Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PIP3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate PDOI:http://dx.doi.org/10.7554/eLife.26640.001 Upon stimulation, PIP3 levels peak rapidly followed by a prompt return to basal levels (3 degradation are the tumor suppressor phosphatase and tensin homolog (PTEN), which removes the 3-phosphate (3 P) and the SH2-containing inositol 5-phosphatase (SHIP), which dephosphorylates the 5 P to generate PIP2. Deregulation of PIP3 levels is one of the most frequent causes of tumorigenesis Pl levels . The twoigenesis .2, is further metabolized to PI(3)P, a reaction catalyzed by inositol polyphosphate 4-phophatases (INPP4). Accumulation of PIP2 in a case where the INPP4 reaction is rate limiting can be oncogenic, since PIP2 can still partially activate the Akt pathway (The SHIP 5-phosphatase (5-Ptase) family consists of two members, SHIP1 and SHIP2. SHIP1 expression is limited to hematopoietic cells and spermatocytes, whereas SHIP2 is ubiquitously expressed . SHIP1 p pathway , but is  pathway .2+-dependent inositol phosphatases with specificity for 5 P dephosphorylation of inositol rings. These 5-Ptases have different substrate recognition specificities (2+-dependent DNase I and Apurinic/Apyrimidinic (AP) endonucleases  substrate. Together, our data provide novel structural and mechanistic insights on a C2 assisted functioning of SHIP2, and by homology likely of SHIP1.Here, we report the crystal structure of SHIP2 containing the 5-Ptase and a C2 domain (Ptase-C2), revealing that the two domains tightly associate via an extensive interface. We show that, both the Ptase and Ptase-C2 regions of SHIP2 bind phosphatidylserine (PS), which likely localizes the rigid Ptase-C2 region close to its substrate. Importantly, even in the absence of a localization effect, the C2 domain provides activating signals to the Ptase active site, resulting in a significant increase in catalytic turnover. Combining enzyme kinetics, molecular dynamics (MD) simulations and mutagenesis we identify distinct regions at the domain interface with different effects on SHIP2 activity. Hydrophobic Ptase-C2 interactions increase SHIP2 turnover with the lipid chain containing PIP12121 space group with eight molecules per asymmetric unit  was crystallized and diffraction data collected to 1.96 Å. The protein crystallized in the P22  at the tip of the C2 domain P3-diC8 and in all cases we observe a behavior close to conventional Michaelis-Menten enzyme kinetics P3-diC8, whereas Ptase-C2 displays ~ 4 fold faster kinetics with PIP3-diC8 compared to IP4 P3-diC8, where the absence of the C2 domain decreases the KM ~ 2 fold P3 P3.The effect of the C2 domain was further investigated by mutagenesis. In order to disrupt the hydrophobic core of the domain interface we mutated F593 and L597 to aspartates (FLDD mutant). Further, we mutated R649 in the Ptase to alanine (R649A) to prevent polar interactions with E862 and F855 in the C2 domain . Crystal3,4,5)P3 . Togethe4 kinetics and increases activity ~40% with PIP3-diC8  PS-diC16 in the vesicles increases SHIP2 activity ~2–3 fold for both, Ptase and Ptase-C2 P3-diC8 , both foPtase-C2 . The PS-Ptase-C2 . In summIn order to obtain insight on the path of communication between the C2 domain and the active site we performed unbiased molecular dynamics (MD) simulations of the SHIP2 Ptase in presence or absence of the C2 domain. Simulations were started in the doubly bound L4-out conformation, where R682 interacts with D613 and D615 . The simulations indicate that the C2 domain induces several dynamic changes in the Ptase domain, as can be seen from root mean square fluctuations (RMSF) throughout the simulation . Not surOn the other hand, once R682 detaches from D613/D615 we observe a more sustained release of L4 in MD simulations where the C2 domain is present. While in absence of the C2 domain R682 experiences only brief releases followed by immediate rebinding, in the Ptase-C2 simulation R682 remains fully detached from D613/D615 for ~1 μs of the 4.35 μs simulation . Indeed,2+ and substrate failed to reveal clear electron density for the substrate. Therefore we generated a model based on the homologue INPP5B crystal structure containing the PI(4)P product P3-diC8 or IP4 substrates bound according to our model shown in To understand how L4 dynamics could affect catalysis, we next analyzed the mode of substrate binding in SHIP2. Co-crystallization experiments using the catalytically inactive SHIP Ptase-C2 D607A mutant with Mg product  and insi product . We mode product . In this product  and 5C a product . In this2+ ion and a phosphate group P3-diC8 P3 kinetics , R691A, D613A/D615A, R682A, N684A as well as active site mutants D607A and R665A . All mut)P3-diC8 , Table 3kinetics  and 7B–Ckinetics . R691 likinetics  and its kinetics , Table 3  into the SHIP2 sequence and transiently expressed the full-length proteins in HEK293 cells . Express3 levels , the SHIP enzymes lag far behind in terms of a detailed understanding of their functioning and regulation. Surprisingly, even though the Ptase domains of PTEN and SHIP2 bear no homology at the sequence or structural level, they conceptually share an important feature in that they are rigidly linked to a C2 domain P2 domain . Despite2 domain . It appe2 domain . Assumin2 domain  and taki2 domain . Althougactivity . A similactivity . The facactivity  suggests3 substrate. The fact that the R649A mutant, which presumably retains hydrophobic Ptase/C2 interactions, only removes C2 effects on IP4 but not PIP3 kinetics, indicates that R649 communicates with the PIP3 headgroup and hydrophobic interactions with PIP3 lipid chains. Since the PIP3 lipid chains are predicted to interact with L2 P with L2 , this su4, whose cellular roles are only emerging for example in T-cell signaling  and an open (L4-out) conformation  and our ormation . With thormation  facilitaormation , might a2 activity of SHIP2 (3 (2+ coordination compared to other 5-Ptases. An asparagine in INPP5B (N275) providing a Mg2+coordination is replaced by a glycine in SHIP2 (G434) and the coordination is likely provided by a water molecule  and the other at a more buried site (B-site)  . Similar(B-site) , we obseng water . As sugge A-site , hence tucleases .SHIP1 shares an overall 45% sequence identity with SHIP2 (65% within the 5-Ptase and 43% in the C2 domain), suggesting that many of the mechanisms described here are conserved among the two enzymes. Residues at the Ptase-C2 domain interface are mostly conserved, suggesting that the interface as well as a possible communication to L2, which is fully conserved, are also present in SHIP1. The polar path is partially conserved with R649 replaced in SHIP1 by H632, D613/D615 are with D593 and E598 similar and R682 is substituted by K665. The equal length of L4 suggests that it will be able to sample a similar space and likely K665 provides the 3 P specificity in SHIP1.In conclusion, our study provides important insights into how the C2 domain assists SHIP2 catalysis via a combination of membrane positioning, active site stabilization and allosteric signaling, which together is crucial for efficient cellular functioning . Since SThe experimental details regarding the cloning, expression and purification of human SHIP2 Ptase-C2 have been described in . The Pta3, 20% (w/v) PEG3350, 0.025% (v/v) CH2Cl2). Crystals were cryoprotected in the precipitant solution with 25% ethylene glycol and then flash-frozen. SHIP2 Ptase-C2 D607A (5.5 mg/mL) in the presence of PIP3-diC8 and Mg2+ (0.450 mM and 0.750 mM respectively) and SHIP2 Ptase-C2 FLDD (5.32 mg/mL) crystallization conditions were initially rescreened. For SHIP2 Ptase-C2 D607A mixed with PIP3diC8 and Mg2+ conditions were optimized to 0.1 M Bis-Tris propane, pH 7.0, 0.4 M KSCN, 20% (w/v) PEG3350 for SHIP2 Ptase-C2 D607A mixed with PIP3diC8 and Mg2+. The crystals were flash-frozen in precipitant with additional 25% ethylene glycol, 0.220 mM of PIP3diC8 and 0.750 mM of Mg2+. For SHIP2 Ptase-C2 FLDD crystals were grown in 50 mM Hepes pH 7.1, 17.5% PEG 1000 and cryopreserved in additionally 25% ethylene glycol  or 0.1 M PCB buffer pH 7 , 25% PEG 1500 and cryopreserved in additionally 25% ethylene glycol .The crystallization conditions for the Ptase-C2 have been previously described . Briefly12121 space group with unit cell parameters a = 136.04 Å, b = 175.84 Å, c = 176.89 Å. The Matthews coefficient is 2.54 Å3.Da−1 corresponding to 8 molecules in the asymmetric unit (asu) with a solvent content of 51.7% (human SHIP2 Ptase model (PDB: 3NR8) was used as search probe for molecular replacement (MR) using Phaser (2+ and a PO43- were placed in the active site of 6 molecules in the asu (monomers C-H). SHIP2 Ptase-C2 FLDD crystallized in two different space groups, one in P21  with 2 molecules per asu and the other in I2  with 1 molecule per asu. The Ptase-C2 WT model was used in Phaser  and ID29 (λ = 0.976 Å) at European Synchrotron Radiation Facility  and BL13-XALOC-BL13  using a Pilatus 6M detector. XDS  was usedof 51.7% . The cryg Phaser , which ig Phaser  and manug Phaser . Translag Phaser . Final Rg Phaser  was usedn Phaser  to provin Phaser  and furtCircular dichroism was used to assess and compare the overall protein fold of the different constructs. Far-UV CD spectra between 250 and 200 nm of protein at 0.25 mg/mL were run at 20°C for each sample.Tm’s were measured using a ThermoFluor assay. Protein at 1–10 μM was mixed with SYPRO Orange and subjected to a temperature gradient of 0.5°/min from 20 to 95°C and fluorescence recorded at 570 nm.2, washed and developed using an HRP conjugated anti-GST antibody.For the PLO assay dipalmitoyl phosphatidylserine or dipalmitoyl phosphatidylcholine  were spotted on a nitrocellulose membrane, dried and blocked with 5% skimmed milk. The membranes were then incubated for 1 hr with 2.5 μg/ml of GST fused SHIP Ptase or Ptase-C2 in blocking solution in presence or absence of 1 mM CaCl2+ were performed with 0.5 mM CaCl2. For all data shown in For SPR measurements vesicles containing 30% PS  and 70% PC  or 30% PC  and 70% PC (chicken egg) were prepared at a final total lipid concentration of 1.5 mM. Organic solvent was removed by rotary evaporation for 1.5 hr at 45°C. The lipid film was resuspended in SPR running buffer  and subjected to six cycles of freeze-thaw and passed 15 times through a membrane with 100 nm pore size, using a mini extruder (Avanti Polar Lipids). SPR experiments were performed on a Biacore X100 instrument . A L1 sensor chip was washed with two 1 min injections of a 2:3 (v/v) isopropanol: 50 mM NaOH solution at a flow rate of 10 µl/min and then coated by injecting PS containing vesicles in the active flow cell (Fc2) and PC vesicles in the reference flow cell (Fc1), for 15 min at a flow rate of 2 μl/min, followed by two 1 min injections of 10 mM NaOH solution at 10 μl/min to remove loosely bound vesicles and for stabilization. SHIP2 Ptase or Ptase-C2 proteins were injected for 2 min at a flow rate of 30 μl/min in running buffer. SPR responses plotted in 2, 1 mM TCEP, pH 7 in a total volume of 25 μL. The reaction was quenched