The oncogenic CCDC6-RET fusion protein is a dual ATP- and ADP-dependent kinase

Domain: chemistry
Published: 2026-03-06
Editorial outcome: major_revision

Abstract
Gene fusion products involving protein kinases are known drivers in human cancers and actionable targets for personalized therapy, yet the structural and molecular determinants that control their function are largely unexplored. Here we show that a CCDC6-RET fusion protein, a driver and therapeutic target in lung and thyroid cancers, is a highly active dimeric kinase. Time-resolved mass spectrometry together with a robust biochemical and biophysical characterization reveal that CCDC6-RET functions as a dual ATP- and ADP-dependent kinase able to bind both nucleotides and to use them as phosphoryl donors. We also identify a crosstalk between the C-terminal and the activation segments controlling both the processing and the catalytic activity of the fusion protein. Furthermore, a 3D-Estructural assembly of a CCDC6-RET homodimer was generated combining single particle electron microscopy, small-angle X-ray scattering and in silico molecular dynamics simulations. Our structural model together with cross-linking mass spectrometry data demonstrated that CCDC6-RET in the inactive state forms a face-to-face dimer characterized by intermolecular-crosslinked activation segments. Upon nucleotide binding the catalytic domains swing apart and fast activation loop phosphorylation could be driven by a mechanism in cis. Our work uncovers the molecular and structural determinants that control the mechanism of CCDC6-RET autoactivation.

Introduction
Following the paradigm of the Philadelphia chromosome and the BCR-ABL1 fusion in chronic myelogenous leukemia (CML) 1 2, gene fusions products involving protein kinases are known drivers in human cancers and actionable targets for personalized therapy 3. Yet, the structural and molecular determinants that control their function and (oncogenic) mechanism of action are paradoxically elusive in most of the cases. In 1985, a proto-oncogene named Rearranged during Transfection (RET) was discovered in NIH3T3 cells transfected with DNA isolated from human lymphoma cells, to cause malignant transformation 4. The characterization of the isolated RET cDNA clones revealed a carboxy-terminal domain with high homology with members of the tyrosine kinase gene family preceded by a hydrophobic sequence characteristic of a transmembrane domain, suggesting that the RET oncogene encoded a cell-surface receptor 5. The characterization of the human 6 and mouse 7, 8 proto-oncogene orthologs revealed the amino acid sequence (primary structure) of the RET protein. Shortly after, a RET fusion product was discovered in papillary thyroid carcinoma (PTAC), it was named RET/PTC1 and its N-terminal partner protein identified as H4(D10S170) 9, 10. The RET/PTC-1 oncogene was a fusion product containing the N-terminus of the H4 gene, currently known as CCDC6 (Coiled-Coil Domain Containing protein 6, aa 1-101) fused to the tyrosine kinase domain of c-RET 10. CCDC6-RET fusions are detected in up to 30% of papillary thyroid carcinomas 10, 11. Although the prevalence of CCDC6-RET fusions in PTC shows significant geographic variation 12, there is a strong correlation with radiation exposure. In 1986, after the Chernobyl nuclear power disaster, a sharp increase in the incidence of PTC was observed in children and adolescents of contaminated regions of Belarus, Russia, and Ukraine with near 60% of post-Chernobyl PTCs children patients harboring a RET fusion 13. Up to date, more than twenty RET fusions products have been described in PTC 3. The most common RET fusions products are CCDC6-RET (RET/PTC1) and NCOA4-RET (RET/PTC3), which together account for about 90% of RET fusion-positive cases in thyroid cancer 12. In all these cases, the DNA-rearrangements generate the fusion of the tyrosine kinase domain of RET to the 5' portion of different genes. Breakpoints in the RET gene are consistently located at intron 11, so the fusion starts from exon 12 to the stop codon, resulting in a protein chimera containing the tyrosine kinase domain but lacking the juxtamembrane segment and extracellular domains of RET 12. A common molecular hallmark in PTC is the constitutive activation of the MAPK (Mitogen-Activated Protein Kinase) cascade. Indeed, besides RET and other RTKs, common PTC-drivers are gain-of-function mutations of RAS and BRAF kinase, respectively. The mutually exclusive nature of these lesions supports the notion that they act along a common signaling axis driving the oncogenic phenotype in PTC 14. RET fusions are also found in 1-2% of lung adenocarcinoma (LADC) 15, 16, 17. The most common RET fusion products in non-small cell lung carcinoma (NSCLC) are KIF5B-RET, NCOA4-RET, and CCDC6-RET respectively. KIF5B (Kinesin-1 heavy chain) is by far the most common RET fusion partner in NSCLC, being detected in up to 70%–90% of the cases 15. In NSCLC, RET fusions are mutually exclusive with other driver mutations, such as ALK or ROS1 rearrangements or EGFR or KRAS mutations, suggesting again common signalling mechanisms driving the oncogenic phenotype, and the definition of a potential molecular subtype 18.

Clinically, there is a strong interest in the development of next-generations RET tyrosine kinase inhibitors for the development of personalized therapies in NSCLC and PTC19 20. Crucially RET fusions (CCDC6-RET, NCOA4-RET, and the newly described CDC123-RET) have been shown to mediate acquired resistance mechanism in lung adenocarcinomas (LADC) to EGFR or ALK tyrosine kinase inhibitors 2, 21, 22. Understanding the structural, molecular and dynamic determinants that control the catalytic function and signalling of these oncogenic RET fusion products will uncover druggable vulnerabilities. Such vulnerabilities can be therapeutically exploited for the design and development of next-generation inhibitors for targeted and personalized therapies that are able to overcome acquired resistance by secondary mutations in RET-driven cancers 19, 20, 23, 24, 25, 26. In this study we produced a recombinant CCDC6-RET gene fusion product using a baculovirus expression system. Here, we performed a detailed functional and structural characterization of the CCDC6-RET fusion protein by state-of-the-art biochemistry, mass spectrometry and structural biology tools that allowed us to generate a 3D-structural model of a CCDC6-RET homodimer for single particle analyses and mechanistic validation. Our findings uncover the structural and molecular determinants that control a RET oncogenic fusion´s mechanism of autoactivation, revealing unexpected and striking features not previously envisioned with important implications for drug discovery. Our data provides at the same time a solid framework for the structural and functional study of other RET fusion products.

Results
1. Expression, purification and quality check of a recombinant CCDC6-RET fusion product.
We have produced a recombinant CCDC6-RET fusion product (Fig. 1a) composed by fragments of residues 1-101 of CCDC6 and residues 713-1072 of RET using a baculovirus expression system in Sf9 insect cells. Protein purification was achieved by tandem immobilized metal affinity (Ni2+) and glutathione-conjugated gravity flow chromatography and in-gel 3C-protease digestion followed by size-exclusion chromatography, if required (Fig. 1b). This is an updated version of a previously published single-step affinity protocol using glutathione-conjugated resin for the purification of recombinant RET kinase and intracellular domains expressed in insect cells using a baculovirus system 27, 28, 29. The tandem double affinity chromatographic step allowed us to obtain highly pure and monodisperse protein with no traces of DNA or nucleotide contamination as indicated by the values of absorbance at 280 and 260 nm and their optimal ratio (Fig. 1c), resulting in a recombinant protein yield of 0.3-0.4 mg per liter of culture. Further quality check over a range of different protein concentrations by mass photometry shows that CCDC6-RET is a 101.2 ± 2.2 kDa dimer in solution (Fig. 1d). Binding of RET tyrosine kinase inhibitors BLU-667 20 and Ponatinib 30 was evaluated by DSF measuring changes in intrinsic fluorescence (IF) upon a temperature gradient (Fig. 1e and f). Binding of both compounds to CCDC6-RET resulted in a significant shift of the inflection temperature (T) of 12.2 and 19.8 °C, respectively indicative of a robust thermal stabilization (Fig. 1f). Altogether, these results assured the quality, monodispersion and catalytic integrity of the protein sample prior to its functional and enzymatic characterization. Initial functional characterization showed that CCDC6-RET functions as a highly-active dimer in solution displaying fast autophosphorylation kinetics (Fig. 1g) in agreement with the K value for ATP 139.3 ± 11 M compared with the RET kinase domain (KD, 429 ± 43 M) construct 27, see Fig. 1h and i.

2. Mass spectrometric characterization of CCDC6-RET autophosphorylation.
Next, we applied high-resolution time-resolved liquid chromatography tandem mass spectrometry (LC-MS/MS) to monitor the dynamic changes of the autophosphorylation reaction (0-30 min) on CCDC6-RET in vitro. Fast autophosphorylation kinetics were observed in most of the identified phospho-sites, which recapitulated the results from the WB data using phospho-specific antibodies (Fig. 2a). A total of seventeen phospho-sites were mapped, of which fourteen were the facto autophosphorylation sites as indicated by their time-dependent saturation kinetics during the reaction (Fig. 2b, c and table 1). CCDC6-RET autophosphorylates on both serine (e.g. S904 and S909) and tyrosine residues (Fig. 2c, f and table 1). CCDC6-RET autophosphorylates not only on sites located on the catalytic core of the fusion, but also on discrete sites of the N-terminal and coiled-coil region of CCDC6, some of which were not previously described. We identify among other several phospho-sites on the amino terminal and coiled-coil region of the gene fusion product: Ser 46, Ser 52, Tyr 80 and Ser 98 (Fig. 2c, d). Mutating Ser 46, Ser 52 and both residues simultaneously to alanine does not cause any defects on the phospho-tyrosine activity as seen in in vitro autophosphorylation reactions (supplementary Fig. 1a-c). In the same line, a CCDC6-RET fusion with a Y80F or a S98A mutation appeared to be catalytic competent (supplementary Fig. 1d and e). Autophosphorylation sites on the catalytic core of the RET fusion were also identified (Table 1 and supplementary tables 1-4). These phospho-sites are located at different structural and functional motifs of the RET kinase domain: Tyr 806 and Tyr 809 (hinge), Tyr 826 (kinase insert), Ser 896, Tyr 900, Ser 904, Tyr 905, Ser 909 (activation loop) and Tyr 1015, Tyr 1029, Ser 1021, Ser 1034 and Tyr 1062 (C-terminal segment), see also Fig. 2c, e, f and g. Some of these phospho-sites showed high levels of phosphorylation in the basal state which decreased over time during the autophosphorylation reaction. This is the case for activation loop Tyr 900 and C-terminal Ser 1034; both phospho-sites are located within peptides containing in addition other bona fide autophosphorylated residue whose levels increase one order of magnitude during the reaction. See the example of activation loop tyrosines 900 and 905 (Fig. 2c, f and g). In other cases, phospho-sites appeared only in doubly or triply phosphorylated peptides (e.g. Ser 896 and Ser 1021). A complete list of the CCDC6-RET phospho sites consistently identified across the four independent experiments is shown on main table 1 (see also supplementary tables 1-4). Quantifications of representative high occupancy autophosphorylation sites from three independent experiments are shown in supplementary Fig. S 2. Next, we targeted C-terminal phospho-sites for site-directed mutagenesis to test whether they are required for catalytic activity. The mutation of residue Tyr 1015 to phenylalanine (YP1015F) did not cause any detrimental effect on tyrosine phosphorylation as seen in the time course autophosphorylation assays (supplementary Fig. 1g). The same applied to a Y1062F and S1034A mutants (supplementary Fig. 1g and h).
As negative controls in the phospho-proteomic experiments we used adenosine monophosphate (AMP), and biphosphate (RADP) nucleotides (supplementary Fig. 3). We found a striking increase of CCDC6-RET tyrosine phosphorylation in the presence of ADP and not by AMP. These unexpected results were a clear sign of ADP-dependency by the CCDC6-RET fusion product, which we explored further (see next result section).

3. CCDC6-RET is a dual ATP- and ADP-dependent kinase.
Next, we performed a direct functional comparison of CCDC6-RET with a RET 713-1072 aa fragment, the catalytic core of the fusion product, as a control (Fig. 3a). First, we performed enzymatic assays to calculate the catalytic rates and enzymatic constants for ATP using an excess of peptide as an exogenous substrate (Fig. 3b). Under these experimental conditions, CCDC6-RET was a more active enzyme than the catalytic core of the RET fusion (aa 713-1072) as indicated by their corresponding K (133.25 ± 13 M vs 429.28 ± 77 M) and a six-fold increase in the catalytic efficiency constant (k /K ,) by the oncogenic chimera (Fig. 3b-d). We next performed in vitro time course autophosphorylation assays (0–30 min) using ATP (1 mM), MgCl (2 mM) with 1 M final protein concentration. Autophosphorylation was monitored by Western blotting (WB) using RET total and phospho-specific antibodies against Tyr 905, Tyr 1015, Tyr 1062 and a total phospho-tyrosine antibody (Fig. 3e). Fast CCDC6-RET autophosphorylation kinetics reaching saturating levels at early time points (1 min) contrasted with slower rates of autophosphorylation by the 713-1072 construct, see e.g. Tyr 905 and Tyr 1015 phosphorylation (Fig. 3e). We also evaluated the binding of ATP and ADP to both constructs respectively by monitoring changes of IF (Fig. 3 h-j). Titration experiments using a range of increasing concentrations of both nucleotides allowed us to calculate an apparent K for ATP (186.8 ± 38 M) and ADP (1057.0 ± 157 M) respectively (Fig. 3j). These results together with the MS data (supplementary table 2) prompted us to explore further whether CCDC6-RET is a dual ATP- and ADP-dependent kinase that can use in addition to ATP, ADP as a phosphoryl donor. We found that in contrast to a 713-1072 construct, CCDC6-RET efficiently auto-phosphorylates in the presence of ADP (Fig. 3f and g), although with slower rates than with ATP, in accordance with the affinity values obtained (Fig. 3j). Together these data demonstrate that CCDC6-RET is a highly active dimeric kinase in solution with fast kinetics of autophosphorylation that functions as a dual ATP- and ADP-dependent kinase able to bind to and use both nucleotides as phosphoryl donors.

4. ADP regulates CCDC6-RET phospho-tyrosine activity in vitro.
To explore further the ADP dependent activity of CCDC6-RET, we performed ATP and ADP titration experiments in which the phospho-tyrosine activity of CCDC6-RET was monitored under increasing concentrations of nucleotide (range 10 to 5000 M) using a total phospho-tyrosine and a RET Tyr 905 phospho-specific antibodies (Fig. 4a). Increasing CCDC6-RET phospho-tyrosine activity was observed at low ATP concentrations (10 to 50 M) reaching saturation levels at 1000 M (Fig. 4a). In the case of ADP, increasing CCDC6-RET phospho-tyrosine activity reached a maximum at 1000 M, which followed a marked decrease in activity at 2500 and 5000 M concentrations. These data suggested a potential inhibitory role of ADP at high concentrations, which we explore further in ADP-ATP competition assays (Fig. 4b). Pre-treatment of CCDC6-RET with increasing concentrations of ADP (10-5000 M) followed by treatment with ATP (1 mM) for 15 minutes resulted again in a detrimental effect on the activity induced by ATP at higher ADP concentrations (2500 and 5000 M). These data again demonstrate a regulatory and competing role of ADP at higher concentrations resulting in the inhibition of the CCDC6-RET activity induced by ATP. These data were also recapitulated in time course autophosphorylation experiments (0-30 min) where the activation of CCDC6-RET by ATP was completely abrogated over the complete time course (even at late time points) by pretreatment with 5 mM ADP (Fig. 4c). In the case of pretreatment with 1 mM ADP, a consistent detrimental impact at early time points (1 min) with 1 mM ATP stimulation was observed (Fig. 4d). In these sets of experiments, we used a catalytic dead HRD/A mutant (D874A) of CCDC6-RET which did not respond to either ATP or ADP (Fig. 4b and c). To prove direct  phosphate transfer of ADP by CCDC6-RET in the autoactivation mechanism we used ADP radiolabeled on the -phosphate (-ADP-P33) in autophosphorylation experiments (Fig. 4e). In parallel, we used radiolabeled ATP (-ATP-P32) as an internal control. In these in vitro experiments using radioactive nucleotides, we found that CCDC6-RET efficiently autophosphorylates in the presence of ADP, although with slower kinetics than with ATP, as anticipated (Fig. 4e).
We generated structural models for an active CCDC6-RET homodimer (accession codes: ma-pcoh8 and supplementary file CCDC6-RET_ADP2.pdb) in complex with ADP, Mg2+ and a Tyr-containing peptide substrate (Fig. 4f) that revealed a plausible structural basis for ADP-dependent catalysis (Fig. 4f). In this process, activation loop Arg 912 would play a pivotal role fixating the -phosphate group of ADP by displaying an extended sidechain that is fully compatible with peptide binding and adoption of a near-attack conformation for the phospho-transfer reaction. Importantly, the backbone amides from activation-loop Iso 913 and Pro 914 form the necessary hydrogen bonds with +1 and +2 residues of the phosphorylatable tyrosine-containing peptide substrate. In addition to the prototypical interactions required for ATP -phosphate transfer, namely, the salt bridge between 3 K758 and C E775, the coordination of Asp 892 (DFG motif) with Mg2+, and the positioning of Asp 874 in the HRD motif for proton abstraction from the tyrosine hydroxyl group; a key cation-pi interaction between Arg 878 and Trp 917 is also in place for the correct arrangement of the Michaelis complex. Taken together, our biochemical and structural data support the notion of an ADP-dependent activity by CCDC6-RET.

