these full-length proteins, or peptides shown in this figure, and the results of what kind of experiment are shown? Even ERK 1 appear to have some phosphorylation. What does "modified TxY " mean?We agree, these data were quite unclear before. We quantified tryptic peptides in this analysis. We have updated the Materials and methods section and manuscript to clarify these points . We have also reanalyzed all of our data and presented the fraction of pT-E-Y, T-E-pY, and pT-E-pY (e.g. where pT is phosphothreonine) for all experiments. We include the precise sequences and modifications of all peptides in Source data 1.3) The part of the manuscript pertaining to Point No. 4 in the "Other Important Points" in the original review, still needs further clarification in the manuscript. If this result is puzzling and currently cannot be explained unambiguously with a model, it is important to articulate this for the reader.The original question was: 4. What is the explanation of a much higher activity of AncERK1-2 with the two mutations in the presence of MEK relative to AncERK1-2 in the presence of MEK? Could the authors also include the data for the reverse experiment, AncERK1-5 M89Q 44Rdel, both in the absence and presence of MEK, in Figure 3?Our reply was: This is a good question. We have added experiments to address this issue. Adding MEK to the AncERK1-2 Q89M ins44R doesn’t increase the kinase activity much. Adding MEK to AncERK1-2 increases activity ~6 fold, but this leads to < 10% of the activity of the active mutant. We believe that this inefficiency is due to the known high specificity of MEK for ERK1 and 2. We speculate that MEK doesn’t recognize AncERK1-2 as efficiently, accounting for the lower efficiency of activation of AncERK1-2.We tried experiments adding MEK to AncERK1-5  and saw only a slight potentiation. We also added MEK to AncERK1-5 M89Q del44R and again saw only an ~2-fold increase in activity. It is likely that AncERK1-5 is an even worse substrate for MEK than AncERK1-2. We have added these experiments to Figure 3—figure supplement 5. We have now added this discussion to the subsection “Shortening of linker loop and mutation of gatekeeper residue led to tight regulation in the ERK1-2 family”.4) The MD simulations are done on ERK2 , but all the experiments in the manuscript are performed on ERK1 . Why is this?This is because we have crystal structures of unphosphorylated ERK2 but not of ERK1. We chose to focus our analysis on unphosphorylated kinase as we decided the initial autophosphorylation would be most relevant. Furthermore, there was previous MD simulation work and mechanistic suggestions on ERK2 autophosphorylation , hence we decided it would be most valuable to extend this research direction on the same system.We have performed biochemical experiments with ERK2 and obtain similar results as with ERK1, i.e. the same gatekeeper and insertion mutations lead to increased activity due to higher basal phosphorylation. Therefore, we believe that results that we obtain with the ERK2 structure will be relevant to both ERK1 and ERK2. We have added the ERK2 experiments to Figure 5—figure supplement 1. We have also now explained our reasoning in the manuscript .There is difficulty in seeing how the MD figures provided now help explain why the mutants of ERK1 autophosphorylate the TxY motive in cis much more efficiently than wtERK1. In Figure 5 all proteins have a bimodal distribution for the plotted distances . If one of these two substates is interpreted to be the active conformation, one would not expect the experimental result of a big difference in phosphorylation state between these proteins. The authors correctly note that there is no difference I domain closure either. These points should be clarified.We have performed the MD simulations to explore some of the structural changes influenced by the mutants and suggest hypotheses for further mutational studies. While these simulations were extensive and long , they were still not long enough to sample and capture some of the conformational changes that occur on a millisecond scale, such as domain closure. Hence, the data clearly do not describe the true  equilibria of the systems, rather it is informative about the neighborhood  of the inactive conformation. While not offering definitive explanations as to the full autophosphorylation activation pathway, the data show the relaxation processes of the mutated systems that would eventually lead to the global equilibrium. Hence, it is not surprising that we observe only small changes to the conformational ensembles at these limited timescales, and with more sampling we expect the differences to keep increasing until we have described the full activation mechanism leading to the experimental magnitude of change in activity. However, knowledge of the initial stages of this relaxation process towards the global equilibrium, particularly the patterns of residue-residue contact disruptions, would provide enough information to make informed hypotheses about mutations that can further tweak the behavior of the protein.To maximize the understanding of the structural changes seen in the current datasets, we have now developed Markov state models (MSM) trained on the inter-residue distances corresponding to contacts most relevant to distinguishing the conformational ensembles of the four protein systems from each other. MSMs help integrate information from multiple simulations to reveal the system’s kinetically distinct macrostates, whose populations can be perturbed by mutations. The MSMs revealed how different structural changes caused by the mutants are kinetically coupled with each other. Again, these insights will allow for generation of further mutational hypotheses to further study structural aspects of the autophosphorylation activation process.In addition to the mutants promoting formation of active-like conformations, cis-autophosphorylation requires that the phosphorylation sites be able to intramolecularly reach the active site – a mechanism of activation lip destabilization leading to that has been previously proposed . We have analyzed our data according to this proposed mechanism, and found the presence of small populations of conformations where the phosphorylation site tyrosine can access the active site aspartate in all systems. We did not observe other changes proposed by the mechanism, instead we analyzed the patterns of contact changes in our simulations at the activation lip and indicate crucial residues for future mutational studies.We have updated the text to indicate the observations and limitations of our current MD simulation. The results of these simulations will be used in future studies as seeds for adaptive sampling to generate MSMs describing all relevant activation steps involved, and to generate first guesses at reaction coordinates for enhanced sampling. We note that a description of the full activation mechanism would be a significant research project in itself, and is far outside the scope of this work.