by addition of 5 μL of 0.5 M EDTA, pH 8. Subsequently, 25 μL of the reaction was mixed with 100 μL of Malachite Green solution (Echelon Biosciences) and left to incubate for 15 min at room temperature and the optical density was measured at 620 nm. Each data point is measured at least in triplicates. For activity assays shown in Activity measurements were performed using a Malachite Green phosphatase activity assay. Proteins were incubated for 2 min at 23°C with the substrate in a reaction buffer containing 20 mM Hepes, 150 mM NaCl, 2 mM MgCl3 vesicles were prepared at a total lipid concentration of 4.5 mM, containing either 10% (mol/mol) PIP3 , 30% (mol/mol) PS  and 60% (mol/mol) PC  or for control vesicles PS was replaced by 30% (mol/mol) PC . The organic solvent was removed by rotary evaporation for 90 min at 45°C. The lipid film lipid was subsequently resuspended in 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, pH 7 and sonicated for 35 min. To calculate the dilution of vesicles in experiments shown in 3-diC16 concentration, present in the outer leaflet of vesicles, was considered.For activity measurements with vesicles two sets of PIPHEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and supplemented with antibiotics. The cells were transiently transfected with the empty vector (pOPINJ) or vector expressing GST fused full-length SHIP2 . The transient transfection was performed using polyethylenimine . After 43, Mg2+ and catalytic waters were based on the INPP5B crystal structure bound to PIP2  and based on AP endonucleases P3 and a net charge of −5 for protein bound IP4/PIP3 were based on previous studies  were applied to the protein and the lipid; 500 steps of steepest descent and 500 steps of conjugate gradient minimization were carried out. In the second stage the lipid harmonic restrains were removed and another 500 steps of minimization were carried out. In the last stage, all restrains were removed and the entire system was minimized for 2000 steps. The resulting configurations were used for MD production: the lipid systems were simulated for a total of 300 ns each, and the non-lipid simulations for 3 × 1.5 μs each (three independent simulations for each system). The first 50 ns of each simulation were considered equilibration time and omitted from the analysis. All minimization and MD production were performed using version 4.6 of the molecular simulations package GROMACS  were modelled with ModLoop  and enerucleases . Lipid p studies . For Pta studies . The sys studies . The siz GROMACS  with the GROMACS  with the GROMACS . The iso GROMACS  and a tiα of the 5-Ptase domain from the total 4.35 μs MD simulations, using the g_rmsf and g_rms tools from the GROMACS package. RMSDs were calculated per residue, using the L4-out structure as reference, and averaged over time. For plots shown in RMSF and RMSD were calculated on the Cα atoms, applying g_covar and g_anaeig tools from GROMACS package on the full MD trajectories, to calculate, diagonalize and analyze the covariance matrix respectively. PCA identifies collective motions associated with the largest variance and the lowest frequencies shown in the first few eigenvectors, thus, we analyzed the first eight PC of each simulation.PCA was performed on all CCluster analysis was performed on all non-hydrogen atoms using g_cluster with the gromos algorithm  and a RM In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by John Kuriyan (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Evzen Boura (Reviewer #3).Thank you for submitting your article "Structural basis for interdomain communication in SHIP2 providing high phosphatase activity" 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.Previous work has established the structure of the SHIP2 phosphatase domain and its relationship with other members of the 5-phosphatase family. This manuscript has established the structure of a pptase-C2 domain construct and examined the role of the C2 domain in catalysis. The current study represents a significant advance in understanding SHIP2. The structure shows that the C2 domain makes an extensive interaction surface with the phosphatase domain. A similar interaction of the C2 domain with the catalytic domain has been observed in other lipid-modifying enzymes, such as PLCδ1 and PTEN. The manuscript establishes that the C2 domain acts as an allosteric activator by stabilising the pptase domain and contributing to membrane binding. The manuscript outlines two distinct pathways of stabilisation contributing to the effect of the C2 domain, a polar pathway manifest on activity with the soluble substrate IP4 and a hydrophobic pathway that specifically affects interactions with the acyl chains of PIP3.The combination of structures and molecular dynamics provides a compelling model for the catalytic cycle of SHIP2 encountering substrate in lipid membranes, with the L4 loop changing conformation between an initial encounter state and a state in which substrate is engaged in the active site. This is an important contribution to our understanding of this family of enzymes. It also provides a detailed picture of how the C2 domain of SHIP2 participates in membrane binding and catalysis.cat and KM as being indicative of allostery (reviewer 1).Several points made by the reviewers (see below) should be taken into account in the revised manuscript. The reviewers have found the allosteric mechanism for the effect of the C2 domain based on crystallography and MD are convincing, but it would be helpful if you could provide a movie that portrays this mechanism. In the revision, you should take account of the suggestions by the reviewers, in particular, in the discussion of an effect on kAn important concern that has been raised by the reviewers is that the lipid blot demonstration of lipid affinity is not adequate for the work. These blots are subject to a range of artifacts. It would be better if you could measure the affinity using lipid vesicles and the FRET assay suggested by reviewer 3. This assay has been described by a number of workers . Also, the methods should be revised to make clear exactly what lipids are being used and how the lipids are prepared for the assay. While the reviewers believe that assays with substrate in lipid vesicles would be an informative addition to the manuscript, it may be that this is beyond the scope of the present work, and this should not be regarded as essential.In an earlier study of several phosphatases (Nordlund and colleagues), the L4 loop of SHIP2 was referred to as the P4-interacting motif (P4IM). It would be helpful for readers familiar with the previous study if the current manuscript also at least mentions this nomenclature.It is claimed at the end of the manuscript that the cellular assays presented show that the C2 domain has an additional role, likely in membrane localization. This statement should be clarified, or if the statement is not based on the results, this should be explained.2+ independent manner" This is misleading. The C2 domain on its own was never tested. It could be that the interaction depends entirely on the pptase domain, but that the pptase is only oriented and stabilised by the C2 domain. The statement consistent with the results would be "both the Ptase only and the Ptase-C2 constructs bind PS in a Ca2+-independent manner."The manuscript claims that "both, the Ptase and C2 domains of SHIP2 bind PS in a Ca2+, which in turn binds to membranes or lys/arg residues that bind directly to the negatively charged lipids. 2+ does not affect binding. It would be helpful if you briefly discuss this point.Common membrane-binding mechanisms are asp/glu residues binding CaFor assistance to color blind readers, it would be helpful if the explanatory keys in In The WT X-ray dataset appears to be the same data set that was described in the earlier crystallisation report, however, the I/σ and highest resolution shell limits are not the same. It appears that the data have been reprocessed, and this is not surprising. However, it should be made clear how the resolution limits were chosen. Since the I/σ for the highest-resolution shells differ for the various datasets, it does not seem that this was the decisive criterion. The correlation coefficients cc1/2 and cc* have been proposed as a criterion to help make resolution cutoff in a statistically reliable manner. The table should also include cc1/2.Reviewer #1:Previous work has established the structure of the SHIP2 phosphatase domain and its relationship with other members of the 5-phosphatase family. The current study by Lietha and colleagues has established the structure of pptase-C2 domain construct and examined the role of the C2 domain in catalysis. A previous report by the same group described crystallisation and diffraction of the pptase-C2 construct and demonstrated its enzymatic activity. The current study represents a significant advance in understanding this enzyme. The structure shows that the C2 domain makes an extensive interaction surface with the phosphatase domain. A similar interaction of the C2 domain with the catalytic domain has been observed in other lipid-modifying enzymes, such as PLCδ1 and PTEN. The authors establish that the C2 domain acts as an allosteric activator by stabilising the pptase domain and contributing to membrane binding. The authors are suggesting two distinct pathways of stabilisation contributing to the effect of the C2 domain, a polar pathway manifest on activity with the soluble substrate IP4 and a hydrophobic pathway that specifically affects interactions with the acyl chains of PIP3.The study provides a compelling model for the catalytic cycle of SHIP2 encountering substrate in lipid membranes, with the L4 loop changing conformation between an initial encounter state and a state in which substrate is engaged in the active site. This is an important contribution to our understanding of this family of enzymes. It also provides a detailed picture of how the C2 domain of SHIP2 participates in membrane binding and catalysis. This is particularly useful, since a similar catalytic domain/C2 module is present in other lipid-modifying enzymes.In a previous study by Nordlund and colleagues, structures of SHIP2, OCRL, and INPP5B pptase domains were determined and compared. In this previous study, it was shown that a motif referred to as the P4-interacting motif (P4IM) interacts with 3- and 4-phosphates of the substrates. In the manuscript of Lietha and colleagues, the same feature is referred to as loop L4. To simplify things for the readers, it would be helpful if the authors of the current manuscript use the P4IM notation or at least point out that this is contained in L4. The presence of the C2 domain results in a large change in the conformation of the L4 loop, and the authors propose that this change likely constitutes an important component of the catalytic cycle of SHIP2 on membranes.cat without much change in KM suggests that the activation is allosteric. The most common definition of allosteric is an effect on activity due to interaction outside the catalytic site. This can be an effect on KM , or it can