5. Activation loop Tyr 905 is required for CCDC6-RET catalytic activity.
It is known that the activation loop of protein kinases in the unphosphorylated state can function as an autoinhibitory module by sterically blocking substrate access into the active site 31. Based on our own results indicating that the C-terminal segment can also restrict the access of the substrate into the catalytic cleft (supplementary Fig.4), we aimed to explore further a potential crosstalk between the activation loop and the C-terminal segments in the mechanism of CCDC6-RET autoactivation and signalling. For this purpose, we generated a series of activation loop mutants in full-length and C-terminal deleted constructs (CT). First, we produced single and double activation loop tyrosine mutants in a full-length construct (Y900F, Y905F and Y900/905F) and evaluated their catalytic activity in time course autophosphorylation experiments (Fig. 5a). None of the evaluated mutants had a significant detrimental impact on the tyrosine phosphorylation activity compared with the WT control in autophosphorylation experiments (Fig. 5a). However, in enzymatic assays using a peptide substrate we found that Tyr 905 was required for enzymatic activity (Fig. 5c). Of note, a full-length CCDC6-RET Y900F mutant construct resulted consistently in very low yield of soluble protein after purification (supplementary Fig. 5). The very low levels of expression made it difficult to work at comparable levels due to its short life and that it was not possible to concentrate it to reach workable concentrations. Intriguingly, such detrimental effect on protein yield was rescued by the concomitant mutation on Tyr 905 in the case of a double activation loop mutant (Y900/905F), see Fig. 5a. On the contrary, when we evaluated the function of the complete series of activation loop mutants in a C-terminal deleted context (CT) we observed that the Y900F mutant was efficiently expressed to the same levels as the WT protein. Interestingly, we also observed a catalytic dependency on activation loop Tyr 905 in the CT constructs (Fig. 5b). We found that in autophosphorylation and enzyme kinetics experiments, where we evaluated the K for ATP in the presence of an excess of peptide substrate, a Y905F and double Y900/905F mutants have a loss of function effect, contrary to the Y900F, which was catalytic competent (Fig. 5d). Hence, these data uncovered a catalytic dependency on Tyr 905 by the CCDC6-RET fusion product. Interestingly, these data were recapitulated although to a lower extent by a S909A mutant, which also has a compromised enzymatic activity towards a peptide susbtrate (supplementary Fig. 6). Taking these data together, we conclude that the CCDC6-RET activation loop Tyr 905 controls the catalytic activity and signalling of the CCDC6-RET fusion protein.

6. In silico reconstruction of a CCDC6-RET homodimer.
We generated in silico a high-confidence structural reconstruction of a CCDC6-RET homodimer. First, we used the Waggawagga coiled-coil prediction server 32. Coiled-coil predictions are based on the identification of periodic heptad repeats, which can be represented by a net- or helical wheel-diagrams. This bioinformatic tool enables the discrimination between coiled-coil-domains and single -helices (SAH). In the case of CCDC6-RET there is a high confidence parallel homodimer coiled-coil interface from residues 56-101 of each protomer (Fig. 6a and d), as indicated by both the coiled-coil probability marcoil (p-value 100) and very low SAH-score (0.0333). The coiled-coil inner hydrophobic core was clearly defined by the heptad repeats (Fig. 6b and c), and several salt-bridge interactions (strong) involving Glu 58 and Arg 62, Lys 74 and Glu 78, and Lys 85 and Glu 89. These contacts were involving Glu 78 and Lys 81 and Glu 89 and Arg 92, and weak coordination between Asp 93 and Lys´96, as depicted in the helical wheel- and heptad-net-diagrams (Fig. 6b and c). We visualized in 3D a parallel homodimeric coiled-coil interface (Fig. 6d) using CCbuilder 2.0 33. In an attempt to disrupt the dimerization interface, we generated a series of mutants at key positions within the coiled-coil, for example Cys 84 (C84A) that can form a disulfide bridge with its counterpart (Fig. 6d), and Ser 98 (S98A) which we found was an unexpected autophosphorylation site, see main table 1 and supplementary tables 1- 4. None of these mutants was able to dissociate the dimer as indicated by the elution volumes observed in SEC analyses compared with the WT (data not shown). These coiled-coil mutants did not affect the activity of the fusion products as detected in autophosphorylation experiments (Supplementary Fig. 1e and f), suggesting a tight dimerization constant by the interface. Next, we applied artificial intelligence to obtain a high-confidence 3D-predictive model of a monomeric CCDC6-RET fusion protomer using AlphaFold (AF id: J7M8C2). By the superimposition of two monomers of the predicted fusion protein onto the dimeric coiled-coil interface (Fig. 6d) we generated a two-fold rotational symmetry dimer (Fig. 6e) in which the catalytic domains are facing away symmetrically from the coiled-coil stem. The high confidence and feasibility of the predictive 3D-model was supported by careful inspection of key structural features, see supplementary Fig. 7a-d: i) the correct position and configuration of secondary structural elements and functional motifs of an active kinase defined overall by the correct alignment of the regulatory (R-) and catalytic (C-) spines and DFG-in configuration 28; ii) the hydrophobic patch formed by contacts between the C helix (i.e. PIF-like pocket) and the coiled-coil stem; iii) analogy to the domain arrangement found in bacterial histidine kinases where the catalytic domain is place to the left of the symmetry plane of the coiled-coil interface defined in those cases by a four helix bundle instead 34, and iv) in good agreement with two independent and high-confidence (pLDDT scores of 79.08 and 87.74, respectively) solutions provided by AlphaFold multimer (ColabFold) and AlphaFold3. These represent different configurations and functional states depending on the specific situation of the catalytic domains with respect to the coiled-coil interface (supplementary Fig. 7e and f).

7. Architecture of a CCDC6-RET homodimer by single particle EM.
For EM, freshly purified CCDC6-RET was placed on home-made carbon-coated 400 mesh copper grids and negatively stained. An analysis of the EM field showed a relatively uniform distribution of particles (Fig. 7a). Reference-free 2D averages obtained with Relion 35 revealed particles of similar size but varying shapes and orientations, clearly indicating the presence of a two-fold symmetry (Fig. 7b). A similar 2D-classes were obtained with ScryoSPARC 36, see supplementary Fig. 8b). To compare the in silico reconstructed CCDC6-RET homodimer with the experimental data, we filtered the in silico model to 15 Å and generated its retroprojections (supplementary Fig. 8a). The generated 2D projections show good agreement in size and shape with the reference-free 2D averages of a CCDC6-RET homodimer (Fig. 7 b and c, and supplementary Fig. 8b), with a thinner stem (coiled-coil interface) in the middle and two globular arms segregated from the stem facing at opposite almost perpendicular directions. An initial 3D model was built and further refined using cryoSPARRC to produce the low-resolution EM map (Fig. 7d and e, and supplementary Fig. 8c). The reliability of the map is supported by the strong agreement between the reference free 2D classes and the projections of the map and the even distribution of Euler angles (supplementary Fig. 8d). The 3D-map confirms the dimeric and twisted arrangement of the globular catalytic domains towards the left of the 2-fold rotational axis from the coiled-coil stem. This comes in line with the catalytic domain configuration seen in bacterial histidine kinases 34, where the catalytic core of the DHp-CA module is placed to the left of helix b. When the density map is contoured at a threshold chosen to encompass a molecular weight of approximately 110 kDa (calculated molecular weight of CCDC6-RET), the map generally has good connections between adjoining domains but lacks density for the whole coiled-coil interface and N-terminal part of the fusion. Importantly, the independent dimeric models from AlphaFold fit well into the low-resolution EM map, supporting the accuracy of the AlphaFold prediction (see Fig. 7e and supplementary Fig. 7a-f and 8c).
To obtain further information on the molecular shape of a CCDC6-RET homodimer in solution, low-angle X-ray scattering (SAXS) data of a CCDC6-RET-CT construct were collected (supplementary Fig. 9a). Pair distance distributions exhibited fine features consistent with a multidomain protein sample. Ab initio envelopes were generated for CCDC6-RET-CT using DAMAVER consistent with the pair distance distributions showing an elongated shape at the root of the coiled-coil and a more globular architecture at the catalytic portion of the fusion resulting in an inverted T-shape object (Supplementary Fig. 9b and c) with a radius of gyration (Rg) of 55.07 ± 0.18 Å and D of 195 Å. These measurements are in accordance with the reference-free 2D-particles and 3D-envelop obtained by electron microscopy and also the 3D-in silico reconstructed particles considering the IDRs (see Fig. 6 and 7). Of note, the CCDC6-RET-CT construct generated an extra volume or ¨bump¨ adjacent to one of the two catalytic domains of the fusion, which was tentatively attributed to the dynamic motion of both catalytic domains, as we can fit in another catalytic domain molecule within the extra volume. The CCDC6-RET-CT structure was modeled using a high confident Alpha-Fold (AF id: J7M8C2) prediction of a monomeric chimera without the amino- and carboxy-terminal IDRs. The initial 54 amino acids were then manually built into the SAXS envelope (supplementary Fig. 9 d). The fitting of the theoretical curves derived from this CCDC6-RET-CT model against the experimental curves gave a χ2 value of 2.2, indicating a good quality of the data, see supplementary table 5. The high-quality SAXS data were therefore sufficient to generate a preliminary model for a CCDC6-RET dimer assembly, defining clear pairwise interdomain angles of approximate 90° between the coiled-coil interface and the catalytic domains and a potential solution for the extra volume observed in the SAXS envelop (supplementary Fig. 9d). The coiled-coil and catalytic domains angle appears to be in the range observed for the DHp-and CA domains in bacterial histidine kinases 34, 37, 38. The SAXS envelops fits well all the in silico generated CCDC6-RET assemblies and the 3D-map from the EM single particle analyses, capturing the dynamic transitions between the different assemblies’ states and the dynamic motion of the catalytic domains from the coiled-coil stem. These data also suggest some degree of asymmetry between catalytic domains and the dimer interface (supplementary Fig. 9d).

8. Cross-linking MS analyses of inactive and autophosphorylated CCDC6-RET.
Cross-linking MS is a technique that captures conformational changes in solution by mapping structural details of individual proteins and their complex assemblies based on the coordination of amino acid pairs that are positioned in close proximity to each other by using chemical crosslinkers 39. These covalent connections then serve as basis for deriving distance constraints within the protein(s) different conformational and functional states. To provide experimental restraints for the 3D-reconstruction of the different functional states of a CCDC6-RET homodimer (i.e. unstimulated and autophosphorylated forms), we performed in-solution cross-linking with the homobifunctional cross-linker DSBU (disuccinimidyl dibutyric urea) 39. CCDC6-RET cross-linking was performed under unstimulated and saturating ATP and MgCl (60 min) conditions. A non-crosslinked sample was used as an internal control (Fig. 8a). Three independent replicates of each set of samples were subjected to high resolution LC-MS analysis. In parallel, a LC-MS analysis of non-crosslinked sample was performed to assess the purity and homogeneity of the CCD6-RET preparation. This analysis demonstrated a highly pure and homogenous recombinant protein preparation (Fig. 8a). Size exclusion chromatography of the crosslinked samples (both unstimulated and autophosphorylated) revealed the same molecular size (as indicated by the same elution volume) than the non-crosslinked one, which excludes the formation of artefactual oligomeric and/or high molecular weight species during the treatment with the crosslinker (Fig. 8a, inset). We identified a total of 148 and 131 crosslinked counts by Merox in the resting (i.e. inactive, unstimulated) and phosphorylated states, which resulted in the final identification of 55 and 46 crosslinked peptides, respectively (Fig. 8b). Next, we classified the specific crosslinked peptides for each of the functional states resulting in the identification of 13 specific crosslinked peptides for the unstimulated form (i.e. resting) and 9 for the autophosphorylated (i.e. active) protein, respectively, as shown in the Venn diagram (Fig. 8c). Overall assessment of cross-linked residues in both states revealed significant changes upon phosphorylation (Fig. 8d). Initial inspection of the identified cross-links for each of the functional states represented in circular views (Fig. 8d) pointed at the activation loop as a hotspot defining the unstimulated or resting state of CCDC6-RET, which involved a series of crosslinked peptides containing activation loop Lys 907 that disappear in the active phosphorylated state of the protein (Fig. 8d and e). In particular, we pay attention to three crosslinked peptides that coordinated Lys 907 (peptide DVYEEDSYVK*R) from the activation loop with other residues from the activation loop itself or its vicinity, for example Ser 909 (S*QGRIPVK), Ser 896 (ISDFGLS*R) and also Lys 889 (MK*ISDFGLSR) just at the beginning of the activation segment defined by the DFG motif (Fig. 8d and e and supplementary Fig. 11). When we look at the distances between pairs at the activation segments (e.g. K907-S909 and K907-S896) in a CCDC6-RET dimer assembly corresponding to the unstimulated (i.e. resting) estate (Fig. 8f), we observed that distances between pairs were compatible with intermolecular crosslinks rather than intramolecular ones considering the size and range of the of the DSBU crosslinker molecule (12.5-27.5 Å). These results are in line with a face-to-face arrangement of both catalytic domains defined by intermolecular crosslinked activation segments within the dimer in the inactive state (Fig. 8e-f). In the presence of nucleotide and MgCl , the catalytic domains swing apart, as indicated by the loss of activation loop crosslinked peptides, adopting a configuration in which both kinase domains are facing towards opposite directions from the stem of the coiled-coil. Furthermore, we showed on the two 3D-structural models the specific crosslinked peptides that defined the two functional and structural states, and the percentage of crosslinks within distance for each of them (Fig. 8g). The crosslinked peptides that defined the topology of the two functional and conformational states were clearly distinct, with one main central node of crosslinked peptides arising from activation loop Lys 907 in the inactive (unstimulated) state. In the case of the active (auto-phosphorylated) state, there were two main nodes identified, one at Lys 965 just below the susbtrate binding pocket, and another at a Lys 747 on the 2 of the G-loop. Mapping all the detected crosslinks in both structural models revealed a percentage of crosslinks within distance (27.5 Å cutoff) of 63.5 and 61.9 % for the unstimulated and phosphorylated states, respectively (supplementary Fig. 11 d and e). The subset of distance-compatible crosslinks and their clustering on specific structural elements is consistently observed across replicates and functional conditions, supporting further the internal consistency of the dataset. These data together demonstrate significant conformational changes during the mechanisms of CCDC6-RET autoactivation, and at the same time validate the internal consistency of the XL-MS dataset and its suitability as a structural restraint for the proposed models.

9. MD simulation analyses of inactive and active (phosphorylated) CCDC6-RET.
We generated structural models for an active CCDC6-RET homodimer (accession codes: ma-pcoh8 and ma-b7la0) in complex with ATP, ADP, Mg2+ and a Tyr-containing peptide substrate (Fig. 9b) that provided a plausible structural explanation for ADP-dependent catalysis (Fig. 4f). To assess the dynamic feasibility of the proposed structural models and peptide binding modes, MD simulations (300 ns) were run for the unstimulated (resting) and phosphorylated (active) states of CCDC6-RET. In the latter case, either ATP or ADP was placed on each of the active sites in complex with Mg2+ in addition to the phosphorylatable tyrosine-containing peptide (Fig. 9a). The results obtained support our proposed model in terms of stabilizing interactions and conformational changes that define both states. Importantly, the results also allowed us to identify the key residues and secondary structural elements implicated in catalysis that define the dual-specificity on Tyr and Ser substrates in the same active site (Fig. 9b). For the Tyr-containing substrate, we find the prototypical interactions required for catalysis with an open G-loop, defined by the side chain of Glu 734 forming a salt bridge with the side chain of A-loop Arg 912 (Fig. 9b, upper inset). In addition to the catalytic salt bridge (3 Lys 758- C Glu 775), the carboxylate of Asp 874 (HRD motif) is well positioned for proton abstraction from the hydroxyl of the phosphorylatable Tyr residue. This key interaction in complemented by the side chains of Arg 878 (held in position by means of a key cation- interaction with the indole ring of Trp 917) and Asp 879 (6-7 loop) to ensure proper -phosphate transfer (Fig. 9b, upper inset). In the case of the Ser specificity, we observed that for the Ser residue in the phosphorylatable peptide to reach deeper into the active site, the activation loop has to undergo a large conformational change that appears to be driven by a critical electrostatic interaction between the ATP's -phosphate and the side-chain guanidinium of Arg 912 (Fig. 9b, lower inset). In this setting, Ile 913, Pro 914 and Lys 916 form key contacts with main and side chain residues flanking the phosphorylatable Ser site.
Next, to identify and quantify the key contacts between chains that define the two specific configurations of CCDC6-RET, we used the PDBsum server 40. Because of a more compacted arrangement of both kinase domains and their interface, a larger number of amino acids interacting from both chains A-B were found in the case of the inactive form (60-59 aa) than the phosphorylated state (37-36 aa). This was the result of a much tighter interface found in the inactive (3084-3136 Å) than in the phosphorylated form (2106-2121 Å), as indicated also by the larger number of salt-bridges and hydrogen bonds found (Fig. 9c and supplementary Fig. 12). These MD simulation data together with the biochemical and structural information supported our proposed model and conformational switch that defines a transition from the inactive to the active (phosphorylated) state in CCDC6-RET during the process of autoactivation and signalling (Fig. 9d).

Methods
Plasmids.
A modified pBac-PAK3 plasmid (Invitrogen) containing an N-terminal 3 x His and a GST (Glutathione S-transferase) tags followed by a Human Rhinovirus (HRV)-3C protease cleavage site was used to express a codon optimized CCDC6-RET fusion product composed by fragments of residues 1-101 of human CCDC6 and 713-1072 of human RET 27, 28, 29. A construct lacking the C-terminal segment of RET (RET aa 713- 1013, i.e. CT) was also generated. These plasmids were used to create different point mutants by site-directed mutagenesis.