Diabetes mellitus is a chronic condition associated with the metabolic impairment of insulin actions, leading to the development of life-threatening complications. Although many kinds of oral antihyperglycemic agents with different therapeutic mechanisms have been marketed, their undesirable adverse effects, such as hypoglycemia, weight gain, and hepato-renal toxicity, have increased demand for the discovery of novel, safer antidiabetic drugs. Since the important roles of the sodium-glucose cotransporter 2 (SGLT2) for glucose homeostasis in the kidney were recently elucidated, pharmacological inhibition of SGLT2 has been considered a promising therapeutic target for the treatment of type 2 diabetes. Since the discovery of the first natural SGLT2 inhibitor, phlorizin, several synthetic glucoside analogs have been developed and introduced into the market. Furthermore, many efforts to find new active constituents with SGLT2 inhibition from natural products are still ongoing. This review introduces the history of research on the development of early-generation SGLT2 inhibitors, and recent progress on the discovery of novel candidates for SGLT2 inhibitor from several natural products that are widely used in traditional herbal medicine. Diabetes mellitus is a chronic condition characterized by hyperglycemia, due to the metabolic impairment of insulin production/secretion and/or utilization in human body. It plays a pivotal role in the development of several important cardiovascular, neurological, and renal complications, which are the main underlying reasons for morbidity and mortality in diabetic patients . The IntType 2 diabetes mellitus, also called non-insulin dependent diabetes mellitus (NIDDM), is the most common type of diabetes and its pathogenesis is associated with various mechanisms. Due to the complexity of this disease and the maintenance of compliance of the diabetic patients, many kinds of oral antihyperglycemic agents have been developed and are now available, such as sulfonylureas, meglitinides, biguanides, thiazolidinediones, α-glucosidase inhibitors, and dipeptidyl peptidase-IV (DPP-IV) inhibitors. Nevertheless, some undesirable adverse effects caused by the administration of these medications, including hypoglycemia, gastrointestinal symptoms, weight gain, and hepato-renal toxicity, have increased demand for the discovery of safer antidiabetic agents with novel therapeutic mechanisms .After the renal glucose reabsorption kinetics were firstly demonstrated in the late 1930s , the twoNatural products have been widely used for the regulation of normal physiological conditions and the treatment of various chronic diseases, as a form of pharmaceuticals and dietary supplements. To date, numerous studies have demonstrated that natural product extracts and/or their active phytochemicals showed various antidiabetic properties, such as insulinotropic effect, peroxisome proliferator-activated receptor γ (PPARγ) activation, AMP-activated protein kinase (AMPK) pathway activation, α-glucosidase inhibition, glucose transporter 4 (GLUT4) expression/translocation, and protein tyrosine phosphatase 1B (PTP1B) inhibition, with lower side effects . This reKi) values of 151 and 18.6 nM, respectively.Phlorizin, a dihydrochalcone isolated from the bark of apple trees in 1835, is known to be the first natural product substance with SGLT inhibitory activity . Due to In spite of its sufficient inhibitory effects against SGLTs, phlorizin was ultimately considered to be inappropriate for further development as an antihyperglycemic medication due to some critical drawbacks. First, as described above, phlorizin inhibits both SGLT1 and SGLT2 with low therapeutic selectivity. The inhibition of SGLT1, which is primarily localized in the small intestine, can cause several gastrointestinal side effects, such as diarrhea, dehydration, and malabsorption. Second, phlorizin is poorly absorbed in the small intestine, due to its low oral bioavailability. Third, phloretin, a β-glycosidases catalyzed hydrolytic metabolite of phlorizin, strongly inhibits the ubiquitous glucose transporter 1 (GLUT1), which then may obstruct glucose uptake in various tissues ,17,24,25O-glucoside analogs, and an orally-available selective SGLT2 inhibitor T-1095 was developed  versus SGLT1 [IC50 (nM): 1391]. Dose-dependent glucosuric response and decreased fasting and postprandial blood glucose levels were also observed after oral administration of dapagliflozin to rats   , canagliT2: 2.2] ,55,56,57T2: 2.2] . In receT2: 2.2] . Other cT2: 2.2] .Although many SGLT2 inhibitors are now available and extensively used, many efforts to find new active compounds from natural products as viable candidates for novel SGLT2 inhibitor, like phlorizin, are still ongoing. The chemical structures and assay results of several major natural compounds are described in Sophora flavescens (S. flavescens) is one of the most popular and important traditional Chinese medicines. The root of this species (known as “Kushen”) is widely used for the treatment of many diseases, including fever, dysentery, jaundice, leukorrhea, pyogenic skin infection, scabies, swelling, and pain. To date, over 200 constituents have been isolated from S. flavescens, and the major component of this plant were alkaloids and flavonoids . In addition, compound 8 also showed moderate inhibition for SGLT2 [IC50 (μM): 15.3 ± 1.44]. Since the study was designed as a simple screening