be an effect on Vmax (V-type), or come combination of these. However, simply an effect on kcat does not suggest an influence directed from outside the active site. A kcat-only effect could be obtained by forming additional interactions with the transition state, but not with the substrate, but this would not be an allosteric effect. What the authors mean is that if the C2 domain has any influence at all, it must be allosteric, since the C2 domain is not near the active site. This should be restated. There is also a grammatic problem with the sentence in its current form (should be "suggests" instead of "suggesting").In the first paragraph of the subsection “The C2 domain affects SHIP2 activity” it states that changing kIn the last paragraph of the subsection “The C2 domain affects SHIP2 activity”, describing In the aforementioned paragraph, it is proposed that PS orients the enzyme on PIP3-containing membranes. This is an interesting proposition that could be tested if the substrate were PIP3 in vesicles, however, the authors are using di-C8 PIP3, which is soluble and not necessarily entirely present in membranes. It is hard to assess what is happening in In the last paragraph of the subsection “Probing the communication path by mutagenesis”, the authors state that their cellular results confirm that C2 interactions observed in the crystal structure are important for SHIP2 activity in cells and that the C2 domain has an additional role, likely in membrane localization. It is not clear how these results show that the C2 domain has an additional role in cells. This should be clarified or the statement removed.Reviewer #2:These authors examine interdomain regulation for the SHP2 phosphatase. In particular, how does the C2 domain affect the phosphatase domain? They report a number of x-ray crystal structures as well as circular dichroism spectra and phosphatase kinetics. Finally, they refine the results with molecular dynamics simulations.They find two conformations: loop L4 in and out. They propose that the C2 domain influences this conformational change. They do a good job with a schematic for their mechanism in 2+ or lys/arg residues that directly to the negatively charged lipids. 2+. But 2+ does not affect binding. This says to me that 1) 2) 3) Reviewer #3:eLife. However, there is one major issue  that should be improved before final acceptance. The additional control experiments should not take more than a day or two of a skilled experimentalist.Le Coq et al. Report the crystal structure of a SHIP2 construct that, unlike previously reported structures, contains the membrane targeting C2 domain. The reported structural analysis together with molecular dynamics simulations reveals how the C2 domain (and thus membrane binding) allosterically controls the enzymatic activity of SHIP2. The findings are important, most of experiments are well done, manuscript is well written and the reasoning is sound and square. The manuscript clearly deserves to be published in 2+. Many reliable standard assays exist. Since SHIP2 contains trypthophan residues I suggest to use a FRET based assay (W residues as donors and dansyl-PE as acceptors) to measure binding.1) A protein lipid overlay assay is a useful screening technique, however, no conclusions should be derived from this method as it is notoriously known to be artifact prone. The authors need to verify their results using liposomes with/out PS and with/out Ca3).2) Biochemical (enzymatic) analysis is done using soluble substrates only. To explore the effect of allosteric regulation these data need to be compared with membrane embedded substrates P Several points made by the reviewers (see below) should be taken into account in the revised manuscript. The reviewers have found the allosteric mechanism for the effect of the C2 domain based on crystallography and MD are convincing, but it would be helpful if you could provide a movie that portrays this mechanism.We now provide 5 videos:cat and KM as being indicative of allostery (reviewer 1).In the revision, you should take account of the suggestions by the reviewers, in particular, in the discussion of an effect on kWe have corrected our statement according to the comment of reviewer 1 .An important concern that has been raised by the reviewers is that the lipid blot demonstration of lipid affinity is not adequate for the work. These blots are subject to a range of artifacts. It would be better if you could measure the affinity using lipid vesicles and the FRET assay suggested by reviewer 3. This assay has been described by a number of workers .As requested, we performed binding experiments with lipid vesicles. We initially tested the FRET assay suggested by reviewer 3. Under replies to reviewer #3 we explain and show data indicating that this assay is not suitable to measure SHIP interactions with PS vesicles.We therefore performed lipid vesicle interaction studies by surface plasmon resonance using a Biacore X100 instrument and a L1 sensor chip with immobilized PS vesicles. These data are shown in Also, the methods should be revised to make clear exactly what lipids are being used and how the lipids are prepared for the assay.We apologize for not specifying the type of lipids used in activity assays. In the previous submission we used soluble short chain lipids PS-diC8 or PC-diC8. This is now clearly stated in the main text , While the reviewers believe that assays with substrate in lipid vesicles would be an informative addition to the manuscript, it may be that this is beyond the scope of the present work, and this should not be regarded as essential.3 vesicles has a larger activating effect than soluble PS-diC8.We agree that this provides an informative addition and have performed activity assays with substrate in vesicles . These dIn an earlier study of several phosphatases (Nordlund and colleagues), the L4 loop of SHIP2 was referred to as the P4-interacting motif (P4IM). It would be helpful for readers familiar with the previous study if the current manuscript also at least mentions this nomenclature.In the fifth paragraph of the Discussion we now refer to the P4IM terminology. However, since 2 residues of the P4IM are located outside L4 (for details see replies to reviewer #1”) and because Arg682 in SHIP2 likely has an important role in P3 substrate interactions, we believe it would be misleading to use the P4IM terminology for SHIP throughout the manuscript.It is claimed at the end of the manuscript that the cellular assays presented show that the C2 domain has an additional role, likely in membrane localization. This statement should be clarified, or if the statement is not based on the results, this should be explained.This statement was based on the observation that deleting the C2 domain has a larger cellular effect than the FLDD mutations. However, we agree this statement could be misleading. We now simply state: “This confirms that C2 interactions observed in the crystal structure are important for SHIP2 activity in cells.”2+ independent manner" This is misleading. The C2 domain on its own was never tested. It could be that the interaction depends entirely on the pptase domain, but that the pptase is only oriented and stabilised by the C2 domain. The statement consistent with the results would be "both the Ptase only and the Ptase-C2 constructs bind PS in a Ca2+-independent manner."The manuscript claims that "both, the Ptase and C2 domains of SHIP2 bind PS in a CaWe have rewritten the statement as suggested (subsection “The C2 domain of SHIP2”).2+, which in turn binds to membranes or lys/arg residues that bind directly to the negatively charged lipids. 2+ does not affect binding. It would be helpful if you briefly discuss this point.Common membrane-binding mechanisms are asp/glu residues binding Ca2+ .In order to show higher significance that is not related to differences in SHIP expression, we performed 5 additional experiments (each in triplicates), therefore the data we now present in The WT X-ray dataset appears to be the same data set that was described in the earlier crystallisation report, however, the I/σ and highest resolution shell limits are not the same. It appears that the data have been reprocessed, and this is not surprising. However, it should be made clear how the resolution limits were chosen. Since the I/σ for the highest-resolution shells differ for the various datasets, it does not seem that this was the decisive criterion. The correlation coefficients cc1/2 and cc* have been proposed as a criterion to help make resolution cutoff in a statistically reliable manner. The table should also include cc1/2.scala (used to cut data) and newer versions of aimless (reported in the table) caused the high I/σ values (see replies to reviewer #3 for a more detailed explanation). We have now reprocessed the data to higher resolution and cut the data at a mean (I/sd), reported in aimless, of ~2.We apologize for this and thank the reviewers for pointing this out. Indeed, the data were reprocessed since it was published in the Protein Journal. Different definitions of I/σ in older versions of CC(1/2) values are now included in Reviewer #1:[…] In a previous study by Nordlund and colleagues, structures of SHIP2, OCRL, and INPP5B pptase domains were determined and compared. In this previous study, it was shown that a motif referred to as the P4-interacting motif (P4IM) interacts with 3- and 4-phosphates of the substrates. In the manuscript of Lietha and colleagues, the same feature is referred to as loop L4. To simplify things for the readers, it would be helpful if the authors of the current manuscript use the P4IM notation or at least point out that this is contained in L4. The presence of the C2 domain results in a large change in the conformation of the L4 loop, and the authors propose that this change likely constitutes an important component of the catalytic cycle of SHIP2 on membranes.In Tresaugues et al. (2014) the P4-interacting-motif (P4IM) is defined to be composed of the INPP5B residues Tyr502, Lys503, Arg518, and Lys516. Of these, only Arg518 (Asn684 in SHIP2) and Lys516 (Arg682 in SHIP2) are contained within loop L4. As suggested we point out that residues Asn684 and Arg682 in SHIP2 correspond to residues in the P4IM . However, since 2 residues of the P4IM are located outside L4 and because Arg682 in SHIP2 likely has an important role in P3 substrate interactions, we believe it would be misleading to use the P4IM terminology in SHIP throughout the manuscript.cat without much change in KM suggests that the activation is allosteric. The most common definition of allosteric is an effect on activity due to interaction outside the catalytic site. This can be an effect on KM , or it can be an effect on Vmax (V-type), or come combination of these. However, simply an effect on kcat does not suggest an influence directed from outside the active site. A kcat-only effect could be obtained by forming additional interactions with the transition state, but not with the substrate, but this would not be an allosteric effect. What the authors mean is that if the C2 domain has any influence at all, it must be