Site-directed mutagenesis. Site-directed mutagenesis was performed by the Q5-site directed mutagenesis kit (New England Biolabs) following manufacturer instructions and the indicated non-overlapping primers:
RET_Y900F- forward 5’- CCGTGACGTGTTCGAGGAGGACT -3’
RET_Y900F- reverse 5’- CTCAGTCCGAAGTCGGAG -3’
RET_Y905F- forward 5’- GGAGGACTCCTTCGTCAAGCGTT -3’
RET_Y905F- reverse 5’- TCGTACACGTCACGGCTC -3’
RET_Y1015F- forward 5’- CGTCGTGACTTCCTGGACCTG -3’
RET_Y1015F- reverse 5’- CTTGACCATCATCTTCTCCAG -3’
RET_Y1062F- forward 5’- AACAAGCTCTTCGGTCGTATCTC -3´
RET_Y1062F- reverse 5’- CTCGATCCAGGTGGAGGG -3´
RET_S1034A_forward 5´- CGACGGACTGGCCGAGGAGGAGA -3´
RET_S1034A_reverse 5´- TCGTAGATCAGGGAGTCGGAGG -3´
RET_S98A_forward 5’-GCGCAAGGCTGCGGTGACTATCG -3´
RET_S98A_reverse 5’- AGGTCGCGGTTTTCC -3
CCDC6_S52A forward 5’- AATCGTGATCGCCCCATTCCGTC -3´
CCDC6_S52A reverse 5’- CCACCAGATTTTCCACCG -3´
CCDC6_C84A_forward 5’- CAAGCTGAAGGCCAAGGCTCTGC -3´
CCDC6_C84A_reverse 5’- TAGGTTTCCAGCTCGATC -3´
CCDC6_Y80F_forward 5´- GCTGGAAACCTTCAAGCTGAAGST -3´
CCDC6_Y80F_reverse 5´- TCGATCTTCAGCACCTTG -3´
RET_D874A forward 5´- GGTGCACAGAGACCTGGPCCGCCA -3´
RET_D874 reverse 5´- AGCTTCATCTCGGCCAGGTACTGCATG -3´
Alternatively, other mutations were generated by using overlapping primers:
RET_S909A forward 5’- CGTCAAGCLGTGCCCAGGGTCGTA -3
RET_S909A reverse 5’- TACGAICCCTGGGCACGCTTGACG -3´
CCDC6_S46A forward 5’ - CGGTGGAAAAGCTGGTGGAATCGTGATCTC -3´
CCDC6_S46A reverse 5’- GAGATCACGATTCCACCAGCTTTTCCACCG -3´

Heterologous protein expression in insect cells.
We used the flashBACTM system (Oxford Expression Technologies) for recombinant protein expression using a baculovirus system 27, 28, 29 following the manufacturer recommendations. Briefly, Sf9 insect cells (Oxford Expression Technologies) were seeded in a 6 wells/plate at a density of 0.3-0.5 x 106 cells/mL in serum free Sf900-II media (Gibco) supplemented with 200 μL of Gentamycin (50 mg/mL) per liter. Next, the transfection reaction was prepared in a sterile Eppendorf tube containing antibiotic-free media Sf900-II (up-to 1 mL) and the transfection mixture composed by: 2.5 μL of flashBacTM linear viral DNA (20 ng/ L), 1 L (500 ng) of transfer plasmid (pBacPAK3) and 5 μL of transfection reagent (Fugene) following manufacturer instructions. After 5-6 days, the supernatant of the transfected cells was collected to have the recombinant baculovirus stock (P1). The P1 baculovirus was inoculated (1:25) in a suspension culture of 25 mL of complete Sf900-II media at a density of 1.5-2 x 106 cells/ml and incubated shaking at 90-95 rpm for 3-4 days. After this time, the supernatant was recovered obtaining the P2 baculovirus stock. After testing the expression of the protein, the P2 baculovirus was used to inoculate another 100 mL suspension culture of Sf9 cells as indicated before at a 1:100 dilution. After 3-4 days (72-96 hours), the supernatant became the P3 baculovirus stock. Both, the P2 and P3 baculovirus were used as baculovirus stocks for protein production.

Protein purification.
Protein purification was achieved by in tandem immobilized metal ion affinity (Ni+2) and Glutathione-conjugated gravity flow chromatography and in-gel 3C-protease digestion followed by size exclusion chromatography if required. This optimized protocol resulted in a highly pure and monodisperse recombinant fusion product. Inoculated insect cell cultures (72-96 hours) were harvested by centrifugation at 1800 g (Eppendorf Centrifuge 5810 R) during 10 min at 4ºC. Pellets were stored at -80 ºC or used directly for protein purification. Pellets were resuspended in lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 10 mM Benzamidine, 0.2 mM AEBSF, 2 mM TCEP). After sonication on ice (20 cycles 9 seconds on/ 3 seconds off and 37% amplitude), crude lysates were centrifugated at 33000g/48000 g (JA 25-50 rotor, Beckman Coulter Avanti J-25) at 4 ºC for 45 min. Clarified lysate underwent a final sonication step of 10 sec followed by filtration with a 45 m filter (Jet Biofilter) with a syringe. Crude lysate, clarified lysate (supernatant) and insoluble material (pellet) samples were mixed with 5X Laemmli sample buffer (Thermo Fisher Scientific) and run on SDS-Polyacrylamide gel electrophoresis (SDS-PAGE). First, the clarified lysate was incubated with a previously equilibrated Nickel (Ni2+) chelate resin. After two hours incubation on rotation in a cold room, the flow through (FT) was separated and beads were washed with Buffer A (20 mM Tris pH 7.5, 500 mM NaCl, 2 mM TCEP) with additional 20 mM Imidazole. Protein elution was done with Buffer B (20 mM Tris pH 7.5, 500 mM NaCl, 400 mM Imidazole, 2 mM TCEP) using a filter spin column (SC 1000-1KT, Lot SLCD4489, SIGMA-ALDRICH) by centrifugation at 6000g (Eppendorf centrifuge 5415D) during 2 min. Fractions were checked by SDS-PAGE stained with Coomassie previously to the second purification step. Positive fractions eluted from the His-affinity step were incubated with Glutathione conjugated Sepharose beads (Glutathione Sepharose® 4 Fast Flow, Cytiva) for several hours at 4 ºC. Next, the GST-tagged protein was separated by centrifugation at 160g (Eppendorf Centrifuge 5810 R) and washed 4-5 times with 10 mL of GST-Buffer (20 mM Tris pH 7.5, 500 mM NaCl, 2 mM DTT) prior to in gel digestion (3-4 hours) with an GST-3C protease (200 g/L culture) and elution (2-3) using a filter spin column at 6000g for 2 min (Eppendorf centrifuge 5415D). Eluted fractions were checked by SDS-PAGE gel stained with Coomassie. Protein concentration and purity were measured by absorbance using a Nanodrop. When required, a size exclusion chromatography (SEC) step using AKTA pure protein purification system (GE Healthcare) was performed. The sample was concentrated up to 2 mL with a Millipore concentrator with a 30 kDa cut-off by centrifugation at 800g (Eppendorf Centrifuge 5810 R) at 4 ºC. The concentrated sample was injected into a Superdex 200 16/60, 10/300 or 3.2/300 columns (GE Healthcare). The input of the column together with the collected fractions were analyzed by SDS-PAGE followed by Coomassie staining, and the protein concentration was measured by absorbance.

SDS-PAGE, western blotting and antibodies.
Proteins were electrophoretically separated on 12% SDS-PAGE gels run in the Mini-PROTEAN system with electrophoresis buffer (25 mM Tris pH 8.3, 192 mM glycine, 0.1% SDS) by using a BIO RAD PowerPac Basic at a constant voltage of 150-200 mV for 30-45min. Pre-stained dual colour markers (Precision Plus Protein Standards 10-250 kDa, BIO-RAD and Protein Ladder PS10 Plus 11-180 kDa, GeneOn) were used as molecular weight standards. For protein staining, gels were gently shacked in Coomassie staining solution (10% acetic acid, 40% absolute ethanol and 50% MilliQ water with 1g/L Brilliant blue 250 R) for 30-60 min. Then the gels were distained for 1-2 hours with a distaining solution (10% acetic acid, 50% absolute ethanol, 40% MilliQ water). Proteins were transferred from SDS-PAGE gels onto nitrocellulose 0.2 m (Amersham) or PVDF 0.45 m (Millipore) membranes using the Mini-PROTEAN system (BIO RAD). Before transference, PVDF membranes were activated first in ethanol 100% for 10 min. Membrane were wet-transferred in transfer buffer (25 mM Tris pH 8.8, Glycine 190 mM and ethanol 10%) on ice at constant 100-200 V for 60 min. Next, transferred membranes were blocked in TBS (10 mM Tris pH 8, 150 mM NaCl) supplemented with 5% (w/v) skimmed powder milk and gently shacked for 30 min. After blocking step, membranes were washed in TBS-T (10 mM Tris pH 8, 150 mM NaCl Tween-20 0.1% v/v) for 3-4 times. Then they were incubated with constant agitation (Duomax 1030, Heidolph) O/N at 4ºC with the following primary antibodies diluted in TBS-T supplemented with 5% BSA (w/v): phospho-Tyr (pTyr-100, CST #9411), RET (D3D8R, CST #14698), phospho-Ret Tyr 905 (CST #3221), phospho-Ret Tyr 1015 (Abcam # 74154) and phospho-Ret Tyr 1062 (Abcam # 51103) at 1:5.000 or 1:10.000 dilution. After overnight incubation with the primary antibody, membranes were washed with TBS-T for 3-4 times and incubated with the secondary antibody for 60 min with gently shaking protected from light. Secondary antibodies used were anti-rabbit IgG (H+L) conjugated at 800 nm (DyLight 800 Conjugate #5151, CST) and anti-mouse IgG (H+L) conjugated at 680 nm (DyLight 680 Conjugate #5470, CST). These secondary antibodies were diluted 1:20.000 in TBS-T with 5% (w/v) skimmed powder milk. After 60 min of secondary antibody incubation, membranes were washed with 20mL of TBS-T for 3-4 times. Finally, proteins were detected in the membranes with the Odyssey CLx scanner (LI-COR Biosciences) and the images were analyzed with the Image Studio lite software.

Differential Scanning Fluorimetry.
To determine the thermal stability of the recombinants proteins we apply a direct method of Differential Scanning Fluorimetry (DSF) based on intrinsic fluorescence (IF) detection using a Tycho NT.6 instrument (NanoTemper Technologies). Briefly, a protein sample (1-2 M, 12L) was loaded into a glass capillary tube and heated from 35 ºC to 95 ºC at a rate of 30 ºC /min. The inflection temperatures (T) were calculated by the instrument based on the 350/330nm fluorescence ratio. We measure the increment in thermal stability in the absence or presence of different ligands and inhibitors, as indicated in the text.

Mass Photometry.
To determine the molecular mass distributions (40 kDa to 5 MDa) of single molecules we applied light scattering to detect individual, unlabeled molecules in dilute solutions using a MPone instrument (Refeyn). All the events were recorded with Refeyn Acquire MP software and analyzed with Refeyn Discover MP software to obtain mass distribution of protein samples.

In vitro phosphorylation assays.
For autophosphorylation experiments, recombinant protein 1-2 M (final concentration) was used in 20 mM Tris pH 7.5, 500 mM NaCl, 2mM DTT and 2 mM MgCl . We performed a time-points reaction triggered by the addition of ATP (1 mM): unstimulated, 1, 5, 15, 30 min. To stop the phosphorylation reaction samples were mixed with 5X Laemmli sample buffer and denaturalized at 95ºC during 2-5 min in a thermoblock (AccuBlock, Labnet). In the case of phosphorylation experiments using intact substrate surrogates (catalytic dead) a 1:3 molar excess of the substrate to the enzyme was employed in the reaction. For the nucleotide titration experiments the phospho-tyrosine activity of CCDC6-RET was monitored under increasing concentrations of nucleotide (from 10 to 5000 M) after 1-15 min stimulation.
For the time course autophosphorylation assays using radiolabeled nucleotides we prepared an in vitro time course autophosphorylation reaction as follow: for ATP (P32, Revvity) we performed a 0-30 min time course assay in elution buffer 20 mM Tris pH 7.5, 500 mM NaCl, 2 mM DTT and 4 mM MgCl complemented with kinase (2.5 M), ATP (2 mM) and ATP P32 (50 Ci). In the case of the radioactive assay with ADP (P33, Hartmann Analytics), for the time course of the autophosphorylation reaction (0-60 min) buffer was complemented with kinase (10 M), ADP (1 mM) and ADP P33 (60 Ci). Samples were mixed with 5X Laemmli sample buffer and denaturalized at 95ºC during 2-5 min before being run on a 4-12% precast bis-tris polyacrylamide gels (Invitrogen, Thermo Fisher Scientific). Electrophoresis was run at 200 V, after which the gels were introduced in a exposure cassette with a phosphor screen for 2-4 hours first, and then overnight for a longer exposure if required. Screens were developed in an Amersham Typhoon Biomolecular scanner using the phosphor imaging option using 4000 volts and 100 µM of pixel size resolution.

Enzyme kinetic assays.
Rates of phospho-tyrosine activity by recombinant CCDC6-RET and its variants were determined by a continuous ATP-coupled pyruvate kinase essay based on the consumption of NADH in the absence and presence of different peptides substrates at increasing concentrations of ATP 28, 29, 30. The enzyme-substrate solution (1 mM Tris-Cl pH 7.5, 1 mM MgSO , 400 mM phosphoenol pyruvate (PEP), 100 mM NADH, 2450 U/mL Pyruvate kinase, 2.26 mg/mL Lactate dehydrogenase) was prepared at different concentrations of ATP (mM): 0, 0.08, 0.16, 0.32, 0.65, 1.25, 2.5 and 5. Unless otherwise stated, the final protein concentration used in these assays was 0.2-1 M, and the concentration of the peptides used EAIYAAPFAKKK and the RET A-loop DVYEEDSYV were 1.35 and 0.55 mM, respectively. The experiments were performed in a 384 well plate (Greiner bio-one) and the NADH consumption was read at 340 nm wavelength during 120 cycles of 30 sec each by using a microplate reader (CLARIOstar Plus, BMG LABTECH). NADH consumption curves were obtained, and slopes were calculated by using de CLARIOstar Data Analysis software. Finally, by using Prism software, catalytic rates and kinetic constants were obtained by fitting the data to the Michaelis-Menten equation.

Mass Spectrometry.
For liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) samples (2.5-5 M) were reduced and alkylated in 15 mM TCEP and 30 mM CAA for 30 min at room temperature in the dark. Proteins were then digested overnight at 37 °C with trypsin (Promega) in 1 M urea, 50 mM Tris-HCl pH 8.0, using an estimated protein-to-enzyme ratio of 100:1. Digestion was quenched with 0.1% TFA and resulting peptides were desalted using C18 stage-tips. LC-MS/MS analysis was performed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled to an UltiMate 3000 RSLCnano system. Digested peptides were loaded into a trap column (Acclaim™ PepMap™ 100 C18 LC Columns 5 µm, 20 mm length) for 3 min at a flow rate of 10 µL/min in 0.1% formic acid, and then separated on an EASY-Spray PepMap RSLC C18 column (2 µm, 75 µm x 50 cm, Thermo Fisher Scientific) operated at 45 °C using a 60 min effective gradient (buffer A: 0.1% FA; buffer B: 100% ACN, 0.1% FA) at a flow rate of 250 nL/min. The gradient used was, from 4% to 6% B in 2 min, from 6% to 33% B in 58 min, plus 10 additional min at 98% B.
Peptides were ionized using an EASY-Spray source at 1.5 kV with a capillary temperature of 300 °C. The mass spectrometer was operated in a data-dependent acquisition mode (DDA using a top 15 method with an intensity threshold of ≥ 4.5 x 104, a dynamic exclusion of 5 or 10 sec and excluding unassigned, +1 and > +6 charged ions. Full MS scans (350–1500 m/z) were acquired at 70,000 resolution (FWHM at 200 m/z), followed by MS/MS scans at 35,000 resolution using higher-energy collisional dissociation (HCD, NCE 27). The isolation window was 2.0 Th. Ion targets were 3×10⁶ for MS (max IT 25 ms) and 1×10⁵ for MS/MS (max IT 110 ms).
Raw files were processed with Maxquant (v 1.6 and higher) using the default settings against a database containing the sequences of the recombinant proteins. Carbamidomethylation of cysteines was set as a fixed modification whereas oxidation of methionines, acetylation and phosphorylation of serines, threonines and tyrosines were set as variable modifications. Minimal peptide length of seven amino acids and a maximum of two tryptic missed cleavages were allowed. Results were filtered at 1% FDR at both peptide and protein levels. Raw data were imported into Skyline. Quantitative analysis was performed in Skyline. Label free quantification of phosphopeptides was performed by integrating the extracted ion chromatogram (XIC) of the isotopic envelope. Only interference-free peaks were used for quantification. Phosphopeptides intensities were normalized by the intensity of the non-modified peptides of the target protein.
Data shown are from four independent autophosphorylation experiments with two technical replicates each (see supplementary tables 1-4). Phospho sites that are consistent across the different datasets are summarized on main table 1.