assessment and the inhibitory activity was established only for SGLT2, the conformational importance of the inhibitory activity and selectivity against SGLT2 was not discussed.More recently, Yang et al.  reportedAcer nikoense (A. nikoense) is native to and widely distributed in Japan, and its stem bark extracts are used in Japanese folk medicine for the treatment of hepatic disorders and eye diseases  and SGLT2 , while other isolated compounds (including one acyclic diarylheptanoid) did not show sufficient inhibition of either SGLT.Morita et al.  evaluateC-11 position  of acerogenin A/B, compounds 11 (acerogenin C), 12, and 13 acquired increased, selective inhibitory activity against SGLT2 versus SGLT1 compared to their precursors at the same tested concentration. Moreover, another derivative of acerogenin A, characterized by dihydroxylation at the C-11 position (14) showed the most potent inhibitory activities against both SGLTs, with non-selectivity. These results suggest that the position and/or presence of a hydroxyl group at C-9 or C-11 and their stereochemistry may not be a key structure for SGLT inhibition. The modification of the diarylheptanoid ring system (such as amide substitution or unsaturated C–C bond formation) resulted in a decrease in SGLT inhibition, indicating that the ring conformation of acerogenins and its derivatives can affect the inhibitory effect of each constituent against SGLTs.Notably, the ketone derivatives at the Alstonia macrophylla (A. macrophylla), also called hard alstonia or hard milkwood, is native to Southeast Asia regions such as Indonesia, Malaysia, the Philippines, Thailand, and Vietnam. This plant has been traditionally used as a general tonic, aphrodisiac, anticholeric, antidysenteric, antipyretic, emmenagogue, and vulnerary agent in Thailand  and alstiphyllanine D . Meanwhile, other ajmaline- and macroline-type alkaloid compounds showed no inhibitory activities against SGLT1 and/or SGLT2. The results from the structure-activity relationship (SAR) approach suggested that the presence of an ester side chain at the C-17 position may play a pivotal role in SGLT inhibitory activity. When further SAR study using eight derivatives was assessed, the hydroxyl derivative in alstiphyllanine D (17) improved the selectivity for SGLT2 more than the others, although the absolute IC50 value was slightly increased .Arai et al.  isolatedGnetum gnemonoides (G. gnemonoides) is a kind of tropical lianas, widely distributed in the Southeast Asia-Pacific region including Malaysia, Indonesia, Philippines, New Guinea, and Bismarck Archipelago. Although there is no scientific report on the medicinal effectiveness of G. gnemonoides yet, it is known that stilbenes isolated from the Gnetum species possess biological properties such as hepatoprotective, antioxidant, antimicrobial, and enzyme inhibitory activities . They also newly discovered two dihydroflavonol-C-glucosides, noidesol A and B, but these compounds showed no SGLT inhibitory potential.Shimokawa et al.  isolatedSchisandra chinensis  is native to Northern China and the Russian Far East, and its fruits are traditionally used as an anti-aging, antitussive, sedative, and tonic agent [S. chinensis contains various phytochemicals including polyphenols, lignans, and triterpenoids, and its pharmacological effects on various organ systems have been extensively assessed [ic agent . S. chinassessed .Qu et al.  recentlyThe effects of three common lignan compounds isolated from F8, deoxyschisandrin, schisandrin B (γ-schisandrin), and schisandrin on SGLT inhibition were finally investigated. However, none of them showed inhibitory activities against either SGLT, suggesting that these major lignans are not the primary components for the SGLT inhibition of SCF.Since the important roles of SGLT2 for renal glucose reabsorption and systemic glucose homeostasis in the human body were elucidated, the inhibition of SGLT2 has been considered a promising therapeutic target for the treatment of type 2 diabetes mellitus. From the beginning of the 21st century, several SGLT2 inhibitors derived from a natural active compound phlorizin, began to be marketed and widely used as monotherapy or in combination with other oral antihyperglycemic agents. Although the therapeutic efficacy of these early-generation SGLT2 inhibitors are well established and most of the reported adverse events associated with drug administration tend to be mild and reversible, we should also not rule out the possibility of unrecognized, unexpected side effects which may be fatal and life-threatening to diabetic patients. It is reported that additional adverse reactions, such as ketoacidosis, urosepsis, pyelonephritis, acute kidney injury, anaphylaxis, and angioedema, have been identified as an early post-marketing experience of canagliflozin and empagliflozin use ,73. FurtIn this review, various active compounds from several natural products with different chemical and biological characteristics from phlorizin and its synthetic analogs were summarized. Although the recent researches on discovering novel SGLT2 inhibitors from natural products are in its beginning stage and the study results are limited to the in vitro screening of SGLT inhibitory activity and SAR approach, further investigations will provide better molecular information on the discovery and development of next-generation SGLT2 inhibitors which are more effective and safer than conventional drugs.