allosteric, since the C2 domain is not near the active site. This should be restated. There is also a grammatic problem with the sentence in its current form (should be "suggests" instead of "suggesting").In the first paragraph of the subsection “The C2 domain affects SHIP2 activity” it states that changing kWe thank the reviewer for pointing this out. We now simply state “The fact that the distant C2 domain has specific and differential effects on catalysis of the two substrates, suggests the presence of an allosteric communication between the domain interface and the active site.”In the last paragraph of the subsection “The C2 domain affects SHIP2 activity”, describing We apologize for not specifying the type of lipids used. We now state in the main text of the Results section , Ideally, it would be helpful if the authors had used long-chain PIP3 incorporated into unilamellar vesicles for at least some of their assays, in order to strengthen their conclusions for the behavior of SHIP2 in cells.As suggested, we performed additional experiments with long-chain PIP3 embedded in vesicles containing 30% PS or PC. The new data are shown in In the aforementioned paragraph, it is proposed that PS orients the enzyme on PIP3-containing membranes. This is an interesting proposition that could be tested if the substrate were PIP3 in vesicles, however, the authors are using di-C8 PIP3, which is soluble and not necessarily entirely present in membranes. It is hard to assess what is happening in As explained above, we have now performed such studies and show the results in In the last paragraph of the subsection “Probing the communication path by mutagenesis”, the authors state that their cellular results confirm that C2 interactions observed in the crystal structure are important for SHIP2 activity in cells and that the C2 domain has an additional role, likely in membrane localization. It is not clear how these results show that the C2 domain has an additional role in cells. This should be clarified or the statement removed.We agree this statement could be misleading. It was based on cell biology data showing that deletion of C2 has a much stronger effect than the FLDD mutation. However, new data presented in Reviewer #2:[…] This study includes a very large number of experiments and addresses an important question. However, there are a number of points that could be improved. My suggestions are as follow:2+ or lys/arg residues that directly to the negatively charged lipids. 2+. But 2+ does not affect binding. This says to me that 1) 2+  We have clearly labelled the curves that show a different behavior. According to the new numbering this is now in 3) Actually, we do detect a very weak band corresponding to endogenous SHIP2 in the control lane. However, since SHIP expression with the pOPINJ vector adds an N-terminal GST tag P P2 has still partial activity towards Akt); 2) What the relative contributions are of SHIP and PTEN in PIP3 degradation under the chosen condition and cellular context and how these activities compare to PI3K activity generating PIP3; 3) How efficiently is the overexpressed SHIP2 targeted to the membrane under the experimental conditions? We believe any of these factors could cause a limited effect of the expressed SHIP proteins on reducing Akt phosphorylation.Regarding the limited decrease in pAkt, we believe there could be several reasons explaining this behavior. As described in the Introduction, the effect of SHIP on Akt phosphorylation is complex and likely depends on several other factors, including: 1) Whether the SHIP product PIPSecond, the deletion of the C2 domain clearly reduces activity, but the FLDD mutation has only a minute affect, which they say is statistically significant. They do not explain the statistical analysis. How was the error for PAkt propagated with the error in Akt?We do not apply error propagation of the pAkt and total Akt measurements, because the two quantities are not “uncorrelated”. The typical error propagation for a ratio Q according to the formula:. Admittedly, the only partial reduction in pAkt levels upon SHIP expression limits the window showing clear significance and the FLDD mutations having only a partial reduction in activity further limits the significance. Nevertheless, after 8 triplicate experiments (n=24), our results do still show significance between WT and FLDD mutants. Taking all experiments together the total SHIP expression levels are no longer smaller for the FLDD mutant , making it unlikely that differences in pAkt levels are due to different SHIP expression levels. We would also like to point out that in the revised manuscript we include activity data with PIP3 embedded in vesicles  that furWe now clearly state in the Materials and methods section how we analyze the statistics for cell biology experiments, i.e. that standard errors are calculated on values of pAkt/Akt ratios and significance is calculated using a two-tailed unpaired Student t test assuming Gaussian distribution.Reviewer #3:Le Coq et al. Report the crystal structure of a SHIP2 construct that, unlike previously reported structures, contains the membrane targeting C2 domain. The reported structural analysis together with molecular dynamics simulations reveals how the C2 domain (and thus membrane binding) allosterically controls the enzymatic activity of SHIP2. The findings are important, most of experiments are well done, manuscript is well written and the reasoning is sound and square. The manuscript clearly deserves to be published in eLife. However, there is one major issue  that should be improved before final acceptance. The additional control experiments should not take more than a day or two of a skilled experimentalist.2+. Many reliable standard assays exist. Since SHIP2 contains trypthophan residues I suggest to use a FRET based assay (W residues as donors and dansyl-PE as acceptors) to measure binding.1) A protein lipid overlay assay is a useful screening technique, however, no conclusions should be derived from this method as it is notoriously known to be artifact prone. The authors need to verify their results using liposomes with/out PS and with/out CaAs requested, we performed binding experiments with lipid vesicles. We initially tested the suggested FRET assay using energy transfer between excited tryptophans and dansyl-PE. However, instead of a FRET induced increase at the dansyl emission in presence of protein, we observed a decrease see . The reaon/koff values from SPR sensorgrams. Binding and dissociation appear to follow non-trivial kinetics, which we were not able to fit with standard models. On the other hand, plotting steady-state values versus protein concentration suggests that KD values are significantly above the highest protein concentration used (10µM).In an alternative approach, we performed lipid vesicle interaction studies by surface plasmon resonance (SPR) using a Biacore X100 instrument and a L1 sensor chip with immobilized PS and PC vesicles. These data are shown in 2+ only with Ptase-C2, increasing the binding by ~10%. We do not claim that this modest effect is biologically significant, but as discussed in the subsection “The C2 domain of SHIP2”, this effect might result from the low conservation of Ca2+ binding residues in CBL1 and 3 of the SHIP2 C2 domain.Injecting higher protein concentrations caused irreversible deposition of protein on the surface making responses unreliable. Interestingly, using injections at 5 µM protein we detected a weak but significant effect of Ca3).2) Biochemical (enzymatic) analysis is done using soluble substrates only. To explore the effect of allosteric regulation these data need to be compared with membrane embedded substrates PAs suggested, we performed enzyme activity assays with vesicles containing PIP3. These data are presented in


A defining feature of eukaryotic cells is the presence of various distinct membrane-bound compartments with different metabolic roles. Material exchange between most compartments occurs via a sophisticated vesicle trafficking system. This intricate cellular architecture of eukaryotes appears to have emerged suddenly, about 2 billion years ago, from much less complex ancestors. How the eukaryotic cell acquired its internal complexity is poorly understood, partly because no prokaryotic precursors have been found for many key factors involved in compartmentalization. One exception is the Cdc48 protein family, which consists of several distinct classical ATPases associated with various cellular activities (AAA+) proteins with two consecutive AAA domains.Here, we have classified the Cdc48 family through iterative use of hidden Markov models and tree building. We found only one type, Cdc48, in prokaryotes, although a set of eight diverged members that function at distinct subcellular compartments were retrieved from eukaryotes and were probably present in the last eukaryotic common ancestor (LECA). Pronounced changes in sequence and domain structure during the radiation into the LECA set are delineated. Moreover, our analysis brings to light lineage-specific losses and duplications that often reflect important biological changes. Remarkably, we also found evidence for internal duplications within the LECA set that probably occurred during the rise of the eukaryotic cell.Our analysis corroborates the idea that the diversification of the Cdc48 family is closely intertwined with the development of the compartments of the eukaryotic cell.The online version of this article (doi:10.1186/s12862-016-0790-1) contains supplementary material, which is available to authorized users. In contrast to prokaryotes, which generally consist of a single intracellular chamber surrounded by a plasma membrane, eukaryotic cells are subdivided into various functionally distinct internal membrane-bounded compartments, including the nuclear membrane, the endoplasmic reticulum (ER), the Golgi apparatus, lysosomes, endosomes, and the cell membrane. Material exchange between compartments of this vast endomembrane system occurs by membrane-enclosed vesicles that bud off from one membrane and specifically fuse with an acceptor compartment after moving along cytoskeletal tracks.N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. These tail-anchored membrane proteins operate via a fundamental mechanism: their sequential assembly into tight membrane-bridging complexes pulls the two membranes together. Their activity is orchestrated by various other conserved factors including Sec1/Munc18 (SM), Rab, and tethering proteins [The central machine involved in the vesicle fusion process in each trafficking step is composed of soluble proteins . During proteins –9. IntriN-ethylmaleimide-sensitive factor (NSF), which is mostly present as singleton in all eukaryotes. This essential factor was originally discovered on the basis of its role in ER-Golgi trafficking [Breaking apart SNARE complexes requires the activity of the ATPase fficking . Togethefficking , 12. FroN-terminal domain that interacts with the SNARE complex and the SNAP adaptor protein [Drosophila, as mac-1 in Caenorhabditis elegans, or as Rix7 in