Crosslinking mass spectrometry.
Both phosphorylated (1 mM ATP, 2 mM MgCl , 30 min) and un-phosphorylated CCDC6-RET (6 µM) were crosslinked with 200-fold molar excess of disuccinimidyl dibutyric urea (DSBU, Thermo Fisher Scientific) in 20 mM HEPES pH 7.5, 500 mM NaCl, 1mM TCEP. All the crosslinking reactions were performed at RT for 50 min and then quenched with 20 nM NH HCO . The cross-linked samples in triplicate were vacuum-dried and resuspended in 4% SDS in 50 mM Tris/HCl pH 8.0. Reduction and alkylation were performed with 15 mM TCEP and 25 mM chloroacetamide 30 min at 45 ºC, respectively. Samples were then trypsinized (16 hours, 37 °C) at an enzyme-to-substrate ratio of 1:50 following an automated SP3 protocol 31. Digested samples were resuspended in 1% (v/v) formic acid, 0.5% DMSO for LC-MS/MS analysis. Peptides were analysed by LC-MS/MS in an Exploris480 mass spectrometer (Thermo) coupled to a Vanquish Neo LC system. Peptides were loaded into a trap column (PepMap™ Neo, 5 µm C18, 300 µm x 25 mm) and then separated on an Easy-Spary™ PepMap Neo™ analytical column (C18 2 µm 75 µm x 500 mm) maintained at 50 ºC. An 85-minute gradient was used at a flow rate of 400 nL/min, with solvent A consisting of 0.1% formic acid (FA) and solvent B consisting of 100% acetonitrile (ACN) with 0.1% FA. The gradient ramped from 2% to 25% solvent B over 50 minutes, then from 25% b to 45% solvent B over 20 minutes, followed by a 9-minute wash at 98% Buffer B.
The mass spectrometer was operated in a data-dependent mode, with an automatic switch between MS and MS/MS scans using a top 15 method. Intensity threshold ≥ 5 x 103, dynamic exclusion of 20 sec and excluding charges (+1 and ≥ +8). MS spectra were acquired from 400 to 1500 m/z with a resolution of 60,000 FWHM (200 m/z). Ion peptides were isolated using a 0.7 Th window and fragmented using higher-energy collisional dissociation (HCD) with a stepped normalized collision energy of 29, 31 and 33. MS/MS spectra resolution was set to 60,000 (200 m/z). The ion target values were 3 x 106 for MS (maximum IT of 25 msec) and 1 x 105 for MS/MS (maximum IT set to auto, according to the transient length). Aliquots of both the cross-linked and pre-cross-linking samples was digest with trypsin in parallel. Two hundred nanograms of the resulting non-cross-linked peptides were analyzed using a 37-minute gradient on a Vanquis Neo, coupled with an Orbitrap Astral. The Orbitrap Astral was operated under a standard Data-Independent Acquisition (DIA) protocol to optimize protein detection and enhance quantification. Subsequently, DIANN analysis was performed on the pre-cross-linking sample, querying a custom-built FASTA database that included the host expression organism (Sf9 and the baculovirus) the CCD6-RET fusion protein construct, and the UniProt sequences of 525 proteins commonly identified as contaminants over the past decade in proteomic analyses of diverse samples. The proteins identified by DIANN were sorted by intensity and examined for potential contamination. For the cross-linked samples, the top 313 most abundant proteins in the non-cross-linked sample, based on the sum of all precursor intensities as calculated by DIANN, were selected. These proteins were then used to construct a customized FASTA database for MeroX searches. This number was deemed sufficient to model decoy hits and control the false discovery rate (FDR), as MeroX uses a built-in target-decoy strategy at the crosslink spectrum match (CSM) level for reliable error estimation. For MeroX analysis of cross-linked samples, raw files acquired from the Orbitrap Exploris 480 were converted to MZML files using the ProteoWizard ms.convert program. The search parameters for MeroX were configured as follows: protease sites R/P and K/P, with a maximum of three missed cleavages, with K blocked as XL-site. Carbamidomethylation of cysteine (C) was set as a fixed modification, while oxidation of methionine (M) and phosphorylation of Ser, Thr, and Tyr were treated as a variable modification. DSBU was chosen as the cross-linker, and MeroX's default parameters were used, considering dead-end reactions with H O and NH , since the cross-linking reaction was quenched with excess ammonium bicarbonate. The precursor and fragment ion precision were set to 5 ppm (MS1) and 10 ppm (MS2), respectively. Identification focused on b and y ion types, with a minimum of three fragments per peptide. Charge states of +2 and higher were selected, and deisotoping was enabled. Precursor mass correction was disabled. RISEUP was chosen as the analysis mode, allowing for one missing ion. Neutral losses were enabled, with a maximum of one loss per fragment. For scoring and FDR calculations, a prescore of 10%, an FDR cutoff of 1%, and a score cutoff of 50 were applied. Given the use of a customized database based on comprehensive sample analysis, the MeroX cRAP database was excluded from the searches.
Finally, each fragmentation spectra were manually inspected and validated. Circular views and further processing of crosslinked residues were done using XiVIEW 32. For cross-linked peptide quantification, raw data were imported into Skyline. Label free quantification of identified cross-linked peptides was performed using the extracted ion chromatogram of the isotopic distribution. Only peaks without interference were used for quantification and manually inspected for correct area integration. The sum of the ion areas was collapsed for each cross-linked peptide and normalized using the total ion intensity of each sample. Data shown are from one experiment with triplicate samples with two technical replicate each. Crosslinked samples were tested by SEC to monitor the formation of dimers versus high-molecular weight species.

Negative staining and electron microscopy.
CCDC6-RET (0.7 M) was placed onto freshly glow-discharged (GloQube, Quorum Technologies) home-made carbon-coated 400 mesh copper electron microscopy (EM) grids. The conditions for glow discharge were: 15 mA, 45“, 0.1 bar. The samples were then incubated for 5 sec at RT. The grids were consecutively placed on top of three distinct 50 μL drops of MilliQ water, striped gently with filter paper for 2 sec and laid on the top of two different 50 μL drops of 1% uranyl acetate and stained for 1 min. Finally, they were stripped gently for 5 sec and air dried. Prepared grids were visualized on a Tecnai G2 Spirit transmission EM (Thermo Fisher Scientific) with Lanthanum hexaboride cathode operated at 120 keV. Images were recorded at 61 320 x nominal magnifications on a 4k x 4k TemCam-F416 CPMOS camera (TVIPS), with pixel size of 2.5 Å/pix. For data collection, the open-source software SerialEM (bio3d.colorado.edu/SerialEM) was used under a Massachusetts Institute of Technology (MIT) license. For data processing, open sources software cryoSPARC 33 and Relion were used 34.

Small angle X-ray scattering.
SAXS experiments were conducted at the beamline B21 of the Diamond Light Source (Didcot, UK)35. CCDC6-RET∆CT (40 L, 5 mg/mL) was injected at room temperature into a Superdex 200 Increase 3.2 column connected to an in-line Agilent 1200 HPLC system. The buffer used for the SEC column was the same protein buffer (20 mM Tris pH 7.5, 500 mM NaCl, 1mM DTT). The eluted samples were exposed for 300 s in 10 s acquisition blocks using an X-ray wavelength of 1 Å, and a sample to detector (Eiger 4M) distance of 3.7 m. The data covered a momentum transfer range of 0.0032 < q < 0.34 Å-1. The frames recorded immediately before elution of the sample were subtracted from the protein scattering profiles. The Scåtter software package (www.bioisis.net) was used to analyze data, buffer-subtraction, scaling, merging and checking possible radiation damage of the samples. The R value was calculated with the Guinier approximation assuming that at very small angles q < 1.3/R . The particle distance distribution, D , was calculated from the scattering pattern with GNOM and shape estimation was carried out with DAMMIF/DAMMIN, both programs included in the ATSAS package 36. The proteins molecular mass was estimated with GNOM. Interactively generated PDB-based homology models were made using the program COOT by manually adjusting them into the envelope given by SAXS that could match the experimental scattering data. This was computed with the program FoXS 37.