Saccharomyces cerevisiae) and cytosolic ribosome maturation  [Thermoplasma acidophilum (VAT)) is present in archaea and in some eubacteria [When searching for the origins of the disassembly machinery, one does not need to enter unknown territory, as NSF is one of the founding members of the large superfamily of ATPases associated with various cellular activities (AAA+) (reviewed in –18) that protein –26. NSF  protein . Initial protein –31). Its protein , 33. Theevisiae) , and regevisiae) . Intriguevisiae) , 20, 36.bacteria ), a detaAlthough the Cdc48 family has successively emerged as a single clade in earlier phylogenetic studies , 36–41, We started by collecting approximately 600 AAA domain-containing sequences of established Cdc48 family members . In geneOur exhaustive classification revealed that in eukaryotes, the Cdc48 family consists of eight different types, whereas generally only one type is found in prokaryotes. An overview of the domain structure of the different eukaryotic family members is given in Fig. N-domain and two distinct AAA domains in tandem. A detailed arrangement of the secondary structural elements of Cdc48 is given in Additional file N-domains. In addition, Yta7 also possesses a bromodomain within its second D-domain. Bromodomains are interaction modules that specifically recognize ε-N-lysine acetylation motifs, a modification found mostly in histones. More subtle changes occurred in the D-domains of other family members. For example, the D1-domain of NVL has an insert approximately 50 amino acids long before the terminal helix of the domain [By and large, the domain arrangement of several family members have remained similar to that of Cdc48, comprising an e domain . Furthere domain  and NVL In order to resolve the phylogenetic relationships of the Cdc48 protein family, we first calculated the phylogenetic trees of the individual D-domains. To reduce bias and to minimize computational effort, we generated a reduced list of species that represent all known major lineages of the eukaryotic phylum. To root the tree, we included several Archaea sequences .N-domain consisting of two smaller subdomains [N-domains. To complement our survey, we examined the evolutionary history of this domain as well. Taking advantage of our sequence collection, we made specific HHMs for each family member with a canonical N-domain. As well as the proteins listed above, our list of family members that have an obvious canonical N-domain included Spaf, Spaf-like, and Pex6. We then calculated a phylogenetic tree for all well-conserved N-domains  while Spaf/Drg1 has been maintained more widely. On the other hand, we noted that both factors were found to interact in a large-scale screen  and it sN-domains. Although several family members have an αβ N-domain, the N-terminal region of NVL is mostly α-helical. Interestingly, the N-terminal domain of NVL contains a nuclear localization signal that steers the factor into the nucleus to work in early steps of ribosome biogenesis, whereas Cdc48 is found in the cytosol and the nucleus.Another putative pre-LECA duplication might have led to the rise of the two factors Cdc48 and NVL. The D-domains of these two factors are conserved, but they comprise two structurally different types of As mentioned above, the LECA possessed at least eight distinct members of the Cdc48 family. If we take the internal duplications into account, this set can be arranged into five distinct basic types  that are likely to have worked in an earlier phase of the evolution of the eukaryotic cell. According to our analysis, NSF, Pex1/6, and Yta7 have diverged more, probably to adapt to novel functions, whereas Cdc48/NVL and Spaf/Spaf-like remained more archetypical.de novo from the ER [The internal duplications within the Cdc48 family must have occurred in earlier evolutionary stages of the eukaryotic cell. For example, it has been recognized that the machinery for peroxisomal protein import is homologous to the machinery that degrades ER-associated proteins (ERAD) , 65, 66.m the ER –69.S. cerevisiae. We were unable to detect Spaf-like in all the alveolates that we inspected. Because of the lack of functional data, it is currently impossible to correlate these losses to a change in a particular cellular process. The same can be said for the conspicuous absence of the chromatin-interacting factor Yta7 in alveolates, for example.To detect losses and gains in extant eukaryotic lineages, we calculated individual phylogenetic trees for each of the family members. We found that the Cdc48 family member Spaf-like has been lost independently in several lineages. For example, although we found Spaf-like in the genome of a few basal fungi, it has been lost in all other fungi including Giardia and Microsporidia. Their genomes seem to encode only for Cdc48, NSF, and NVL. Notably, we found these three factors to be present in every eukaryotic genome, suggesting that they represent the minimal set for eukaryotic cell function, while the other factors appear to be expendable under certain conditions.As expected, drastic losses occurred in parasitic lineages. An account is given in Table Encephalitozoon cuniculi (Microsporidia), Giardia lamblia (Diplomonads), Entamoeba histolytica (Archamoebae), and Trichomonas vaginalis (Trichomonads) as well as several apicomplexans [Blastocystis hominis, suggesting that this intestinal parasite does not have peroxisomes. Furthermore, we did not find Pex1 and Pex6 in many apicomplexans, corroborating earlier accounts [Eimeria and Toxoplasma. Both factors are also present in the genome of the chromerids Chromera velia and Vitrella brassicaformis, which are photosynthetic relatives of apicomplexan parasites [It had been noticed earlier that some parasitic protists do not possess peroxisomes, such as the amitochondriate eukaryotes mplexans , 70, 71.arasites .Trichinella and Trichuris. This raises the possibility that these nematode lineages might have lost their peroxisomes as well. Furthermore, we did not find both peroxins in the free-living tunicate Oikopleura dioica. Similar observations have been reported recently [Recently, the absence of key peroxisomal markers in several genomes of parasitic flatworm were reported , 74. Indrecently . HoweverIn our analysis, we came across an already established multiplication of Cdc48 that has occurred only in eukaryotic lineages that have engulfed a red algae. The secondary endosymbiosis of another eukaryotic cell, followed by several reductions into a so-called red complex plastid, has taken place in cryptophytes, alveolates, stramenopiles, and haptophytes (CASH) lineages (reviewed in –80). In In order to take a closer look at Cdc48’s phylogenetic distribution, we calculated a tree of Cdc48 from CASH lineages and archaeplastida. In agreement with previous studies , 81–83, N-terminal signal sequence and are thought to reach their destination through the host cell’s ER, which is connected with the outermost membrane of the red algae. Interestingly, the host genome of the cryptophyte Guillardia theta encodes for two Cdc48 variants, one of which, sCdc48, carries an N-terminal signal sequence. According to our phylogenetic analysis, one sCdc48 from G. theta is a SELMA component, while the other Cdc48 is working in the ERAD machinery of the host cell. A third Cdc48, a SELMA Cdc48, is encoded by the so-called nucleomorph, which is a residual cell nucleus of the engulfed red algae endosymbiont within the PPC of cryptophytes [In most lineages, SELMA components are encoded in the host nucleus, which is a good example of endosymbiotic gene transfer. SELMA components carry an tophytes  . With this extended dataset, we refined our hierarchical classification and further re-trained the HMMs. These models were then used to supplement the dataset by scanning various other sequence resources . The vast majority of identified domains matched one subgroup significantly better than any other (see Additional file For phylogenetic reconstructions, we used a combination of three different programs (IQ-TREE , 105, Rahttp://bioinformatics.mpibpc.mpg.de/aaa/index.jsp to provide access to our results. It is divided into three sections. The first section provides access to our collected information, which can be searched for groups, species, and protein names. The second section allows users to submit new sequences to our HMM models. We have implemented the expectation value cut-off to reflect the strict and soft bounds for each family (see Additional file We have implemented a web-based interface called AAA Database at


Drosophila) and T27A3.1 (C. elegans). HAP1, TRAK1 and TRAK2 have a degree of common function, particularly regarding intracellular receptor trafficking. However, TRAK1, TRAK2 and Milton (which have a “Milt/TRAK” domain that is absent in human and rodent HAP1) differ in function to HAP1 in that they are mitochondrial transport proteins, while HAP1 has emerging roles in starvation response. We have investigated HAP1 function by examining its evolution, and upstream gene promoter sequences. We performed phylogenetic analyses of the HAP1_N domain family of proteins, incorporating HAP1 orthologues (identified by genomic synteny) from 5 vertebrate classes, and also searched the Dictyostelium proteome for a common ancestor. Computational analyses of mammalian HAP1 gene promoters were performed to identify phylogenetically conserved regulatory motifs.Huntingtin-associated Protein 1 (HAP1) is expressed in neurons and endocrine cells, and is critical for postnatal survival in mice. HAP1 shares a conserved “HAP1_N” domain with TRAfficking Kinesin proteins TRAK1 and TRAK2 (vertebrate), Milton , and myc-associated factor X (MAX) in the region of the TATA box. CDX1 and CDX2 are intestine-enriched factors, prompting investigation of HAP1 protein expression in the human duodenum. HAP1 was localized to singly dispersed mucosal cells, including a subset of serotonin-positive enterochromaffin cells.We found that as recently as marsupials, HAP1 contained a Milt/TRAK domain and was more similar to TRAK1 and TRAK2 than to eutherian HAP1. The Milt/TRAK domain likely arose post multicellularity, as it was absent in the We have identified eutherian HAP1 as an evolutionarily recent adaptation of a vertebrate TRAK protein-like ancestor, and found conserved CDX1/CDX2 and MAX transcription factor binding sites near the TATA box in mammalian HAP1 gene promoters. We also demonstrated that HAP1 is expressed in endocrine cells of the human gut.The online version of this article (doi:10.1186/s12862-016-0780-3) contains supplementary material, which is available to authorized users. Huntingtin-associated Protein 1 (HAP1) is a ~100 kDa protein that was first identified in a screen for proteins that interact with the Huntington’s disease (HD) gene product, huntingtin (HTT) , and assglued AAA), CDX1, and CDX2 (5′TTT[A/G]T), such that the minor allele abolishes the prediction of all of these sites by the MatInspector software. The SNP is well represented in the Encyclopedia of DNA Elements (ENCODE) which provided functional supporting evidence from ChIP-seq experiments that TBP, and associated regulatory factors including RNA polymerase II, bind at this genomic location in a number of human cell lines [The order, position and strand orientation of the transcription factors common to mammalian HAP1 gene promoters were compared between species to identify common patterns. Most notably, there was a site for TATA-binding protein (TBP), also known as the ‘TATA box’, on the sense strand, overlapped on the opposite strand by a site recognized by caudal-type homeobox transcription factors CDX1 and CDX2 . These sites were identically arranged in all HAP1 gene promoters tested here Fig. . A site ll lines . This SNll lines .Fig. 4PrA search of the Genomatix human gene promoter database (112705 sequences) was performed using the ModelInspector program , to idenCDX1 and CDX2 are intestine-specific transcription factors differentially involved in enterocyte proliferation and differentiation. The presence of conserved sites for these factors in HAP1 gene promoters prompted us to investigate HAP1 expression in the intestine. HAP1 protein is known to be present in singly dispersed cells of the mucosal layer of the stomach and duodenum of mice . EnteroeDrosophila) and T27A3.1 (C. elegans). Previous studies have identified similarities in sequence and function between TRAK1, TRAK2 and Milton which possess an additional domain, the Milt/TRAK domain, further carboxyl to the HAP1_N domain. The C. elegans homolog, T27A3.1, has been identified as a homolog of mammalian HAP1 [HAP1 of rodent and human have been recognized as sharing a region of homology (the HAP1_N domain) with TRAK1 and 2 proteins, and invertebrate proteins Milton , HAP1 orthologues in human, rat, mouse, dog, rabbit, opossum, Tasmanian devil, Drosophila. Within the proteome of the early eukaryote Dictyostelium, we found regions of similarity to the HAP1_N domain in actin binding and cytoskeletal proteins but no similarity to the Milt/TRAK domain. We therefore propose that the HAP1_N domain has ancient origins in actin/microtubule attachment and transport functions, and that the Milt/TRAK domain arose post multicellularity but prior to the divergence of protostomes and deuterostomes. Given that HAP1_N proteins with a Milt/TRAK domain  function in targeted transport of mitochondria to distant cellular regions of energy need, a putative ancestral homolog containing HAP1_N and Milt/TRAK domains may have evolved with the development of longer cell types in multicellular organisms.HAP1_N family members with TRAK domains exist in both vertebrates and Xenopus and opossum), than observed between TRAK1 and TRAK2 orthologues. This suggests HAP1 sequences were already diverging (compared to TRAK proteins) prior to loss of the Milt/TRAK domain in eutherians. However loss of the Milt/TRAK domain may have further altered the selective pressure on the eutherian HAP1 domain, as the latter domain has diverged approximately 3 times more in eutherians than in marsupials, since their last common ancestor.In deuterostomes, the number of Milt/TRAK domain-containing HAP1 proteins has increased such that in vertebrates there are three homologs . Since all 5 classes of vertebrate have these 3 paralogs, the increase in paralog number most likely occurred prior to divergence of the vertebrate lineage into these classes. Sequence analyses indicated less similarity amongst the vertebrate Milt/TRAK domain-containing HAP1 orthologues  [via sequence(s) within the first 750aa, spanning the HAP1_N and Milt/TRAK1 domains [To our knowledge, no protein interactions of Milton, TRAK1, or TRAK2 have been specifically attributed to the Milt/TRAK domain, and it remains to be determined how loss of the Milt/TRAK domain, shortening of the carboxyl terminus, and divergence of the HAP1_N domain have altered the function of eutherian HAP1 orthologues. Loss of the Milt/TRAK domain and carboxyl terminus may have affected the ancestral role in mitochondrial trafficking since 47–1116)  and this domains .Hap1 gene leads to defects in mouse cellular models, and causes postnatal starvation in mice. Mouse HAP1 has pro-catabolic attributes . These factors may act upstream of HAP1’s roles in postnatal development [We utilized computational promoter analyses to gain insight into transcription factors and signaling pathways likely to regulate HAP1 transcriptional activity. We found the analyzed mammalian HAP1 gene promoters to be rich in sites for factors involved in the cell cycle, growth, and differentiation. The predominance of transcription factors involved in cell cycle control  suggests a key association of HAP1 with this process. HAP1 has been shown to have an inhibitory effect on the growth rate of breast cancer cell lines, and is under-expressed in breast cancer tissues . This ne150glued , PCM1 [3150glued , 24 and 150glued . Other felopment , 28, neuelopment , neuronaelopment  and neurelopment . Bindingelopment . NRF1 acelopment .Hap1 promoters (opossum and Tasmanian devil) contained two TATA box motifs in close proximity, further upstream from the ORF than in eutherians (the closest motif starting 197 and 238 nucleotides upstream of the ORF). Marsupial Hap1 promoters also lacked XCPE1 sites, that were detected on the reverse strand within the TATA box region of most eutherian promoters tested. XCPE1 sites are general core promoter elements that can enhance transcription activated by other factors (such as NRF1) in in vitro promoter assays [TATA box motifs were present in all mammalian HAP1 gene promoters tested. This motif, named after its consensus DNA recognition sequence (5′ TATAAA), is found in approximately one third of vertebrate promoters . It is tr assays .Notably, sites for MAX, CDX1 and 2, and TBP were found to form a highly conserved promoter model common to all mammalian promoters tested. The model consisted of a TATA box on the sense strand, a site for CDX1 and CDX2 on the antisense strand overlapping the TATA box, and a MAX site shortly upstream. CDX1 and CDX2 are predominantly intestine-specific transcriptional factors involved in intestinal development, proliferation and differentiation, and are differentially expressed along the intestine and in the crypt/villus axis . Myc-assSonic hedgehog (Shh) [glucose-6-phosphatase (G6PC) [calbindin-D9K [clusterin [G6PC gene (which is required for the catabolic processes of gluconeogenesis and glycogenolysis), transcription is activated by CDX1, and this activation is inhibited by CDX2, suggesting the existence of a competitive regulatory mechanism [GO-term enrichment associated with other genes with this promoter model (investigated with and without inclusion of the MAX site) identified biological processes potentially regulated by this combination of factors. In addition to processes in which HAP1 has been implicated, such as microtubule-based movement , nervousog (Shh) , glucosee (G6PC) , calbindndin-D9K  and cluslusterin . In the echanism .rs116737192) was found to occur at a critical nucleotide in the binding motifs for TBP, CDX1 and CDX2 in the HAP1 promoter. Individuals homozygous for the minor allele present at expected Mendelian frequency amongst African populations  in singly dispersed mucosal cells in the human duodenum. Moreover, a subset of these cells were serotonin (5-HT)-containing EC cells, which also express CDX2 [via a unique mechanism involving a small fusion pore [Whilst HAP1 protein has been detected in the stomach and duodenum in mouse , localizess CDX2 . Expressess CDX2 –75, gathess CDX2 –78. Furtess CDX2 , 80, andess CDX2 , likely ion pore , 82. Wheion pore , 26 is cIn this study we have identified eutherian HAP1 as an evolutionarily recent adaptation of a vertebrate TRAK protein-like ancestor. The function of the Milt/TRAK domain, how loss of this domain has affected HAP1 function since divergence from marsupials, and whether/how this provides a selective advantage in eutherians, particularly in the postnatal period, are questions remaining to be addressed. We have identified conserved regulatory elements amongst mammalian HAP1 gene promoters that provide insight into specific transcription factors and biological processes likely to act upstream of HAP1 gene transcription. Finally, prompted by the presence of a conserved promoter model including sites for intestinally expressed transcription factors  in the TATA box region of the promoter, we assessed HAP1 expression in the human small intestine. We detected the protein in singly dispersed mucosal cells, including within a subset of serotonin (5-HT) positive EC cells, with partial intracellular colocalization suggesting a role for HAP1 in peripheral 5-HT secretion.HAP1 genes examined were human (H. sapiens ENSG00000173805), rat (R. norvegicus ENSRNOG00000014819), mouse (M. musculus ENSMUSG00000006930), dog (C. familiaris ENSCAFG00000015920), horse , rabbit (O. cuniculus ENSOCUG00000000605), opossum (M. domesticus ENSMODG00000014659), Tasmanian devil (S. harrisii ENSSHAG00000005125), zebrafish (D. rerio ENSDARG00000074508), chicken , anole lizard (A. carolinensis ENSACAG00000017899) and clawed frog . Protein sequences and domain predictions were obtained where possible from Ensembl, or otherwise from NCBI if a more recent build was available . Protein sequence alignment and identity comparisons were conducted using the Clustal Omega  algorithm [Drosophila Milton HAP1_N domain sequence as the outgroup, and clustering outcomes were tested by bootstrapping (1000 replicates). Protein sequence comparisons were performed using the following sequences , opossum , Tasmanian devil (S. harrisii HAP1: XP_012403736.1), rat (R. norvegicus HAP1: ENSRNOP00000072494.1), dog (C. familiaris HAP1: ENSCAFP00000023416.4), zebrafish , clawed frog , chicken , anole lizard , nematode (C. elegans T27A3.1: T27A3.1a.1), fruitfly (D. melanogaster Milton: FBpp0297338.1). Dictyostelium proteins with regions of similarity to human TRAK1 and Drosophila Milton were identified in proteins encoded by the Dictyostelium genome [via dictyBase [Gene information for synteny analysis was acquired from the Ensembl genome browser  first frlgorithm , by firsm genome , via dicHap1 mRNA sequences from NCBI  were used to identify  associasitivity , and thoDuodenal biopsies were collected from 3 healthy subjects, aged 18–75 with no previous gastrointestinal disease and normal renal and kidney function. Subjects fasted overnight then attended the laboratory at 0800. A small diameter video endoscope  was passed into the second part of the duodenum, and mucosal biopsies collected using standard biopsy forceps. These were immediately placed into 4 % paraformaldehyde for 2 h, cryoprotected ), embedded in cryomolds, and frozen before sectioning at 6–10 μm  and thaw-mounting onto gelatin-coated slides. HAP1 immunoreactivity was detected using a mouse primary antibody  following antigen retrieval according to manufacturer’s instructions . 5-hydroxy tryptamine (5-HT) was subsequently detected using a goat primary . Immunoreactivity was visualized using species-specific secondary antibodies (1:200 in PBS-Tween 20) conjugated to Alexa Fluor dyes for HAP1  and 5-HT . Nuclei were visualized using DAPI mounting media .