Structural modelling and molecular dynamic simulations.
Publicly available X-ray crystal structures of different RET kinase constructs, as well as those of related tyrosine kinase domains from catalytic receptors such as EGFR, FGFR (1-3), and the human insulin receptor, were suitably aligned using the Dali server. The RCD+ server 38 was employed to fill in existing gaps in the structurally disordered regions due to lack of electron density. The ATP molecule and the two coordinating, partially hydrated, magnesium ions (Mg²⁺) were carefully placed in an arrangement compatible with transfer of the -phosphate to a peptide substrate (near-attack conformation), as found in the prototypical cyclic AMP-dependent protein kinase A (PDB ids. IL3R and 4HPU). This experimental information was complemented with models generated by AlphaFold 3 39 for wild-type RET and the oncogenic CCDC6-RET homodimer, both in the absence and presence of adenosine nucleotides, Mg²⁺, and peptide substrates (accession codes: ma-pcoh8 and ma-b7la0). Additional models of CCDC6-RET in the unstimulated form were obtained with AlphaFold (AF id: J7M8C2, for the single chain in the manual model) and AlphaFold multimer Colab (RETPTC1DIMER_a902d_unrelaxed_rank_001_alphafold2_multimer_v3_model_2_seed_000), in the case of the homodimer. The SWISS-MODEL homology modeling server 40 was also used for validation. Energy minimization and molecular dynamics (MD) simulations at 300 K of the systems studied were run in explicit physiological saline solution under periodic boundary conditions using the ff14SB AMBER force field 41 with updated parameters for phosphates 42, TIP3P water molecules and the pmemd.cuda_SPFP engine, as implemented in AMBER 18 running on Nvidia GTX 1080 RTX2080Ti GPUs, essentially as described 43. The cutoff distance for calculating the non-bonded interactions was 9 Å and the smooth particle mesh Ewald method with a grid spacing of 1 Å was used to account for electrostatic interactions. Application of the SHAKE algorithm to all bonds involving hydrogens allowed an integration step of 2.0 fs to be used. Each solvated system was simulated in the absence of any restraints for at least 300 ns during which system coordinates were collected every 0.5 ns for further analysis.
Complementarily, for the respective Michaelis complexes, a time-dependent biasing harmonic potential of 5 kcal mol−1 Å−2 was included -as an extra term in the form of a flat well with parabolic sides- to impose pulsating distance restraints within a range compatible with nucleophilic attack (3.9-4.0 Å) and hydrogen bond formation (2.9-3.1 Å). The molecular graphics program PyMOL v. 2.6 was employed for molecular visualization and figure preparation.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors describe a biochemical and structural study of the CCDC6-RET fusion oncoprotein, which is a highly active kinase consisting of the kinase domain of the RET tyrosie kinase fused to a coiled-coil domain from CCDC6. This extensive work incorporates diverse experimental approaches including enzyme kinetics, mass spectrometry, negative stain EM and SAXS to probe the sturcture and activity of this fusion oncoprotein. While there are some solid novel findings here - the comparison of activity of the intact fusion and isolated kinase domain, time-course mapping of autophosphorylation sites - other findings are not sufficiently fleshed out or conclusively supported by the data presented. In particular, there is a worry that ADP-dependent phosphorylation - one of the more interesting findings presented here - is an artifact of contamination with ATP. The negative stain EM reconstructions and computational modeling of the structure, including with Alphafold models, are fine as far as they go, but I am not convinced that there are important dimer contacts apart from the coiled-coil -- are there contacts between the two kinase domains in the dimer that are important for activity? Contacts between the kinase and coiled-coil from the fusion partner? Additionally distinct structural states for unphosphorylated and phosphorylated dimers are presented based on differences in crosslinking mass spec results, but there is a worry that some of these crosslinks could arise from higher order oligomers rather than intra-dimer interactions. In any case, there is no structure-function work ppresented to test these questions.\nSpecific comments/concerns:\n1. This is clearly a highly active kinase fusion – why is it not already autophosphorylated as purified from insect cells?\nPlease include the reaction progress curves from the coupled enzyme assay for Fig. 1h and 3b as supplemental figures.\nWould be informative with respect to whether there is a lag phase resulting from a requirement of autophosphorylation for full\nactivity.\n2. p.16 Binding tightness – not specific, better to just say “Binding…was evaluated).\n3. Fig. 1f. Does not make sense to label x-axis with delta Ti, instead indicate which inhibitor and concentration for each bar.\n(and legend incorrectly refers back to itself).\n4. With the mass spec autophosphorylation time course experiments, it would be informative to analyze the corresponding\nnon-phosphorylated species in an analogous manner. The fractional decrease in the non-phoshospho peptides would give\na sense of the stoichiometry of phosphorylation of these many sites…are they major sites with high site-occupancy? Or\ndetectable but present on a very low proportion of molecules?\n5. Why is apparent Km[atp] so different in Fig 1h vs. Fig 3b for the intact fusion proteins? Please provide the Km values for\nfig. 3b.\n6. There is a very real worry that the apparent ADP-dependent phosphorylation is just an artifact of contamination of the ADP\nwith ATP. Or that contaminating ATP was carried along with the kinase in the course of enzyme purification. Not clear that\nthe authors have done anything to address this possibility. A few suggestions: 1) measure ATP content of ADP preparation\nusing a luciferase assay. Or better yet measure directly by LC/MS. 2) Test activity with beta-32P-labelled ADP. 3) Test\nphosphorylation of a peptide substrate in a quantitative way, with peptide in excess. If you start with say 500 nmoles of ADP,\ncan you phosphorylate most or all of 500 nmoles of peptide? Or do all of the above!\n7. In figure 4, what are the Km values for full length and truncation? (they look quite similar). How do we know that the\napparent difference in Vmax in not just a difference in enzyme concentration? Legend not reassuring in this respect – reports\n0.2-0.3 uM. Need demonstrated careful measurement of enzyme concentration. At a minimum Coommassie stained gel with\nequivalent amounts used, or better to titrate both with a potent Ret kinase inhibitor – show that x micromoles of inhibitor\ncompletely kills x micromoles of enzyme. Also, would be worth looking at the effect on Km[peptide].\n8. Figure 5 and associated text was very difficult to follow and full of logical leaps. The lack of side-by-side comparable\nexperiments with the A-loop mutants in FL vs deltaCT makes it impossible for me to draw any meaningful conclusions.\n9. Crosslinking-MS data are interpreted as being intra-dimer, inter-molecular crosslinks. But seems no steps were taken to\nensure that none are inter-dimer crosslinks? (For example SEC after crosslinking to remove any higher order crosslinked\nspecies?)\n10. Actually the SDS-PAGE gel with crosslinked protein shows the major species is a smear around 200kDa (presumably\ndimer of dimers?), and there is also a pronounced band above 250 kDa. So not reasonable to interpret crosslinks as intra-\ndimer.\n11. It is clear that phosphorylation changes the pattern of crosslinking, but not clear how the authors arrived at their structural\nmodels. And in any case, given concerns in 9 and 10 above, I feel the structural models are a gross over-interpretation of the\ndata.\n12. What is meant by cis-autophosphorylation? The data suggest autophosphorylation in trans in the context of the dimer –\nno clear concentration dependence of autophosphorylation of the dimer, but there is concentration dependence of a\nmonomeric kinase construct. Is this what the authors mean by “cis” autophosphorylation? I think most would say “the\nprotomers of the dimer autophosphorylate in trans”. Cis auto-phosphorylation implies that a given kinase domain/subunit\nautophosphorylates itself. There is no data that I see here that would test this mechanism (for example preparation of a\ndefined heterodimer with one active and one inactive subunit and a test of whether the active subunit could phosphorylated\nthe inactive partner.", "reviewer_2": "The paper by Plaza-Menacho et al aims at describing the enzymatic activity as well as the molecular structure of CCDC6-\nRET chimeric oncoprotein. The paper is potentially interesting, with data suggesting that: i) CCDC6-RET is a dual\nATP/ADP-dependent kinase; ii) the activation loop and the c-terminal tail of RET interact modulating protein folding and\nactivity; iii) CCDC6-RET autophosphorylation occurs in cis. In addition, they also provide an in silico as well as Cryo-EM\nand crystallographic analysis to generate a structural model of CCDC6-RET homodimer both in the active as well as\ninactive conformation. A major weakness of the paper is that it is not clear which is its principal message since all those\ntopics tackled in the manuscript are extremely important, but none is demonstrated in a complete and convincing manner.\nFor example, the ability by CCDC6-RET to use ADP to phosphorylate itself and substrates is striking news. ADP is\nclassically and paradigmatically unable to provide the beta-phosphate to kinases for phosphorylation. A deeper and more\ndetailed description of this activity, putting it in comparison with wt RET (not just the TK domain which is a poorly active\nkinase) and possibly other chimeric RET fusions, or providing the rate of AMP production (both when supplying ATP or\nwhen using ADP), as well negative controls using other kinases or a kinase dead mutant.\nAnother example of lack of completeness regards the interaction between the C-tail and the activation loop. Is this true also\nfor the other forms of RET, and in particular the longer one RET-51 (they have used the RET-9 isoform in the paper)? Is this\ninfluenced by proteins that bind to the c-tail in cells (SHC, ENIGMA, FRS2, IRS1/2, ect)? Again, is this peculiar to CCDC6-\nRET or is important also for other RET rearrangements and wt RET?\nMy suggestion is to study individually each of these aspects, providing a more comprehensive and analytical research study\nfor each of this topic (maybe using the unpublished data reported in the discussion). I guess that in this way authors would\nmake a definitive and convincing point on each of the finding they have presented in the manuscript.", "reviewer_3": "The paper of Martín-Hurtado et al. uncovers some of the mechanism and molecular function of the CCDC6-RET fusion\nkinase, with important findings on the specificity and autophosphorylation activation. Where the paper is strongest is\nbiochemical characterization, and especially the phosphorylation assays, which clearly demonstrate the dual specificity of\nthis kinase. However, it is severely lacking in structural characterization, not because of lack of experiments (which are\nplentiful) but because the model derived is not sufficiently supported by data and does not explain the authors’ observation.\nSome of the experiments in particular are filtered in ways that inevitably support the proposed model, rather than building a\nmodel from the available data.\nIt is not the lack of resolution per se (if high resolution could not be attained, that is the way it is), but the lack of a proper\nintegrative approach that accounts for the experiments carried out. Thus the model derived does not clarify or explain the\nobservations made in biochemical experiments, and does not come with quantifiable precision/uncertainty. Furthermore, the\nmethods section is lacking some critical details that would help understand some of the experiments and modeling steps, as\ndetailed below.\nOverall, this leads to a major shortcoming in terms of establishing a structure-function relationship and a mechanistic\nunderstanding of what underpins the kinase activity and regulation. In short, the paper has some major findings on the\nspecificity and mechanism of the kinase, but then the model presented does not explain how CCDC6-RET achieves its\nproperties. In particular, little data is presented as to the activation mechanism (it is speculated upon) and as to why this\nkinase would have tyrosine and serine specificity or why it would work on both ATP or ADP given the model.\nThe biochemical and structural data taken by itself is of excellent quality, except for the proteomics and crosslinking MS,\nwhere the experimental analysis and design leaves something to be desidred (see below). The level of support for the\nconclusions- i.e. the generation of the structural model and the interpretation of the connection between biochemistry and\nstructure- is currently weak. If properly characterized however, this paper would provide highly significant findings.\nI would recommend accepting the paper only after major revisions.\nMajor concerns:\n- Phosphoproteomics. It seems that there are 2 full replicates, each of which was injected in technical duplicates, but a\nsingle plot is “representative of 2 independent experiments”, rather than showing all 4 traces. As we are being asked to\njudge dynamics, the reproducibility of the quantification is quite critical, so at least 3 or 4 full replicas are required, even in\nsingle injection, so that proper quantification and error estimation may be carried out. The variance of a double injection is\nlow, as expected, but the authors are claiming this is \"representative” without even showing all 4 traces. The\nphosphoproteomics plot should come with error bars coming from quantification of replicates.\n- In the methods section, state the number of full and technical replicates for both crosslinking MS and proteomics.\n- Quantitative crosslinking MS: quantitation is not performed across replicates, which are needed when comparing between\nconditions. I would expect volcano plots where we can see what residue pairs are significantly changing between apo and\nphosphorylated conditions. See for example Tomko et al. Cell 2015 (https://pubmed.ncbi.nlm.nih.gov/26451487/). The fact\nthat a crosslink is detected only in a particular condition, when variance is not estimated, is not indicative of a unique\nconformation, especially in DDA.\n- Show all detected residue pairs on the structure and provide a distance histogram, as well as coloring the circle plot by\ndistance. I find it hard to believe that almost all crosslinks detected arise from peptides containing the critical tyrosine, and\nthere is almost no coverage of the rest of the protein. Does the crosslinking MS support this model? Only an unbiased\nmapping of all detected residue pairs at 5% FDR (provided the database did at least include contaminants) can show- one\ncannot claim validation after filtering to only have distances within range.\n- The structural modeling is perhaps the weakest part of the paper. In fact, there is no methods section for the modeling part,\nwhich comprises a major part of the paper. The results are interspersed with the steps done to assemble the model. The\nauthors manually generated and adjusted a model by patching a coiled coil alignment and the kinase region. This is then\nplaced in the negative stain density and presented as a good fit, which is not quantified. Similar thing for the SAXS, where\nthe authors delete the disordered regions to rejigger them inside the DAMMIN density, only to then say the resulting model is\na good fit with CRYSOL. Are the disordered regions compact or extended? This is exactly the sort of information SAXS may\nprovide if it is treated as the ensemble solution experiment that it is. The crosslinking MS experiment is similarly presented\nas validating the model, but only after filtering crosslinks to within distance and not plotting the full crosslinking MS dataset\non the structure. As a matter of fact, what is the advantage of using the hand made model over the AlphaFold one (or\nadjusting the alphafold one)? At least AlphaFold has a confidence metric that shows that it is not confident in the coiled coil\nregions. I would encourage the authors to use HADDOCK or the integrative modeling platform (IMP) to integrate SAXS,\nnegative stain, crosslinking MS and basic structure predictions to generate an ensemble of predicitons based on the data.\nOr, the crosslinking MS data can perhaps be used to improve the modeling with AlphaLink2. In both cases, this would then\nlead to a model that can be interpreted as it comes with a precision that encodes for the uncertainty\n(https://pubmed.ncbi.nlm.nih.gov/25173450/) of low resolution and sparse restraints.\n- Figure 5 c, “Enzymatic assay performed with CCDC6-RET  CT, WT and activation loop mutants Y900F, Y905 and\nY900/905F (1 M) incubated with increasing concentrations of ATP at a fixed concentration (1.35 mM) of a c-Abl derived\npeptide (EAIYAAPFAKKK), I would expect to have five curves but only four are shown. Also the activation loop mutants\nused in the essay are presumably from the main text from the protein version lacking the C-terminal domain; please annotate\nit in the legend accordingly to WB figures.\n- Where are all the phosphosites detected on the structure? Does the model explain how some phosphosites are\nphosphorylated before others? Are they near the active site? The pocket is not highlighted in any of the figures. What are the\nmotifs around the phosphosites? Showing the phosphosites, discussing their position and coloring them by time of\nphosphorylation would help address many of these questions. The distinction between cis and trans autophosphorylation\nshould have a correspondence in the model.\nMinor concerns:\n- How do the authors explain the upper and lower bands of the western blot in the phosphorylation assays?\n- Figure 4f, please show the residue number in the circos plot for better clarity.\n- Figures 4 b and d, although clear from figures c and e, state the WT and ΔCT in the legends of the enzymatic assays with\nA-loop and abl peptide.\n- DSBU is a homobifunctional (not heterobifunctional) crosslinker. It has 2 NHS-ester moieties.\n- “In particular, we found that in autophosphorylation and enzyme kinetics experiments, where we evaluated the KM for ATP\nin the presence of an excess of peptide susbtrate, a Y905F and double Y900/905F mutants have a loss of function effect,\ncontrary to the Y900F, that was catalytic competent. “ Please state Figure 5c for better clarity of the reader.\n- Figure 8b, kcat/km ratio instead of only kcat should be indicated on the y axis of the bar plot. Please indicate legend with\nthe different CCDC6-RET concentrations used in the enzymatic activity assay plot in figure 8b.\n- Why not AlphaFold3 but filtered by prediction confidence? What does the AlphaFold3 model look like?\n- In one of the panels showing the model, highlight the ATP binding site and substrate binding pocket, if possible. Does the\nmodel give hints as to why it would phosphorylate serine and tyrosine? Is the pocket similar to other dual specificity\nkinases?\n- Show SEC-MALS data, size exclusion traces if possible. The results of the coiled-coil predictor, which in general may be\nless accurate than AlphaFodl, can be shown in fewer panels.\n- “This residue when mutated appears to have a profound effect on the catalytic activity of the RET fusion product towards a\nsubstrate despite lacking any effect on autophosphorylation (unpublished data).” This is a particularly important point. I\nsuppose the difference between “data not shown” and “unpublished data” is that these findings lie in another paper that is\nalso in review? Can there be at least pointing to a preprint?\nTechnical points:\nCrosslinking MS analysis:\n- For MeroX analysis, please state clearly how you performed the analysis. E.g., Was precursor correction enabled? For mgf\nfile (only MS2 data), the software would do this without knowing the real MS1 data, so either turn off that option or inspect the\nMS1-precursor level of the cross-links. If you export mzML with also MS1 information, then it would be looking at MS1 data to\nperform precursor quadrupolar isolation window correction. For QExactive series of instrument this correction is quite useful,\nnever tested on Exploris 480. Was RISE-UP mode used? How many proteins were used in the fasta? Fusion protein alone\nor with contaminants (cRAP database option in the last panel)? Searching with target-decoy FDR against a single protein\ninevitably leads to poor error estimation, as the decoy database is not large enough to model the noise of random\nmatching.\nAlphaFold:\n- The authors do not state which version of AlphaFold multimer was used or any of the settings with regards to template and\nMSA. It seems the authors may have used colabFold, with relaxation off, given the severe clashes present in the model.\nSimilar steric clashes are also present in the model the authors built, which shows that the coiled coil prediction server may\nhave packed the helices a bit too close together, or at the wrong angle.\nData availability:\n- We were not given access to the proteomics or crosslinking MS data as the authors did not include the reviewer password\nassociated with the deposition.\n- Is the Skyline file containing the phospho-peptide quantification deposited somewhere?\n- Models generated in the study should be deposited in PDB-Dev (integrative models) or ModelArchive (AlphaFold models)", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_6": "This is a complex but very interesting and potentially very important manuscript that describes the cis and trans\nautophosphorylations that take place in a fusion protein where the N-terminus of CCDC6 is fused to the kinase domain of\nRET. This fusion creates a constitutively active dimer that now functions as a driver of tumors in the thyroid. Several RET\nfusions have been reported in the past and these gene rearrangements were found in Chernoble following the nuclear\ncontamination because some of the primary events were found in the thymus where iodine accumulates. While other fusions\nwith other tyrosine kinases have been reported, including BCR-Abl, which was the first protein shown to cause cancer, this\nstudy with CCDC6-RET is important because it delves more deeply into the properties of this fusion protein. In all cases, the\nfusion proteins are thought to be active because the fusion creates a constitutive dimer which is essential for activation of\nany of these tyrosine kinases. Usually a transcient dimer forms as a part of the activation process. This paper first shows\nconvincingly not only that this dimer is constitutively active but also that it phosphorylates many sites that are not typically\nreported to be phosphorylation sites in RET. This includes many novel pTyr sites as well as pSer sites and it also includes\nsite in CCDC6 as well as in RET where CCDC6 is not a normal target for RET. They also show a time frame for these\nphosphorylation events which relies on Mass Spectrometry and is very difficult to capture. With mutagenesis they decipher\nwhich of the sites are likely to be biologically important. The authors also suggest that this kinase may be able to use ADP\nas a phosphoryl donor. Finally with preliminary negative staining and preliminary and preliminary CryoEM they are able to\nbuild a credible model for this fusion protein. Overall, there are some very important things that we can learn from this\nmanuscript even though some of the things are likely to be artifactual. Some of the concerns are serious and would likely\nrequire some additional experiment; however, if the authors could satisfactorily address these concerns, then the manuscript\nshould be re-considered for publication.\nSpecific Concerns:\n1. The mapping of the phosphorylation sites is very detailed and comprehensive and shows very clearly that this fusion\nprotein is hyperactive and is phosphorylating a lot of spurious sites that are not seen with the native RET. It shows global\ndysregulation and also defines lack of specificity. It is important to unambiguously show that this lack of specificity is a\nfeature of this constitutive dimer. This point should be emphasized.