The multi-domain transcriptional coactivators CBP/p300 integrate a multitude of signaling inputs, interacting with more than 400 proteins via one or more of their globular domains. While CBP/p300 function is typically considered in terms of these structured domains, about half of the protein consists of intrinsically disordered regions (IDRs) of varying length. However, these IDRs have only been thought of as linkers that allow flexible spatial arrangement of the structured domains, but recent studies have shown that similar IDRs mediate specific and critical interactions in other proteins. To examine the roles of IDRs in CBP, we performed yeast-two-hybrid screenings of placenta and lung cancer cDNA libraries, which demonstrated that the long IDR linking the KIX domain and bromodomain of CBP (termed ID3) can potentially bind to several proteins. The RNA-binding Zinc-finger protein 106 (ZFP106) detected in both libraries was identified as a novel substrate for CBP-mediated acetylation. Nuclear magnetic resonance (NMR) spectroscopy combined with cross-linking experiments and competition-binding assays showed that the fully disordered isolated ID3 transiently interacts with an IDR of ZFP106 in a fashion that disorder of both regions is maintained. These findings demonstrate that beside the linking function, ID3 can also interact with acetylation substrates of CBP. Their function is mainly based on their interaction with a large variety of transcription factors and other regulatory proteins targeting their intrinsic histone acetyl transferase (HAT) activity on the chromatin and a broad range of partner proteins4. Mutations affecting CBP/p300 genes have been implicated in Rubinstein-Taybi syndrome and several types of cancer, emphasizing their central role in orchestrating gene regulation3.CREB-binding protein (CBP) and its homolog histone acetyltransferase p300 (p300) are multi-domain transcriptional regulators involved in diverse biological functions such as cell cycle, proliferation, differentiation, homeostasis, and apoptosis1. Although intrinsically disordered proteins/regions of proteins (IDPs/IDRs) have frequently been implicated in protein-protein interactions5, the function(s) of the IDRs in CBP/p300 has never been investigated in detail. Rather, it has been suggested that their role is to “connect” the folded, functional elements, enabling CBP/p300 to engage with distinct patterns of chromatin-bound transcription factors in assembling large signaling complexes, such as the enhanceosome6. However, it would be quite surprising if about half of the protein primary sequence would only have the function of “connecting” globular functional domains, and therefore it is likely that a variety of different unexplored functional roles are encoded in those long disordered regions. The N- and C-terminal IDRs in CBP/p300 may also enable long-range structural/functional communication within the protein, as exemplified by the repression of HAT activity by phosphorylation of N-terminal Ser 89 in p3007. HAT activity and substrate specificity are also regulated by the flanking domains of HAT  through intramolecular interactions8.CBP/p300 are about 2400 residues in length and contain several highly conserved domains and motifs, such as zinc-finger motifs PHD, RING, TAZ1, TAZ2 and ZZ, the CREB binding (KIX) domain, nuclear receptor coactivator binding domain (NCBD), bromodomain and HAT domain. Long intrinsically disordered regions  of IDRs and their interplay with globular domains. No specific interaction has been mapped to any of these regions yet11. For ID3, we identified several potential new interaction partners in yeast two-hybrid (Y2H) screenings, among them the Zinc-finger protein 106 (ZFP106). Recent studies suggest that this nuclear protein, ZFP106, is involved in RNA processing and it has been associated with neuromuscular and neurodegenerative disorders15 such as amyotrophic lateral sclerosis (ALS). We identified ZFP106 as a novel substrate of CBP-mediated acetylation. Active, full-length CBP acetylated ZFP106 on several lysines residues located in two main clusters. The transient interaction between the two IDRs was confirmed by a combination of competition-binding assays, cross-linking experiments and NMR spectroscopy. Finally, we determined the regions of ID3 and ZFP106 involved in the fuzzy interaction by employing paramagnetic relaxation enhancement (PRE) NMR spectroscopy.To assess the possible binding and regulatory role(s) of IDRs in CBP/p300, we are conducting systematic studies on the structural state and binding function of the IDRs of CBP, including ID3 (located between the KIX domain and bromodomain), ID4 (located between the TAZ2 domain and NCBD domain)16. In far-ultraviolet (UV) circular dichroism, ID3 has a strong negative peak around 200 nm, typical of random coil proteins, with a slight tendency to local secondary structure formation16  of the 407 residues of ID3, including its 75 prolines (18%), the recently developed NMR strategy to study complex IDPs, which combines 1H and 13C detected high-resolution multi-dimensional NMR experiments22 was necessary  23 calculated from experimental CS values24 suggested short regions of isolated transient α-helices  or extended β-type conformations , highlighting the presence of potential binding sites , the apparent molecular weight (Mw) of ID3 is about 70 kDa, two times its actual Mw, which is often the first indication of structural disordertes Fig. .9, for which two segments showing significant α-helical propensity were detected, indicating that each linker has specific structural and dynamic properties that are likely to have an impact on their function.The structural properties are different from those of ID4SAXS measurements [following removal of potential aggregates by high-pressure liquid chromatography (HPLC)] yielded an average radius of gyration (Rg) of 80 Å of the different conformers in solution Fig. , corresp25, we combined data from SAXS and NMR experiments and calculated an ensemble of possible conformations that ID3 can adopt in solution. To this end, Flexible-Meccano was used with NMR-derived ncSP . C-terminal regions of β propensity are highly conserved and have a conspicuous enrichment of post-translational modification (PTMs) sites, suggesting functional regulation of this region. The DynaMine30 profile also shows peaks in this region   was found in the lung cancer library . Only one copy was found in each library, which is consistent with the low expression levels of ZFP106 in the respective tissue types38. For the detailed characterization of the ID3 – ZFP106 interaction, the shorter fragment 1265 to 1521, denominated as ZFP106-f, was selected.To find out if ID3 mediates protein-protein interactions, we applied a Y2H screening on two different cDNA libraries, one from human placenta (important for its broad spectrum of expressed genes) and another from human lung cancer . We expected that besides general, housekeeping proteins, shared partners in the two different libraries may point to functionally important and potentially disease-associated interaction partners of CBP/p300 mediated by ID3, mitigating the effect of known high false positive hit rates of Y2H. A total of 37 proteins were classified as potential ID3 binders, including 4 candidates that have already been described to interact with CBP or p300 (HIPK135) Fig. . Surpris35) Fig. . Importa1. To probe such a functional consequence of the interaction between CBP and ZFP106, we assayed the acetylation of ZFP106-f with active, full-length CBP. The reaction was performed in HAT assay buffer in the presence of reducing agents, at low protein concentrations. Western blot (WB) analysis with anti-acetylated-lysine antibody showed a band for the 50 kDa ZFP106-f, revealing ZFP106 as a novel substrate of CBP  that can acetylate interacting protein partners, primarily transcription factors or coactivatorsBy MS, no basal acetylation (prior to CBP addition) of the ZFP106-f sample was detected, whereas CBP targeted lysines primarily within two main hot-spots in the C-terminal half of the protein, acetylating only ten lysines out of twenty-four (in about 5–10% of the population) Fig. . For theIn order to determine if the targeting of lysine acetylation of ZFP106-f required specific protein-protein interaction in the context of full-length CBP, we compared the acetylation pattern of ZFP106-f and histone H4 (the classic CBP substrate) by the core domain   and His-tagged ZFP106-f , and also intramolecularly, in multiple different ways, causing the band to appear as a smear of heterogeneous apparent Mw population (as a consequence of the high number of lysine residues in the protein). Specificity of the cross-linking was assessed in our control reaction (C), where under the same conditions no complex formation between GST and ZFP106-f could be observed  perturbations could be observed along the ID3 sequence  can have a larger - and thus observable - contribution to the signal measured44. To outline the ID3 and ZFP106 regions involved in the interaction, PRE NMR experiments were carried out by labeling ZFP106-f at three different positions  with an EDTA(Mn2+)-based paramagnetic tag  measurements. By paramagnetically tagging one of the interaction partners, residues close to the interaction site of the second protein can be detected by decreases in signal intensity. Because PRE effects are averages weighted by the <rtag Fig.   and the other in the C-terminal region  Fig. . However1H-15N NMR spectra  of ZFP106-f can be delineated from the different effects the presence of the three ZFP106-f constructs has on ID3 signal intensities in the 2D tra Fig. . A strontra Fig. . In contInterestingly, the region following the M2 label in ZFP106-f is enriched in lysines and carries a positive net charge, whereas the CTR of ID3 is highly negatively charged Fig. , indicatIn all, these PRE experiments provided evidence for a transient electrostatic interaction between the two proteins mediated by the negatively charged CTR of ID3 and a positively charged lysine-rich patch in the middle of ZFP106-f.6. The function of its long IDRs has been mostly described as that of passive linkers. Here, we focused our attention on possible additional roles of the 407 residues long ID3 located between the KIX domain and bromodomain of CBP.CBP is a multi-domain transcription coactivator that mediates and integrates the action of hundreds of transcription factors and coactivators, which interact with its globular domains and target its activity on the chromatin and/or other proteins1H-detected and 13C-detected high-resolution multi-dimensional NMR experiments22 we assigned about 95% of backbone chemical shifts, providing atomic resolution information on its structural and dynamic features. An