\n2. These phosphorylation sites are mostly cis as would be expected. While the authors made this point earlier with Src, this\nis an important point and will likely be a conserved feature of all of these oncogenic fusion proteins. The biological\nsignificance of these sites in cells where phosphatases are often constitutively expressed and localized at the same site is\ndebatable. In contrast to the cis phosphorylations, which can be quite random and meditated whenever a surface hydroxyl\ngroup comes in close proximity to the ATP, the transphosphorylation sites are required for phosphorylation of heterologous\nprotein substrates so these are likely to be the more physiologically relevant sites. This point should be very clearly made.\n3. The trans phosphorylations require correct organization of the Activation Loop and the authors do show how Y900 and\nY905 are more important for activity than the other sites. How these contribute to organization of the Activation Loop is still\nunclear and also unclear is the chronology. Is modification of Y900 a required precursor step for the phosphorylation of\nY905? Does their data indicate this stepwise process? If so, it should be articulated very clearly.\n4. Importance of targeting. The many random cis auto-phosphorylations suggest that the first cis auto-phosphorylations take\nplace quickly when then the kinase is first activated. Is this a correct assessment of the data? It makes sense that the\nphosphates of ATP, which is initially targeted with low affinity binding by hydrophobic residues that interact with the adenine\nring of ATP, are highly flexible and easily able to phosphorylate any hydroxyl group that comes in close proximity. . In the wt\nkinases the phosphorylation sites are very focused and are organized so that only a specific protein is modified, usually on\na single site. This is because the kinase is recruited to a specific site which contains may contain other proteins such as\nphosphatases and the downstream proteins that will bind to the pTyr. In this case the kinase will only be transiently\ndimerized. This specificity is lost in the fusion protein where the kinase is constitutively activated because it is a dimer but\nclose to the fusion protein rather that its physiological partner. How does this kinase find its right target?\n5. Another point that the authors make is that ADP, as well as ATP, can serve as the donor for phosphorylation. To make this\nclaim I think it is important that the authors show direct transfer of the beta phosphate using 32P-labeled ATP. An old set of\npapers by Ora Rosen, indicated that ATP could be generated directly from a phosphorylated residue at the active site of one\nof the PKA regulatory subunits. While such an observation to my knowledge has not been pursued for other kinases, it is still\npossible that the phosphates from ATP could somehow be “scrambled”. While ADP may stabilize one of the protomers,\nto claim that the phosphate is also transferred from ADP requires the radio-labelled ATP.\n6. Heterodimers. Do the authors know if heterodimers are formed between the fusion protein and wt CCDC6?\n7. Where is the fusion protein localized in cells? Presumably targeting is mediated improperly by the coiled:coil domains of\nCCDC6.\n8. Is the CCDC6 homodimer also expressed in cells? Presumably there is one good copy of both RET and CCDC6. Is this\nhomodimer more stable so that neither free RET nor the CCDC6 dimer are present in these cells that express the fusion\nprotein? If this is so, then this mutation not only creates an oncogene but also abolishes the normal function of both proteins."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made important improvements in the revised manuscript. In particular the new data with radiolabeled ATP\nand ADP makes a convincing case for the ability of the fusion protein to transfer the b-phosphate of ADP in an\nautophosphorylation reaction (although the Figure providewd for review is not helpful in that all traces are identified as ATP\nin the legend). However, a number of issues remain.\n1. It is unclear that there is any physiological relevance to the transfer of ADP. Experiments performed here are at\nconcentrations that are ~100 times greater than cellular concentrations, and given the more efficient phosphorylation with\nATP, unclear that there could ever be a cellular condition in which limiting ATP led to phosphorylation with ADP. As the\nauthors acknowledge, ATP concentrations are ~1000 times that of ADP. The authors suggest that this might differ in certain\nsubcellular environments, but wildly speculative to suggest that it could differ enough to make this relevant to the activity of\nthis RET fusion. That said, utilization of ADP by a kinase is an interesting biochemical result.\n2. Along the same lines, seems wrong to suggest that high concentrations of ADP \"regulate\" the RET fusion, when the\nconcentrations required are ~100x that found in cells (cells would not survive such a concentration of ADP).\n3. the authors us of \"apo\" to refer to the unphosphorylated kinase is confusing. \"Apo\" typically means without a binding\npartner or co-factor. Clearer to refer the the unphosphorylated state as the \"unphosphorylated state\" or perhaps inactive\nstate.\n4. Authors at least imply that utilization of ADP for autophosphorylation is unique to the RET fusion. But not clear that is does\nnot also happen with the isolated kinase domain (which has lower catalytic activity overall) but is at a level that is difficult to\ndetect.\n5. The authors appear to have misunderstood or at least incompletely understood my concern about their conclusions\nregarding cis vs trans autophosphorylation. I think the authors and I would both agree that their data argue against inter-\ndimer phosphorylation as the dominant mechanism. The question is how intra-dimer phosphorylation occurs, which could\neither be in cis (each subunit of the dimer phosphorylating itself) or in trans (one submit phosphorylating the other). In the\nkinase field, cis-autophosphorylation would ordinarily be taken to mean a given kinase domain phosphorylating itself,\nirrespective of its oligomeric state. For example in an RTK dimer, cis autophosphorylation would imply that each subunit in a\ndimer transferred a phosphate to its own activation loop, while trans autophosphorylation would mean each transferred a\nphosphate to the activation loop of the partner subunit in the dimer. I still do not know for certain what the authors are\nconcluding here, but as best I can tell there are two possibilities: 1) they are using cis-autophosphorylation to mean that\nCCDC6-RET dimers are autophosphorylating themselves, without regard to whether this intra-dimer phosphorylation is\nocuring in cis or in trans. This is a correct interpretation of their data, but a mis-use (or at least a misleading use) of \"cis\", for\nthe reasons outlined above. 2) the authors are claiming that intra-dimer phosphorylation occurs in cis - that is, that each\nsubunit in the dimer transfers a phosphate to itself. I think this is what the interested reader would likely take as the authors\nmeaning, but as best I can tell, there is not a single experiment presented to discriminate between cis and trans\nphosphorylation within a CCDC6-RET dimer. The \"gold standard\" experiment the authors now present supports intra-dimer\nphosphorylation, but does not address whether intradimer phosphorylation is occurring in cis or trans. The authors could\nclear all this up by simply avoiding the \"cis\" designation, and concluding that autophosphorylation occurs in an intra-dimer\nmanner, but acknowledging that they have not addressed whether this occurs in cis vs trans.\n6. to my understanding, phosphotransfer with ATPgammaS (or more precisely, transfer of thiophosphate) typically involves\ntransfer of the gamma thiophosphate and not transfer of the beta or beta-gamma phosphate unit as the authors suggest here.\nI think this experiment detracts, rather than adds to the story.\n7.How do the authors reconcile the high-molecular weight species seen in prior SDS-PAGE gel with crosslinked samples\nwith the clean, dimeric peak seen in the SEC experiment provided in the revision? Presumably this was carried out on a\nparallel sample, and not the actual sample used for crosslinking mass spec studies? Was the SDS-PAGE gel of the actual\nsample?", "reviewer_3": "The authors present a much improved version of the manuscript that broadly addresses our concerns, specifically on the\nphosphoproteomics and the crosslinking MS quantitation aspects.\nThere are still 2 areas that present major issues:\n- we requested to have all crosslinks of the datasets mapped on the structure. The authors reply to this by saying that only\nfew crosslinks arise from the site of interest. The issue is that in order to interpret those proposed crosslinks in a structural\ncontext, one would need to understand if the proposed model is validated/consistent with the crosslinking dataset as a\nwhole. What is the percentage of crosslinks within distance? is the sample overcrosslinked (as another reviewer was\nwondering)? Normally this is addressed also with a distogram over the whole dataset. Without this, we have no way of\nassessing what is the relevance and reliability of the specific crosslinks highlighted by the authors\n- Again, alphafold models are presented without PAE plots or coloring by pLDDT. This is discouraged also by DeepMind\nthemselves. Indeed, looking at the ModelArchive deposition, one can see that the confidence of the inter-dimer arrangement\nof the catalytic lobes is low, as well as the pLDDT scores around the active site and in the ADP itself. A model with ATP can\nalso be predicted and the difference in pocket conformation (again, with judgment gauged by inspection of pLDDT) can\nsupport or refute some of the arguments being made. Similarly, the fact that this \"fits well\" with the negative stain data is\npresented with no cross-correlation or %ge inside metrics.\nIn general, while the manuscript has greatly improved in transparency and in overall readability, our objections were that the\nmodel uncertainty is not quantified, the model precision is not quantified, the fit to the data (save for SAXS chi squared) is not\nreally quantified. These remain problematic. Ultimately, this structure does not have the resolution or interpretability to\nexplain why this kinase should use ADP as substrate, for example with mutation of R912 that supposedly plays a key role in\nthe proposed mechanism.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_6": "The authors did an extensive amount of work to address the various specific concerns that were raised by the reviewers. My\nmajor concern was with regard to phosphorylation by ADP. The authors did the critical experiment of using radio-labeled\nADP to show that the bets-phosphate was transferred to the protein. While this may not be physiological, the data\nnevertheless shows that some transfer is possible with this hyperactive oncoprotein although it is not observed in the wt\nRET. If Reviewers 1 2, and 3, in particular, are satisfied with the authors revised manuscript, then I would concur with\npublication.", "reviewer_7": "Dear Authors,\nThank you for submitting the revised manuscript and for addressing some of the previous comments. While the study has\nbeen improved, several important concerns remain that should be clarified to strengthen the significance and rigor of your\nwork.\n1. In your response, you indicate that other RET fusion proteins (e.g., PTC2 and RIα-RET) were used to support the claim\nthat ADP-dependent phosphorylation is a distinctive feature of CCDC6-RET. However, it is important to note that fusion\npartners such as RIα-RET are rare in human cancers, especially when compared to more common and clinically relevant\nfusions such as NCOA4-RET (PTC3) or KIF5B-RET. The use of such uncommon variants as primary comparators limits the\ngeneralizability and biological relevance of your conclusions. To more convincingly demonstrate the uniqueness of CCDC6-\nRET’s biochemical behavior, direct comparative analyses with frequent RET fusions—particularly those with well-\nestablished roles in oncogenesis—are essential. Furthermore, it appears that these critical comparative data are not\nincluded in Figure 4, which diminishes support for this central claim.\n2. The RET gene produces two isoforms, RET9 and RET51, through alternative splicing, which differ in their C-terminal\nsequences and signaling properties. There is accumulating evidence that the RET51 isoform may play a more prominent\nrole in cancer development. However, your study is based primarily on the RET9 isoform. A key unresolved issue is which\nRET isoform is predominantly involved in genomic rearrangements with CCDC6 in actual human tumors. Is CCDC6-RET9\nthe predominant form, or does CCDC6-RET51 also occur?\nWe believe that addressing these issues will significantly enhance the impact and translational relevance of your study."}, "author_response": "Answer to overall assessment reviewer #1:\nWe appreciate the time taken for the careful evaluation of our work by reviewer #1, which\nhas helped us to improve the quality and impact of our revised manuscript. We agree\nwith reviewer# 1 this is an extensive and integrated study including autophosphorylation\nassays, enzyme kinetics, phospho-proteomics and XL-mass spectrometry, negative\nstaining EM and SAXS together with structure-guided in silico analyses to perform the\nfirst structural and functional characterization of the mechanism of CCDC6-RET\nautoactivation. We agree with referee#1 there are solid and novel findings, in particular\nthe biochemical and biophysical characterization of the fusion product versus the\nisolated catalytic core. On the other hand, in order to make clearer and more evident the\ncontacts that define the different functional states of the CCDC6-RET dimer we have\nrefined and complemented the XL-MS and AlphaFold data and provide an in-depth\nstructural and molecular dynamics simulation analyses (300 ns) of each configuration\nand the contacts defining them (see data on new main figures 8 and 9 and\nsupplementary figures 12 and 13). Further, we tested by size exclusion chromatography\nthat the crosslinked samples were dimeric species and not high molecular weight nor\noligomeric species (see data now on new figure 8). To address the main concern by the\nreviewer #1 regarding the ADP-dependent activity by CCDC6-RET (i.e. the artefactual\ncontamination of the ADP source with ATP and the ADP-dependency characterization),\nwe performed a quality control of the purity of our nucleotide stocks by ion exchange\nchromatography before using them in our phosphorylation experiments (see figure 1, on\nthis rebuttal letter). In addition, we have made further experiments: ADP titration and\nADP-ATP competition assays as well as capturing the -phosphate transfer by using\nradioactive ADP (see new figure 4), that altogether demonstrate that CCDC6-RET is a\nkinase that can bind and use ADP to autophosphorylate itself and other substrate\nsurrogates (see Fig. 2 of this rebuttal letter).\n1. This is clearly a highly active kinase fusion – why is it not already autophosphorylated\nas purified from insect cells? Please include the reaction progress curves from the\ncoupled enzyme assay for Fig. 1h and 3b as supplemental figures. Would be informative\nwith respect to whether there is a lag phase resulting from a requirement of\nautophosphorylation for full activity.\nAnswer point 1 (A1): The protein itself comes with some degree of basal phosphorylation\nlevels at some specific sites (see e.g. Tyr 900 and S1034 phospho-sites, see Fig. 2c and\nd). The reaction progress curves from the enzymatic assays do not present any delay or\nlag phase in line with a lack of requirement of the autophosphorylation for full catalytic\nactivity. We also must keep in mind that autophosphorylation kinetics are very fast, and\nmay explain the lack of the lag phase as well. These data are now part of the Fig. 1i and\nFig. 3d, respectively, in the revised version of the manuscript.\n2. p.16 Binding tightness – not specific, better to just say “Binding…was evaluated).\nA2: This has been corrected now in the text (see page 19).\n3. Fig. 1f. Does not make sense to label x-axis with delta Ti, instead indicate which\ninhibitor and concentration for each bar. (and legend incorrectly refers back to itself).\nA3: This has been changed now in the figure of the revised version of the manuscript\naccordingly, see Fig.1f. We have indicated the inhibitor on axis X and on the top of the\nbars we show the increment or differential in T versusthe apo form.\ni\n4. With the mass spec autophosphorylation time course experiments, it would be\ninformative to analyse the corresponding non-phosphorylated species in an analogous\nmanner. The fractional decrease in the non-phospho peptides would give a sense of the\nstoichiometry of phosphorylation of these many sites…are they major sites with high site-\noccupancy? Or detectable but present on a very low proportion of molecules?\nA4: One of the main goals of our study was the identification of the CCDC6-RET\nautophosphorylation sites. We did not look at the stoichiometry initially. However, we\nhave performed some additional analyses about the stoichiometry of activation loop\nphospho-tyrosines as an example of high occupancy phospho-sites. These data are now\nprovided in supplementary Fig. 2.\n5. Why is apparent Km[atp] so different in Fig 1h vs. Fig 3b for the intact fusion proteins?\nPlease provide the Km values for fig. 3b.\nA5: The apparent K for ATP were different between Fig. 1h and 3b due to the\nM\nconcentration of enzyme used in each set of experiments (4-5- fold higher in Fig. 1h, as\nthe figure legend indicated). We have now used data from a set of experiments where\nthe concentration of the enzyme is the same as in Fig. 3b (0.25 M final concentration\nin assay), see now Fig. 1h and i and Fig. 3b.\n6. There is a very real worry that the apparent ADP-dependent phosphorylation is just\nan artifact of contamination of the ADP with ATP. Or that contaminating ATP was carried\nalong with the kinase in the course of enzyme purification. Not clear that the authors\nhave done anything to address this possibility. A few suggestions: 1) measure ATP\ncontent of ADP preparation using a luciferase assay. Or better yet measure directly by\nLC/MS. 2) Test activity with beta-32P-labelled ADP. 3) Test phosphorylation of a peptide\nsubstrate in a quantitative way, with peptide in excess. If you start with say 500 nmoles\n2\nof ADP, can you phosphorylate most or all of 500 nmoles of peptide? Or do all of the above!\nA6: This is a critical point raised by reviewer#1, and in response we provide several\npieces of evidence to support the ADP dependency by the CCDC6-RET fusion product.\nFirst, we performed a quality control of the purity of both nucleotide stocks by ion\nexchange chromatography before our phosphorylation experiments. Both stocks were\nhighly pure with no traces of contamination. Second, we checked by spectroscopy that\nthe protein comes out of the cells already after the second affinity purification step free\non DNA or nucleotide contamination as indicated by the optimal (260/280 nm) ratios (see\nFig. 1c). We have data from other oncogenic RET fusions products that we work in the\nlab (i.e. PTC2, RI-RET). The PTC2 fusion is capable of binding ATP and ADP; however,\nit cannot use ADP efficiently as the phosphoryl donor during the autophosphorylation\nprocess, contrary to PTC1 (see Fig. 4 on page 22 on the rebuttal letter). These data are\npart of an independent and ongoing project by a PhD-student in the lab. Furthermore,\nwe have performed ADP titration experiments and ADP-ATP competition assays to\ndemonstrate a dose-dependent phospho-tyrosine activity by ADP and at the same time\na regulatory function (i.e. inhibition) at high concentrations of ADP. We used also ATPS\nas a phosphoryl donor, based on the premise that CCDC6-RET can hydrolyse the beta-\nbond of the nucleotide derivative, as it does with ADP. In fact, we observed CCDC6-RET\nautophosphorylation in the presence of ATPS, although (but as expected) with slower\nkinetics than ATP, mirroring the autophosphorylation kinetics observed with ADP (see\nsupplementary Fig. 4). Finally, we demonstrate -phosphate transfer in\nautophosphorylation experiments using radioactive ADP. These data are all included on\nFig. 3 and on new Fig. 4 and new supplementary Fig. 4 in the revised version of the\nmanuscript. Last, we have preliminary results using cell extracts of dormant (serum\ndeprived) cells as a substrate source and found that CCDC6-RET activated by ADP\ninduces the phosphorylation of a specific set of substrates compared with the effect seen\nwith ATP, see figure 2 on this rebuttal letter on page 7.\nFigure 1. Quality check (purity) of the different nucleotides (1 mM) in buffer 20 mM Tris pH 8.0, 500 mM\nNaCl, 2 mM DTT, 2 mM MgCl2 was performed by ion exchange chromatography upon a salt (Ammonium\nbicarbonate) gradient, showing very high purity of the stocks.\n7. In figure 4, what are the Km values for full length and truncation? (they look quite similar).\nHow do we know that the apparent difference in Vmax in not just a difference in enzyme\nconcentration? Legend not reassuring in this respect – reports 0.2-0.3 uM. Need\ndemonstrated careful measurement of enzyme concentration. At a minimum\nCoommassie stained gel with equivalent amounts used, or better to titrate both with a\npotent Ret kinase inhibitor – show that x micromoles of inhibitor completely kills x\nmicromoles of enzyme. Also, would be worth looking at the effect on Km[peptide].\nA7: We have now represented the K values for the full-length and C-terminal truncated\nM\nconstructs following the indications of the referee. We measured protein concentration\n3\nby absorbance and checking also on a gel by Coomassie. In these sets of experiments,\nwe used 2.5 M enzyme concentration in the enzymatic assays (0.25 M final cc).\nThe changes in catalytic efficiency, as indicated now in the figures are not driven by the K .\nM\nThese data confirm that constructs lacking the C-terminal segment are more catalytic\nefficient.\n8. Figure 5 and associated text was very difficult to follow and full of logical leaps. The\nlack of side-by-side comparable experiments with the A-loop mutants in FL vs deltaCT\nmakes it impossible for me to draw any meaningful conclusions.\nA8: We agree that this result section was complex and we have tried now to focus and\nsimplify the message towards the catalytic dependency on activation loop Tyr 905 by\nCCDC6-RET. In line with this, in enzymatic assays using an ABL-derived peptide as a\nsubstrate surrogate we found that the activation loop Tyr 905 was also required for\ncatalytic activity in the construct with an intact C-terminal segment. This detrimental\neffect (i.e. dependency) was also seen by an activation loop Ser 909 mutant (S909A)\nalthough to a lower extent, see new Fig. 5 and supplementary Fig. 7. In consequence,\nwe have modified the message of this result section and connected it with the comment\nfrom referee#3 “This residue (i.e. S909A) when mutated appears to have a profound\neffect on the catalytic activity of the RET fusion product towards a substrate despite\nlacking any effect on autophosphorylation (supplemental Fig. 1). This is in fact a clear\nevidence that autophosphorylation and the phosphorylation of an exogenous substrate\n(e.g. a peptide, or an intact substrate surrogate) are not governed by the same\nmechanistic determinants. Thus, despite that both are used as readouts for catalytic\nactivity, they should be treated as independent mechanisms or processes”\nSee discussion on page 36 on the revised version of the manuscript.\n9. Crosslinking-MS data are interpreted as being intra-dimer, inter-molecular crosslinks.\nBut seems no steps were taken to ensure that none are inter-dimer crosslinks? (For\nexample SEC after crosslinking to remove any higher order crosslinked species?)\nA9: The reviewer #1 is correct on her/his observation. First, we performed size exclusion\nchromatography on crosslinked samples and we found that the crosslinked sample\nelutes at the same V (ml) as the un-crosslinked one (11.2 ml), corresponding to a dimer\ne\nin solution. These data demonstrate that the main crosslinked species appears to be\ndimeric as we do not see any oligomers and large molecular weight species in the size-\nexclusion chromatography step (see new Fig. 8a, inset). Besides we are filtering the XL-\ndata (distances) in our high confidence structural models from AlphaFold and MD\nsimulation assemblies to conclude that in the apo form we observed several key\ncrosslinked peptides at the activation loop (K907-S909, S896-K907 and K889-K907)\nwhose distances are compatible with an intermolecular interaction supporting the notion\nof crosslinked activation loop and in line with a face-to-face arrangement of the catalytic\ndomains within the homodimer. Crucially, in the active (auto-phosphorylated) state all\nthe indicated crosslinked peptides involving the activation loop disappeared. These data\ncan be found on new Fig. 8, where we have now repeated the experiment in triplicate\nand have consistent and quantifiable results see page 28-30. Results section main\nmanuscript, Fig. 8 and supplementary Fig. 12).\n10. Actually the SDS-PAGE gel with crosslinked protein shows the major species is a\nsmear around 200kDa (presumably dimer of dimers?), and there is also a pronounced\nband above 250 kDa. So not reasonable to interpret crosslinks as intra-dimer.\nA10: See previous response.\n4\n11. It is clear that phosphorylation changes the pattern of crosslinking, but not clear how\nthe authors arrived at their structural models. And in any case, given concerns in 9 and\n10 above, I feel the structural models are a gross over-interpretation of the data.\nA11: First, as the referee#1 indicates, there is a clear set of specific crosslinked peptides\nthat define both the apo and autophosphorylated (i.e. active) forms of CCDC6-RET (see\nnew Fig. 8 and supplementary Fig.12). From these data we can conclude that there are\nsignificant conformational changes upon CCDC6-RET autophosphorylation and\nautoactivation. Second, by size exclusion chromatography we demonstrate that the\ncrosslinked CCDC6-RET sample corresponds to a dimer species in solution as it has the\nsame elution volume than the untreated CCDC6-RET sample, with no apparent\nformation of oligomers nor high molecular weight species (see response 9). Third, our\nstructural models are based on highly confident AlphaFold predictions for the apo and\nactive (autophosphorylated) CCDC6-RET homodimers, all of them in good agreement\nwith the experimental EM single particle analyses (Fig. 6 and 7 and supplementary\nFig.9). In addition, we have now generated MD simulations assemblies for each of these\nfunctional states of CCDC6-RET (see Fig. 9 and supplementary figure 13). Then we filter\nour XL-MS data, in particular the crosslinked peptides at the activation segment or its\nvicinity (i.e. distances) that are specific for the apo state (model 2 assemble) that\ndisappear in the auto-phosphorylated state. These crosslinks are compatible with\nintermolecular rather than intramolecular distances, which are shorter than the size of\nthe crosslinker itself (BSBU, 12.5 A). These data together align with a face-to-face dimer\nin the apo state that swings apart in the active (i.e. autophosphorylated) state of the\nhomodimer (see Fig. 8 and Fig. 9, and supplementary Fig. 12 and 13)\n12. What is meant by cis-autophosphorylation? The data suggest autophosphorylation\nin trans in the context of the dimer – no clear concentration dependence of\nautophosphorylation of the dimer, but there is concentration dependence of a\nmonomeric kinase construct. Is this what the authors mean by “cis” autophosphorylation?\nI think most would say “the protomers of the dimer autophosphorylate in trans”. Cis auto-\nphosphorylation implies that a given kinase domain/subunit autophosphorylates itself.\nThere is no data that I see here that would test this mechanism (for example preparation\nof a defined heterodimer with one active and one inactive subunit and a test of whether\nthe active subunit could phosphorylate the inactive partner.\nA12. We agree with the referee there is no concentration dependency on the kinetics of\nautophosphorylation of the CCDC6-RET homodimer, in contrast with the concentration\ndependency observed for the isolated kinase core construct (monomeric, RET 713-1072\naa). These data can be interpreted as a lack of interdimeric phosphorylation during the\nCCDC6-RET autophosphorylation process. We have tried to dissociate the dimer by\ndifferent means to evaluate the function of a monomeric form in solution. For this\npurpose, we tried partial denaturation with urea and engineered several mutants of two\nhighly conserved leucine residues that define the coiled-coil interface (L62Q and L64Q).\nUnfortunately, we did not manage to dissociate the monomer from the dimeric form of\nthe fusion product in either of the cases yet. We are trying further with a different set of\nmutants (namely L62D, L64D and L62/64D, work in progress)\nA gold standard method to discriminate between cis- and trans-autophosphorylation is\nto evaluate if an active kinase can phosphorylate intermolecularly an inactive substrate\nsurrogate that mimics its own kinase domain (Cuesta-Hernandez et al., 2023; Reinhardt\nand Leonard, 2023). Time course experiments of CCDC6-RET autophosphorylation in\nthe presence of a substrate surrogate were performed. In this case an amino- and\ncarboxy-terminal deleted and HRD catalytic inactive mutant (CCDC6-RET-HRA,\nD874A) was used as a substrate surrogate (Supplemental Fig.11c). WBs data using a\ntotal phospho-tyrosine and RET activation loop phospho-specific antibodies revealed\n5\nthat CCDC6-RET display a very strong tendency to autophosphorylate itself rather than\nto the inactive kinase substrate (Supplementary Fig. 11c), which is indicative of cis-\nautophosphorylation. Note that the levels and kinetics of CCDC6-RET\nautophosphorylation are unchanged in the presence of the substrate molecule,\nsupporting our interpretation. In fact, in the same experimental setting, evaluation of\nanother protein kinase known to autophosphorylate its activation loop in trans (e.g. c-\nSrc) resulted in the opposite output, highlighting a distinct mode of autophosphorylation.\nIn this case, we observed (contrary to CCDC6-RET) a very rapid and significant\nsubstrate surrogate phosphorylation, which is accompanied by a slow-down in the\nkinetics of autophosphorylation of the active kinase in the presence of the substrate\nmolecule.\nOur interpretation of cis-autophosphorylation on the activation loop is also based on the\nstructural information related to the different functional states: apo (with face-to-face\ncatalytic domains with inter-crosslinked activation loops) and active (auto-\nphosphorylated states) where the catalytic domains and their active sites are facing into\nopposite directions from the stem of the coiled-coil (see proposed model on Fig. 9).\nThis is supported by the XL-MS (Fig. 8) and EM data (Fig. 6 and 7) and structure-guided\nmodelling and MD simulation data (see previous response to point A11 and Fig. 9 and\nsupplementary figure 13).\nWe also acknowledge that the transition from the apo (i.e. resting state) to the active\n(phosphorylated) state may not be driven by nucleotide binding only, and that at the very\nearly stages, an intermolecular phosphorylation process may be triggering the transition\n(see discussion on page 37). Taking this reasoning into consideration, we decided not\nto include the statement of cis-autophosphorylation on the title of the paper.\nThe paper by Plaza-Menacho et al aims at describing the enzymatic activity as well as\nthe molecular structure of CCDC6-RET chimeric oncoprotein. The paper is potentially\ninteresting, with data suggesting that: i) CCDC6-RET is a dual ATP/ADP-dependent\nkinase; ii) the activation loop and the c-terminal tail of RET interact modulating protein\nfolding and activity; iii) CCDC6-RET autophosphorylation occurs in cis. In addition, they\nalso provide an in silico as well as Cryo-EM and crystallographic analysis to generate a\nstructural model of CCDC6-RET homodimer both in the active as well as inactive\nconformation. A major weakness of the paper is that it is not clear which is its principal\nmessage since all those topics tackled in the manuscript are extremely important, but\nnone is demonstrated in a complete and convincing manner.\nAnswer: We understand the comments and suggestions by the referee, and\nconsequently we have tried in the revised version of the manuscript to focus on one main\nmessage. The main goal of our study was to dissect the mechanism of CCDC6-RET\nautoactivation and to provide a plausible biochemical and structural mechanistic model.\nOf course, the dual ATP and ADP dependency is the most critical and novel message of\nthe paper and we have focused during our revision in several sets of experiments that\nconfirms the ADP-dependent activity by CCDC6-RET (see new Fig. 4). In addition, we\nhave refined and validated the XL-MS data with MD simulation and structure-guided\nanalyses (see new Fig. 8 and 9 and supplementary Fig. 12 and 13) that altogether\nsupport the proposed mechanistic model, in which the apo form is defined by a face-to-\nface dimer (with crosslinked activation loops) and in the active (phosphorylated) state\nthe catalytic domains are facing away into opposite directions in a configuration\ncompatible with cis-autophosphorylation at the activation loop.\nFor example, the ability by CCDC6-RET to use ADP to phosphorylate itself and substrates is striking news. ADP is\nclassically and paradigmatically unable to provide the beta-phosphate to kinases for phosphorylation. A deeper and more\ndetailed description of this activity, putting it in comparison with WT RET (not just the TK domain which is a poorly active\nkinase) and possibly other chimeric RET fusions, or providing the rate of AMP production (both when supplying ATP or\nwhen using ADP), as well negative controls using other kinases or a kinase dead mutant.\nAnswer: We agree with the reviewer#2 the ability by CCDC6-RET to use ADP to\nphosphorylate itself and substrates is striking and unexpected. We have performed a\nseries of additional experiments to support these unexpected results (see new Fig. 4):\n1. We performed a quality control check of the purity of the nucleotide stocks to prove\nthat they were highly pure with no traces of contaminations (see Fig. 1 of this rebuttal).\n2. We checked by spectroscopy that the protein come out of the cells already after the\nsecond affinity purification step free of DNA or nucleotide contamination as indicated by\nthe optimal (260/280 nm) ratio (see Fig. 1C).\n3. We have data from other oncogenic RET fusions products that we work with in the\nlab (i.e. PTC2, RI-RET). The PTC2 fusion is capable of binding ATP and ADP,\nhowever and contrary to PTC1, it cannot use ADP efficiently as phosphoryl donor during\nthe autophosphorylation process, contrary to PTC1 (see Fig. 4 of this rebuttal).\n4. We have used ATPS as a phosphoryl donor, based on the premise that CCDC6-RET\ncan hydrolyse the beta-bond of the nucleotide derivative, as it does with ADP. In fact, we\nobserved CCDC6-RET autophosphorylation in the presence of ATPS, although (but as\nexpected) with slower kinetics than ATP, mirroring to some extent the\nautophosphorylation kinetics observed with ADP. These data are now provided in\nsupplementary Fig. 4 in the revised version of the manuscript.\n7\n5. We have performed ADP titration experiments to prove a dose dependent effect in\nautophosphorylation assays resulting in a significant inhibition at 2.5- and 5-mM\nconcentration of ADP; and also, in ADP-ATP competition assays, where we demonstrate\na regulatory function (inhibition) of the ADP at high concentrations (5 mM). See data on\nthe revised version of the main manuscript on Fig. 4 a-d.\n6. We have performed autophosphorylation assays with CCDC6-RET using radioactive\n(P33) labelled ADP to prove -phosphate transfer. These data are now part of the main\ntext of the revised article on Fig. 4 panel e.\n7. We have preliminary results using cell extracts of dormant (serum deprived) cells as\na substrate source where CCDC6-RET activated by ADP induce the phosphorylation of\na specific/different set of substrates compared with ATP, see figure 2 of the rebuttal\nbelow. This work is ongoing and beyond the scope of this manuscript, but it is expected\nto be a follow-up story (Martín-Hurtado et al. in preparation I).\nfor the other forms of RET, and in particular the longer one RET-51 (they have used the RET-9 isoform in the paper)? Is this\ninfluenced by proteins that bind to the c-tail in cells (SHC, ENIGMA, FRS2, IRS1/2, ect)? Again, is this peculiar to CCDC6-\nRET or is important also for other RET rearrangements and wt RET?\nAnswer: We agree with the referee that the connection between the activation loop and\nthe C-terminal segment requires further experimental work. However, we have to\nacknowledge that to complete this task properly we would require of a whole new project\nitself, which is what we are currently working on. We have preliminary results showing\nthat the specific contacts between the activation loop Tyr 900 and the C-terminal\nsegment are specific for the shorter C-terminal splicing isoform 9 (i.e. the Y900F mutant\nis not viable and hardly expresses in insect cells). We have now expressed and purified\nthe complete set of activation loop mutants in the context of the longer C-terminal\nsegment splicing isoform 51 and the Y900F is viable and functional, see Fig. 3 below.\nThese data indicate that the interactions necessary for proper processing and folding of\nthe fusion protein, specifically between the activation loop tyrosine 900 and a key amino\nacid or motif in the C-terminal segment 9 isoform, are localized within amino acids 1063\nto 1072. We are now mapping this small C-terminal sequence by site-directed\nmutagenesis, in order to rescue the defective expression caused by the Y900F mutant\n(work in progress). In addition, we have preliminary results using cell extracts of dormant\n(serum deprived) cells as a substrate source where we compared CCDC6-RET iso9\nactivated by ATP induces the phosphorylation of a specific/different set of substrates that\nare different by those phosphorylated by the CCDC6-RET iso51. This work is ongoing\nand beyond the scope of this manuscript, but it is expected to be a follow-up story\n(Martín-Hurtado et al. in preparation II).\nk Da W T9 W T51 Y F 9 0 0 51 Y F 9 0 5 51 Y F 9 0 0 9 0 5 / 51 W T9 W T51 Y F 9 0 0 51 Y F 9 0 5 51 Y F 9 0 0 9 0 5 / 51 W T9 W T51 Y F 9 0 0 51 Y F 9 0 5 51 Y F 9 0 0 9 0 5 / 51\n21 5500\n1 00 GST-CCDC6-RET\n75\nGST-3C\nD\nFigure 3. Expression, purification and characterization of a CCDC6-RET51 Y900F mutant. Affinity\nchromatography using a glutathione-conjugated resin followed by digestion using a 3C-GST protease to\ncleavage the tags. Indicated fractions were run on an SDS-PAGE and stained with Coomassie: (1)\nrecombinant protein bound to Glutathione-conjugates beads, (2) elution after cleavage with 3C-GST\nprotease and (3) beads after recombinant protein elution. The Y900F in the context of the c-terminal splicing\nisoform 51 is viable and can be produced satisfactorily in insect cells using a baculovirus system (left panel),\nStandard enzymatic assay using a c-ABL derived peptide at increasing concentrations of ATP demonstrated\nthe Y900F mutant contrary to the case of the short isoform 9 is an active protein.\ncomprehensive and analytical research study for each of this topic (maybe using the\nunpublished data reported in the discussion). I guess that in this way authors would\nmake a definitive and convincing point on each of the finding they have presented in the\nmanuscript.\nThe paper of Martín-Hurtado et al. uncovers some of the mechanism and molecular\nfunction of the CCDC6-RET fusion kinase, with important findings on the specificity and\nautophosphorylation activation. Where the paper is strongest is biochemical\ncharacterization, and especially the phosphorylation assays, which clearly demonstrate\nthe dual specificity of this kinase. However, it is severely lacking in structural\ncharacterization, not because of lack of experiments (which are\nplentiful) but because\nthe model derived is not sufficiently supported by data and does not explain the authors’\nobservation. Some of the experiments in particular are filtered in ways that inevitably\nsupport the proposed model, rather than building a model from the available data.\nIt is not the lack of resolution per se (if high resolution could not be attained, that is the\nway it is), but the lack of a proper integrative approach that accounts for the experiments\ncarried out. Thus the model derived does not clarify or explain the observations made in\nbiochemical experiments, and does not come with quantifiable precision/uncertainty.\nFurthermore, the methods section is lacking some critical details that would help\nunderstand some of the experiments and modelling steps, as detailed below.\nOverall, this leads to a major shortcoming in terms of establishing a structure-function\nrelationship and a mechanistic understanding of what underpins the kinase activity and\nregulation. In short, the paper has some major findings on the specificity and mechanism\nof the kinase, but then the model presented does not explain how CCDC6-RET achieves\nits properties. In particular, little data is presented as to the activation mechanism (it is\nspeculated upon) and as to why this kinase would have tyrosine and serine specificity or\nwhy it would work on both ATP or ADP given the model.\nThe biochemical and structural data taken by itself is of excellent quality, except for the\nproteomics and crosslinking MS, where the experimental analysis and design leaves\nsomething to be desidred (see below). The level of support for the conclusions- i.e. the\ngeneration of the structural model and the interpretation of the connection between\nbiochemistry and structure- is currently weak. If properly characterized however, this\npaper would provide highly significant findings.\nAnswer: We agree with referee#3 that the initial derived model did not explain all the\nobservations made in the biochemical and functional experiments. We have now\nprovided more in-depth structural and functional analyses on the activation mechanism;\nin particular those related to the dual ATP- or ADP-dependent activity by CCDC6-RET\n(see new Fig. 4), the XL-MS data (see new Fig. 8 and supplementary Fig. 12) and MD\nsimulation and structural analyses of the key contacts defining each of the functional\nconfigurations, namely the apo and active (phosphorylated) states (see new Fig. 9 and\nsupplementary Fig. 13).\nI would recommend accepting the paper only after major revisions.\nMajor concerns:\n- Phosphoproteomics. It seems that there are 2 full replicates, each of which was injected\nin technical duplicates, but a single plot is “representative of 2 independent experiments”,\nrather than showing all 4 traces. As we are being asked to judge dynamics, the\nreproducibility of the quantification is quite critical, so at least 3 or 4 full replicas are\nrequired, even in single injection, so that proper quantification and error estimation may\nbe carried out. The variance of a double injection is low, as expected, but the authors\nare claiming this is \"representative” without even showing all 4 traces. The\nphosphoproteomics plot should come with error bars coming from quantification of\nreplicates.\nphospho-proteomics four independent autophosphorylation experiments with two\ntechnical replicates each (see supplemental tables 1-4 and supplementary material file\nAllSites_Scaled_Intensity_dataset1.xlsx) and summarized those phospho sites that are\nconsistent across the different datasets on main table 1. In addition, we have quantified\nrepresentative phospho-sites from 3 independent experiments (see new Fig. 2g). In the\ncase of the XL-MS data, we have performed a new experiment in triplicate and\nundertaken a quantitative approach to analyse the specific crosslinks that define both\nthe apo- and phospho-states of CCDC6-RET (see Fig. 8 and supplementary Fig. 12).\n- In the methods section, state the number of full and technical replicates for both\ncrosslinking MS and proteomics.\nAnswer: We have included the number of full and technical replicates for the phospho-\nproteomic (see methods section on page 13) and X_MS data (see methods section on\npage 16). See also response to previous point.\n- Quantitative crosslinking MS: quantitation is not performed across replicates, which are\nneeded when comparing between conditions. I would expect volcano plots where we\ncan see what residue pairs are significantly changing between apo and phosphorylated\nconditions. See for example Tomko et al. Cell 2015\n(https://pubmed.ncbi.nlm.nih.gov/26451487/). The fact that a crosslink is detected only\nin a particular condition, when variance is not estimated, is not indicative of a unique\nconformation, especially in DDA.\nAnswer: We have performed a second experiment in triplicate and opted for showing\nthe data in Venn diagrams for the overall results. We found that while the approach\nproposed by Tonko et al. was deemed generally appropriate, we chose to employ a label-\nfree quantification strategy instead of isobaric labelling and M/I plots, as the latter do not\nadequately account for variability across biological replicates. Crosslinked peptide\nquantification was carried out using the widely adopted Skyline software, utilizing\nextracted ion chromatograms of the isotopic distributions corresponding to all ions\nidentified by MeroX. To ensure accuracy, interfering ions were rigorously filtered, and\npeak area integrations were manually inspected. The most relevant crosslinks for each\nof the functional states (apo and phosphorylated) were visualized (and quantified) using\na heat map, enabling the assessment of Z-score variations across the three biological\nreplicates. Together with a detailed inspection of the distances of the main crosslinked\npairs specific for the apo and phosphorylated states. See new Fig. 8 and supplementary\nFig. 12.\n- Show all detected residue pairs on the structure and provide a distance histogram, as\nwell as coloring the circle plot by distance. I find it hard to believe that almost all crosslinks\ndetected arise from peptides containing the critical tyrosine, and there is almost no\ncoverage of the rest of the protein. Does the crosslinking MS support this model? Only\nan unbiased mapping of all detected residue pairs at 5% FDR (provided the database\ndid at least include contaminants) can show- one cannot claim validation after filtering to\nonly have distances within range.\nAnswer: Not all the crosslinks detected in our XL-MS experiment arise from peptides\ncontaining activation loop tyrosine residues. It is true that the presence or absence of\ncrosslinks involving the activation segment can define the two configuration or functional\nstates (apo and phospho). We have shown specific crosslinked peptides for the apo and\nphosphorylated states in Venn diagrams and circle plots (see new Fig. 8). In addition to\nthe quantification and visualization using a heat map, enabling the assessment of Z-\nscore variations across the three biological replicates. See result section related to the\nnew XL-MS data and analysis on Fig. 8, supplementary Fig. 12 and supplementary\nmaterial file: Skyline_Crosslinked_peptides_quantification_ZSCORE dataset2.xlsx\n12\n- The structural modeling is perhaps the weakest part of the paper. In fact, there is no\nmethods section for the modeling part, which comprises a major part of the paper. The\nresults are interspersed with the steps done to assemble the model. The authors\nmanually generated and adjusted a model by patching a coiled coil alignment and the\nkinase region. This is then placed in the negative stain density and presented as a good\nfit, which is not quantified. Similar thing for the SAXS, where the authors delete the\ndisordered regions to rejigger them inside the DAMMIN density, only to then say the\nresulting model is a good fit with CRYSOL. Are the disordered regions compact or\nextended? This is exactly the sort of information SAXS may provide if it is treated as the\nensemble solution experiment that it is. The crosslinking MS experiment is similarly\npresented as validating the model, but only after filtering crosslinks to within distance\nand not plotting the full crosslinking MS dataset on the structure. As a matter of fact,\nwhat is the advantage of using the hand-made model over the AlphaFold one (or\nadjusting the alphafold one)? At least AlphaFold has a confidence metric that shows that\nit is not confident in the coiled coil regions. I would encourage the authors to use\nHADDOCK or the integrative modeling platform (IMP) to integrate SAXS, negative stain,\ncrosslinking MS and basic structure predictions to generate an ensemble of predicitons\nbased on the data. Or, the crosslinking MS data can perhaps be used to improve the\nmodeling with AlphaLink2. In both cases, this would then lead to a model that can be\ninterpreted as it comes with a precision that encodes for the uncertainty\n(https://pubmed.ncbi.nlm.nih.gov/25173450/) of low resolution and sparse restraints.