ensemble of possible conformations calculated with experimentally derived ncSP scores and SAXS data supported the presence of several potential binding sites with mild local helical or β propensities, within a largely disordered module. The extended and flexible conformations of ID3 are expected to favor functions/interactions that take advantage of its dynamic properties. In accord, the specific binding role(s) of ID3 is supported by our Y2H studies in two cell-lines that uncovered dozens of potential ID3 binding partners. Among those, the transcriptional regulator ZFP106 was identified as a new acetylation substrate of CBP in vitro, which is in line with prior MS-based proteomic studies, which showed acetylation of ZFP106 on some of the lysine residues identified here (cf. PhosphoSitePlus45). The interaction of ZFP106-f with ID3 was confirmed by cross-linking and the observed competition between ID3 and full-length CBP in the acetylation reaction. Specificity of the interaction is underscored by the highly nonspecific acetylation of ZFP106-f by the core domain of CBP, which also strongly acetylates the lysine-rich patch located in the middle of ZFP106-f. PRE experiments, at low and high salt concentration, allowed us to determine the ID3 – ZFP106-f interacting regions, which include an extended negative patch of ID3  and a mostly positive patch of ZFP106-f . These experiments together with the HAT competition assays, in which the C-terminal charged region of ID3 has been deleted  interaction has been demonstrated in focal adhesion dynamics. Here, the interaction of the SH3 domain of Nck2 and the LIM4 domain of PINCH-1 has a Kd of 3 mM, still it is indispensable in integrin signaling, as suggested by genetic evidence48. The importance of transient, lowly-populated states that are still invisible to most structural biology techniques has also been demonstrated in encounter complexes49, RNA-protein recognition50 and the catalytic cycle of dihydrofolate reductase51. However, we cannot exclude the possibility that other domains of CBP or ZFP might be also involved in the interaction and influence affinity. This is also corroborated by the ID3 competition experiments, in which ID3 in a large molar excess  is required to compete with binding and targeting of ID3 region within full-length CBP.An intriguing aspect of our investigation is the transient character of this targeting interaction; such fuzzy and/or weak and transient complexes have been suggested to confer specificity without a structured statecf. Fig. 52, BRCA153, or UPF254. Moreover, the observed acetylation of ZFP106-f fits perfectly with CBP/p300 function in general: CBP/p300 integrate the action of hundreds of transcription factors and coactivators, the function of which is often regulated by acetylation3. In this context, the highly significant enrichment of ID3 binding partners for transcription-regulatory and DNA-binding functions is notable4. Moreover, “in vivo” acetylation of ZFP106 was previously described by Li Y. and colleagues in 2006. High-throughput MS analysis of acetylated proteins present in colorectal carcinoma cell lines (SNU-C2B) identified four acetylation sites in ZFP106  (Record ID: 11618 in PhosphoSitePlus45) within a region that is comprised in our construct (AA1265–1521). Interestingly, in our in vitro experiments those sites seem only to be acetylated by the highly active and nonspecific core domain but not by the full-length CBP.It is worth mentioning that the identified interaction regions are also rather long and may contain specific interaction motifs or charge distribution required for the interaction. Especially, ID3 sequence presents a characteristic and regular distribution of charges that has been conserved during evolution cf. Fig. . Similar55. Recent studies have shown that the nuclear ZFP106 protein that localized in nuclear speckles is required for normal assembly of the mammalian spliceosome, a process in which CBP/p300 also seems to participate57.In a cellular context, ZFP106 and CBP share roles in developmental pathways and diseases such as cancer. They also share a number of interaction partners, such as the tumor suppressors APC (Adenomatous polyposis coli protein) and Smad2 (Mothers against decapentaplegic homolog 2), which suggests that ZFP106 maybe involved in pathways such as TGFβ or Wnt signaling. In all, it seems reasonable that ZFP106 and CBP are part of a larger transcriptional complex that regulates expression of target genes involved in specific cellular processes, such as muscle cell differentiation or maintenance of the neuromuscular signalingIn summary, our results suggest a possible additional role of an IDR in CBP, which may not only lead to a better description of the function of CBP/p300 and ZFP106, but also provides a precedence for addressing the function of long “linker” regions in large multi-domain signaling proteins.SI Materials and methods.A set of homologous ID3 amino acid sequences of various vertebrate species was assembled to study disorder propensities, sequence conservation, and the presence of potential binding sites. Cf. SI Materials and methods.For control purposes, a scrambled-ID3 sequence sharing 21% identity with wt ID3 was generated. , core domain of CBP (residues 1095–1849); ID3 of CBP, (residues 674–1080); ID3-F1 of CBP (residues 674–848); and Cys-to-Ser mutant ID3(C845S) obtained by site-directed mutagenesis of Cys845 of ID3, were cloned from the full-length construct. ZFP106-f (residues 1265–1521 of full-length ZFP106 (UniProt Q9H2Y7)). Scrambled-ID3 (scrID3), ID3delCTR and the three single-cysteine mutants of the ZFP106-f were cloned by GenScript (M1: C1472S (paramagnetic label on C1307), M2: insertion of Cys between Thr1346 – Arg1347 (paramagnetic label on insC1347*), M3: C1307S (paramagnetic label on C1472)). SI Materials and methods. Identified partners were analyzed with IUPred (disorder level) and classified using GO annotation .Y2H analysis was carried out by Hybrigenics Services S.A and described in E. coli. Purification was achieved by a two- or three-step purification protocol including nickel (and Flag for the CBP constructs) affinity followed by size exclusion chromatography.Full-length (FL) CBP and its core domain (CD) were expressed in Sf9 cells, while the rest of the proteins were expressed in 17, which was shown not to alter the secondary structure content of the protein  and the hCSD1 (for control experiment) were mixed with the metal-containing probe (thiol-reactive label pEDTA). The reaction was carried out overnight at RT and labeled proteins were re-purified. For further details, cf. SI Materials and methods.Intact protein masses were measured by direct infusion in a microelectrospray ionization ion trap mass spectrometer (LTQ XL). The mass spectra were deconvoluted using the software ProMass Deconvolution. For further details, cf. SI Materials and methods.Far-UV circular dichroism spectra of ID3, ZFP106-f and the complex (1:1 molar ratio) were recorded on a J-715 spectropolarimeter in reducing buffer of physiological salt concentration. 58 (buffer scattering subtracted), following data processing/analysis using ATSAS suite59 and calculation of the radius of gyration by PRIMUS QT60. For further details, cf. SI Materials and methods.ID3 was injected into a size-exclusion column equilibrated with buffer of physiological salt concentration and SAXS data were collected continuously, selected frames corresponding to the main protein elution peak were averaged using FOXTROTSI Materials and methods and Tables All the experiments and acquisition parameters regarding ID3 sequence-specific assignment, backbone dynamics and, PRE experiments are reported in 13C,15N] labeled ID3 and ID3-F1 in reducing buffer of physiological salt concentration at pH 6.5, with 10% D2O added for the lock. The CS assignment of ID3 has been deposited in the BioMagResBank  as entry 26841. For further details, cf. SI Materials and methods.All the multidimensional NMR experiments for sequence-specific assignment were performed on uniformly  ID3(C845S) and 1 molar equivalent (eq) of single-Cys ZFP106-f mutant EDTA(Mn2+)-tagged (paramagnetic samples). The diamagnetic references were done with wt ZFP106-f with DTT added -tagged version for PRE referencing62). The unrelated protein control experiments were done with 1 eq of wt hCSD1-EDTA(Mn2+) (paramagnetic sample) or wt hCSD1 . The reduced-label control experiments, the  ID3(C845S):ZFP106-f-M2 sample was measured in absence and presence of reducing agents (DTT). All NMR samples were measured at pH 6.5 and 10% D2O for the lock, NaCl was added to a final concentration of 150 mM. For further details, cf. SI Materials and methods.For the PRE measurements, 2D TopSpin 1.3 software or nmrPipe63. NUS data sets were converted with nmrPipe and then processed using either the Multidimensional Fourier Transform (MFT) or the Sparse MFT (SMFT) algorithm. Both programs are available at http://nmr.cent3.uw.edu.pl. Data analysis was done with Sparky64 or CcpNmr Analysis65.NMR data sets were processed using Bruker α, Cβ and N chemical shifts (95% assigned) by using the neighbor-corrected structural propensity calculator (ncSP) tool66. For further details, cf. SI Materials and methods.Secondary structure propensity analysis was calculated from C′, C26 and SCCOMP67 were used to generate a pool of 9876 semi-random conformations (with side-chains modeled). Then, CRYSOL27 and the GAJOE algorithm were employed27 to select subsets of the random pool that that agreed with the experimental SAXS data. For further details, cf. SI Materials and methods.Ensemble models of ID3 were based on the combination of SAXS data and ncSP derived from experimental chemical shift values. Flexible-MeccanoIn vitro ZFP106-f or H4 acetylation assays were performed in reducing buffer of physiological salt concentration at 20 or 30 °C using FL or CD of CBP. For the HAT competition assay, x10, 100 and 150 molar excess (relative to full-length CBP) of ID3, scrID3 and ID3delCTR were added to the acetylation reactions. The reactions were completed to saturation and the samples were analyzed by Western blot by anti-acetylated-lysine (Bioke) and anti-His (Sigma-Aldrich) antibodies. For the identification of acetylation sites, the proteins in solution were desalted and digested with either pepsin or endoglu-C, and analyzed by LC-MS/MS in a microelectrospray ionization ion trap mass spectrometer  as described69. For further details, cf. SI Materials and methods.3 was added to a final concentration of 0.25 mM. For further details, cf. SI Materials and methods.Proteins were mixed in approximate 1:1 ratio (concentrations adjusted by SDS-PAGE). Cross-linking reactions were carried out for 15 min at RT in PBS with 1 mM DTT . Crosslinker BSSupplementary Information