\nAnswer: We have included in the revised version of the manuscript a method section for\nthe modelling part and MD simulation (see page 17-18). Our models have been refined\nfollowing runs of MD simulations for 300 ns. This information can be found on new Fig.\n9 also Fig. 4f, and supplementary Fig. 13 of the revised version of the manuscript.\n- Figure 5 c, “Enzymatic assay performed with CCDC6-RET ÑCT, WT and activation\nloop mutants Y900F, Y905 and Y900/905F (1mM) incubated with increasing\nconcentrations of ATP at a fixed concentration (1.35 mM) of a c-Abl derived peptide\n(EAIYAAPFAKKK), I would expect to have five curves but only four are shown. Also the\nactivation loop mutants used in the essay are presumably from the main text from the\nprotein version lacking the C-terminal domain; please annotate it in the legend\naccordingly to WB figures.\nAnswer: The enzymatic data shown in Fig. 5c corresponds to the comparison of the\nactivation loop Y/F mutants in the context of the construct lacking the C-terminal segment\ncompared with the full-length construct. See updated Fig. 5. A direct comparison\nbetween a full-length and delta C-tail constructs in enzymatic assays using exogenous\nABL-derived and A-loop peptides can be found in supplemental Fig. 4. These data show\nincrease catalytic efficiency by the construct lacking the C-terminus. We have annotated\nthe figure legend when a full-length or a C-tail construct was used accordingly.\n- Where are all the phosphosites detected on the structure? Does the model explain how\nsome phosphosites are phosphorylated before others? Are they near the active site?\nThe pocket is not highlighted in any of the figures. What are the motifs around the\nphosphosites? Showing the phosphosites, discussing their position and coloring them\nby time of phosphorylation would help address many of these questions. The distinction\nbetween cis and trans autophosphorylation should have a correspondence in the model.\nAnswer: The phospho sites are found scattered in the coiled-coil and the kinase domain\nportion of the fusion product. There is no sequential autophosphorylation as such, due\nto very fast kinetics of autophosphorylation otherwise indicative of lack of specificity (see\nanswer to referee#6 comment 1). Already by 1 min we can observe that most of the sites,\nif not all, are already phosphorylated (see Fig. 2). In my opinion, this is an indication (also\nsupported by some of the referees, see e.g. referee#6) that cis-autophosphorylation may\nbe driving the autoactivation process of the fusion product.\n- How do the authors explain the upper and lower bands of the western blot in the\nphosphorylation assays?\nAnswer: CCDC6-RET appears on a Coomassie as a doublet. We have checked indeed\nboth bands by MS and found no difference on the aa sequence nor the PTM status of\nCCDC6-RET.\n- Figure 4f, please show the residue number in the circos plot for better clarity.\nAnswer: we have shown on new Fig. 8 and supplementary material the full crosslinking\nMS dataset with the crosslinks specific for each of the two functional configurations, with\ncircle plots and their residues number and specific protein domains (color coded) in the\ncircus plot.\n- Figures 4 b and d, although clear from figures c and e, state the WT and ΔCT in the\nlegends of the enzymatic assays with A-loop and abl peptide.\nAnswer: These details has been checked and are correct.\n- DSBU is a homobifunctional (not heterobifunctional) crosslinker. It has 2 NHS-ester\nmoieties.\nAnswer: Thanks for the correction, this has been now modified in the text.\n- “In particular, we found that in autophosphorylation and enzyme kinetics experiments,\nwhere we evaluated the KM for ATP in the presence of an excess of peptide substrate,\na Y905F and double Y900/905F mutants have a loss of function effect, contrary to the\nY900F, that was catalytic competent. “Please state Figure 5c for better clarity of the\nreader.\nAnswer: We have now stated figure 5c for better clarity of the readers, following the\nindications by the reviewer.\n- Figure 8b, kcat/km ratio instead of only kcat should be indicated on the y axis of the bar\nplot. Please indicate legend with the different CCDC6-RET concentrations used in the\nenzymatic activity assay plot in figure 8b.\nAnswer: In this particular Fig. 8b we wanted to show the k variation as a direct readout\ncat\nof enzyme velocity and not catalytic efficiency of CCDC6-RET autophosphorylation in\nMichaelis-Menten kinetics at increasing protein concentrations in the absence of any\npeptide substrate, measuring just protein autophosphorylation. This figure is now part of\nthe supplementary Fig. 11, panel c.\n- Why not AlphaFold3 but filtered by prediction confidence? What does the AlphaFold3\nmodel look like?\nAnswer: Initially we did use the AlphaFold multimer ColabFold, with relaxation off (model\n2, apo). In the case of the active dimer assembly shown in new Fig. 4f and Fig. 9 in the\nrevised version of the manuscript we have used the AlphaFold3\n(https://alphafoldserver.com/). In every case, and in order to minimize potential steric\nclashes we performed MD simulations (300 ns) after energy minimization for each of the\nmodels obtained from AlphaFold (see material and methods and new Fig. 9).\n- In one of the panels showing the model, highlight the ATP binding site and substrate\nbinding pocket, if possible. Does the model give hints as to why it would phosphorylate\nserine and tyrosine? Is the pocket similar to other dual specificity kinases?\ndependent activity) and Fig. 9b for the dual specificity on Tyr and Ser.\n- Show SEC-MALS data, size exclusion traces if possible. The results of the coiled-coil\npredictor, which in general may be less accurate than AlphaFold, can be shown in fewer\npanels.\nAnswer: In Fig. 1 panel d we have mass photometry data about the dimeric state of the\nfusion product. Data on figure 6 describe the in silico reconstruction of the manually\nassembled homodimer, which is now supported in the revised version of the manuscript\nby an active dimer assembly provided by AlphaFold3 (AlphaFold server from Google\n(https://alphafoldserver.com/) and MD simulations data on the apo (model 2) and active\n(AF3) forms of the homodimer (see new Fig. 9).\n- “This residue when mutated appears to have a profound effect on the catalytic activity\nof the RET fusion product towards a substrate despite lacking any effect on\nautophosphorylation (unpublished data).” This is a particularly important point. I\nsuppose the difference between “data not shown” and “unpublished data” is that these\nfindings lie in another paper that is also in review? Can there be at least pointing to a\npreprint?\nAnswer: We have data related to the effect of the CCDC6-RET S909A mutant that\ncontrasted with the data previously published by the native protein (see Plaza-Menacho\net al. Cell Reports 2016). The CCDC6-RET S909A mutant (as does the Y905F) can\nautophosphorylate itself (see supplementary Fig. 1). However (and contrary to the effect\nseen in the wild-type kinase domain with or without the JM-segment) it lacks enzymatic\nactivity towards a peptide substrate (see supplementary Fig. 7, on the revised version of\nthe manuscript). These data come in line with results presented in Fig. 5 that support the\nnotion about both autophosphorylation and the phosphorylation of a specific substrate\nsurrogate are defined by specific and independent structural and dynamic determinants.\nWe have now included these data, and make this point clearer in the discussion on page\n36.\nTechnical points:\nCrosslinking MS analysis:\n- For MeroX analysis, please state clearly how you performed the analysis. E.g., Was\nprecursor correction enabled? For mgf file (only MS2 data), the software would do this\nwithout knowing the real MS1 data, so either turn off that option or inspect the\nMS1-precursor level of the cross-links. If you export mzML with also MS1 information, then it\nwould be looking at MS1 data to perform precursor quadrupolar isolation window\ncorrection. For QExactive series of instrument this correction is quite useful, never\ntested on Exploris 480. Was RISE-UP mode used? How many proteins were used in the\nfasta? Fusion protein alone or with contaminants (cRAP database option in the last panel)?\nSearching with target-decoy FDR against a single protein inevitably leads to poor error\nestimation, as the decoy database is not large enough to model the noise of random\nmatching.\nAnswer: An updated figure about the whole XL-MS data can be found in Fig. 8 on the\nrevised version of the manuscript. Regarding this point in particular, an aliquot of both\nthe cross-linked and pre-cross-linking samples, were digested with trypsin in parallel.\nTwo hundred nanograms of the resulting non-cross-linked peptides were analyzed using\na 37-minute gradient on a Vanquis Neo, coupled with an Orbitrap Astral. The Orbitrap\nAstral was operated under a standard Data-Independent Acquisition (DIA) protocol to\noptimize protein detection and enhance quantification. Subsequently, DIANN analysis\nwas performed on the pre-cross-linking sample, querying a custom-built FASTA\ndatabase that included the host expression organism (Sf9 and the baculovirus) the\nCCD6-RET fusion protein construct, and the UniProt sequences of 525 proteins\ncommonly identified as contaminants over the past decade in proteomic analyses of\n15\ndiverse samples. The proteins identified by DIANN were sorted by intensity and\nexamined for potential contamination. For the cross-linked samples, the top 313 most\nabundant proteins in the non-cross-linked sample, based on the sum of all precursor\nintensities as calculated by DIANN, were selected. These proteins were then used to\nconstruct a customized FASTA database for MeroX searches. This number was deemed\nsufficient to model decoy hits and control the false discovery rate (FDR), as MeroX uses\na built-in target-decoy strategy at the crosslink spectrum match (CSM) level for reliable\nerror estimation. For MeroX analysis of cross-linked samples, raw files acquired from the\nOrbitrap Exploris 480 were converted to MZML files using the ProteoWizard ms.convert\nprogram. The search parameters for MeroX were configured as follows: protease sites\nR/P and K/P, with a maximum of three missed cleavages, with K blocked as XL-site.\nCarbamidomethylation of cysteine (C) was set as a fixed modification, while oxidation of\nmethionine (M) and phosphorylation of tyrosine (Y) were treated as a variable\nmodification. DSBU was chosen as the cross-linker, and MeroX's default parameters\nwere used, considering dead-end reactions with H O and NH , since the cross-linking\n2 3\nreaction was quenched with excess ammonium bicarbonate. The precursor and\nfragment ion precision were set to 5 ppm (MS1) and 10 ppm (MS2), respectively.\nIdentification focused on b and y ion types, with a minimum of three fragments per\npeptide. Charge states of +2 and higher were selected, and deisotoping was enabled.\nPrecursor mass correction was disabled. RISEUP was chosen as the analysis mode,\nallowing for one missing ion. Neutral losses were enabled, with a maximum of one loss\nper fragment. For scoring and FDR calculations, a prescore of 10%, an FDR cutoff of\n1%, and a score cutoff of 50 were applied. Given the use of a customized database\nbased on comprehensive sample analysis, the MeroX cRAP database was excluded\nfrom the searches.\nAlphaFold:\n- The authors do not state which version of AlphaFold multimer was used or any of the\nsettings with regards to template and MSA. It seems the authors may have used\ncolabFold, with relaxation off, given the severe clashes present in the model. Similar\nsteric clashes are also present in the model the authors built, which shows that the coiled\ncoil prediction server may have packed the helices a bit too close together, or at the\nwrong angle.\nAnswer: We initially used model 2 (apo) and model 5 (transition) generated by AlphaFold\nmultimer (ColabFold), with relaxation off. To obtained a dimeric model of the active\nconfiguration compatible with autophosphorylation state we built manually a dimer as\nindicated on Fig. 6. In addition, we have now used in the revised version of the\nmanuscript a dimeric assembly of CCDC6-RET in an active configuration (analogous to\nthe manual model) using AlphaFold3 from the Google AlphaFold server\n(https://alphafoldserver.com/). Furthermore, and in order to overcome some steric\nclashes found in the initial AlphaFold models, in this revised version of the manuscript\nwe have included 300 ns molecular dynamic simulations of the dimeric assemblies under\nthe different functional configurations (see Fig. 9 and supplementary Fig. 13). Please\nnote that the model for transition state have been omitted in the revised version of the\nmanuscript, see also discussion on page 37.\nData availability:\n- We were not given access to the proteomics or crosslinking MS data as the authors did\nnot include the reviewer password associated with the deposition.\nAnswer: All the phospho-proteomic datasets and XL-MS data have been deposited in\nthe ProteomeXchange Consortium via the PRIDE partner repository with the dataset\nidentifier: PXD053907\n16\n(https: //proteomecentral. proteomexchange.org/cgi/GetDataset?ID= PXD053907).\nPassword: KlpwHu4rkXby.\n- Is the Skyline file containing the phospho-peptide quantification deposited somewhere?\nAnswer: see answer to previous point.\n- Models generated in the study should be deposited in PDB-Dev (integrative models) or\nModelArchive (AlphaFold models)\nAnswer: We have submitted the active homodimer generated by Alpha-Fold3 server to\nthe repository (please see https://modelarchive.org/doi/10.5452/ma-edq6r/).\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nThis is a complex but very interesting and potentially very important manuscript that\ndescribes the cis and trans autophosphorylations that take place in a fusion protein\nwhere the N-terminus of CCDC6 is fused to the kinase domain of RET. This fusion\ncreates a constitutively active dimer that now functions as a driver of tumors in the\nthyroid. Several RET fusions have been reported in the past and these gene\nrearrangements were found in Chernoble following the nuclear\ncontamination because some of the primary events were found in the thymus where iodine accumulates. While other fusions\nwith other tyrosine kinases have been reported, including BCR-Abl, which was the first protein shown to cause cancer, this\nstudy with CCDC6-RET is important because it delves more deeply into the properties of this fusion protein. In all cases, the\nfusion proteins are thought to be active because the fusion creates a constitutive dimer which is essential for activation of\nany of these tyrosine kinases. Usually a transcient dimer forms as a part of the activation process. This paper first shows\nconvincingly not only that this dimer is constitutively active but also that it phosphorylates many sites that are not typically\nreported to be phosphorylation sites in RET. This includes many novel pTyr sites as well as pSer sites and it also includes\nsite in CCDC6 as well as in RET where CCDC6 is not a normal target for RET. They also show a time frame for these\nphosphorylation events which relies on Mass Spectrometry and is very difficult to\ncapture. With mutagenesis they decipher which of the sites are likely to be biologically\nimportant. The authors also suggest that this kinase may be able to use ADP as a\nphosphoryl donor. Finally, with preliminary negative staining and preliminary and\npreliminary CryoEM they are able to build a credible model for this fusion protein. Overall,\nthere are some very important things that we can learn from this manuscript even though\nsome of the things are likely to be artefactual. Some of the concerns are serious and\nwould likely require some additional experiment; however, if the authors could\nsatisfactorily address these concerns, then the manuscript should be re-considered for\npublication.\nAnswer: We are grateful for the referee´s comments and positive evaluation. We agree\nthis is a complex but very interesting and potentially very important manuscript that\ndescribes for the first time the molecular mechanism of CCDC6-RET autoactivation. We\nhave tried to experimentally address most of the referee’s comments and concerns, in\nparticular the experiments that proved beta-phosphate transfer from ADP during the\nCCDC6-RET autophosphorylation reaction.\nSpecific Concerns:\n1. The mapping of the phosphorylation sites is very detailed and comprehensive and\nshows very clearly that this fusion protein is hyperactive and is phosphorylating a lot of\nspurious sites that are not seen with the native RET. It shows global dysregulation and\nalso defines lack of specificity. It is important to unambiguously show that this lack of\nspecificity is a feature of this constitutive dimer. This point should be emphasized.\nAnswer1: Lack of specificity is supported by the very fast kinetics of autophosphorylation\n(e.g. activation loop) observed and by the proposed cis-driven mechanism which is\ncontrary to the native (wild-type) kinase, which is characterized by a temporal and\nsequential pattern of (intermolecular) autophosphorylation. This is unexpected, as the\nsimplest and more predictive working hypothesis was that dimerization will favour\nintermolecular phosphorylation between catalytic domains. We have emphasized on this\naspect now in the results and discussion sections in the revised version of the\nmanuscript, where we state that this lack of specificity is a feature of this constitutive\ndimer. See page 31 on the revised version of the manuscript.\n20\n2. These phosphorylation sites are mostly cis as would be expected. While the authors\nmade this point earlier with Src, this is an important point and will likely be a conserved\nfeature of all of these oncogenic fusion proteins. The biological significance of these sites\nin cells where phosphatases are often constitutively expressed and localized at the same\nsite is debatable. In contrast to the cis phosphorylation, which can be quite random and\nmeditated whenever a surface hydroxyl group comes in close proximity to the ATP, the\ntransphosphorylation sites are required for phosphorylation of heterologous protein\nsubstrates so these are likely to be the more physiologically relevant sites. This point\nshould be very clearly made.\nAnswer 2: We agree with the referee that most of the autophosphorylation sites would\nbe in cis. This comes in line also with the mechanism of autophosphorylation observed\nin some bacterial hybrid histidine kinases as we indicated and referred in the text (see\nresult section page 28 and discussion on page 33. In the active configuration the catalytic\ndomain of each of the protomers of the homodimer are facing away from the stem of the\ncoiled-coil dimeric interface and this is compatible with cis-autophosphorylation. In the\ncase of c-Src we recently published a study where we dissected a cis-to-trans\nautophosphorylation switch at the activation loop that preceded the intermolecular\nautophosphorylation of its C-terminal segment (Cuesta-Hernández and Contreras et al.\n2023). Whether cis-autophosphorylation is a conserved feature of all of these oncogenic\nRET fusion proteins is something that will depend on the monodispersion state of the\nfunctional assemblies (mono-di-oligomeric state) and the contacts that define each of\nthem. For example, in the case of the PTC2 we found a dynamic equilibrium between\nmonomer and dimer species, and binding to c-AMP favours the dissociation and\nmonomeric state of the fusion product, altering its capacity to autoactivate (in this case\ndelays the process). How this translates to the phosphorylation of heterologous protein\nsubstrates is a critical question and we are currently exploring this aspect by using a\ncellular system where cell extract from starved cells are used as a source of substrates\nfor the recombinant protein to be phosphorylated under different nucleotides stimulation\n(work in progress, see also Fig. 2 on this rebuttal letter).\n3. The trans phosphorylation require correct organization of the Activation Loop and the\nauthors do show how Y900 and Y905 are more important for activity than the other sites.\nHow these contribute to organization of the Activation Loop is still unclear and also\nunclear is the chronology. Is modification of Y900 a required precursor step for the\nphosphorylation of Y905? Does their data indicate this stepwise process? If so, it should\nbe articulated very clearly.\nAnswer 3: This is another interesting and important point raised by the reviewer#6.\nBased on our data shown on Fig. 2, activation loop Tyr 900 comes already\nphosphorylated when we purify the protein. Upon RET autoactivation, levels of Tyr 905\nphosphorylation augment, and as a consequence the levels of mono-phosphorylated Tyr\n900 peptide decreased proportionally along the time course. See also the supplementary\ninformation about the stoichiometry analyses for CCDC6-RET activation loop phospho-\nsites (supplementary Fig. 2). Crucially Tyr 900 appears to be critical for the stability and\nfolding of the RET fusion product (specifically to the short C-terminal isoform 9, see also\nFig. 3 rebuttal letter), as a Y900F mutant is not viable and does not expressed properly.\nStrickinly, this dependency is lost in a construct lacking the C-terminal segment where a\nY900F mutant is viable, expresses properly and functional (see Fig. 5 on main\nmanuscript). The fact that a Y900F mutant of the long C-terminal splicing isoform RET\n51 is also functional and viable support the notion that there are critical contacts between\n(phosphorylated) Y900 and the C-terminal segment of the short C-terminal segment of\nRET that control the fold and stability of the oncogenic fusion (see Fig. 3 rebuttal letter).\n4. Importance of targeting. The many random cis auto-phosphorylations suggest that the\nfirst cis auto-phosphorylations take place quickly when then the kinase is first activated.\nIs this a correct assessment of the data? It makes sense that the phosphates of ATP,\n21\nwhich is initially targeted with low affinity binding by hydrophobic residues that interact\nwith the adenine ring of ATP, are highly flexible and easily able to phosphorylate any\nhydroxyl group that comes in close proximity. In the wt kinases the phosphorylation sites\nare very focused and are organized so that only a specific protein is modified, usually on\na single site. This is because the kinase is recruited to a specific site which contains may\ncontain other proteins such as phosphatases and the downstream proteins that will bind\nto the pTyr. In this case the kinase will only be transiently dimerized. This specificity is\nlost in the fusion protein where the kinase is constitutively activated because it is a dimer\nbut close to the fusion protein rather that its physiological partner. How does this kinase\nfind its right target?\nAnswer 4: The assessment of our data is that the kinetics of autophosphorylation of the\nCCDC6-RET fusion product are very quick and much faster that the sequential\nautophosphorylation observed by the RET kinase and intracellular domains which\nundergo intermolecular autophosphorylation (see Plaza-Menacho et al. Mol Cell 2014).\nIn a physiological context, the referee is correct about its own assessment and\ninterpretation in the case of native kinases that are tightly controlled by transient\ndimerization, this specificity is lost in the case of the fusion protein where the kinase is\nconstitutively activated because it is a dimer but close to the fusion protein rather that its\nphysiological partner. We hypothesize that the C-terminal segment will play a critical role\non this aspect of the signalling process by the oncogenic chimera, as the phosphorylation\nsites (e.g. Tyr 1015, Ser1034, Tyr 1062) will serve as docking for adaptor proteins having\na SH2-, or PTB-binding domain and others. The phospho-proteomic data from the\ncellular extracts comparing the CCDC6-RET c-terminal splicing isoforms (i.e. 9vs 51,\nshort vs long) come in line with this working hypothesis (Martín-Hurtado et al. II in\npreparation). We have to consider that the cellular localization of the fusion protein is\ndifferent from that of the native kinase. CCDC6 is a nuclear protein and interacts with\nDNA most likely by the leucine-zipper motifs found in the ordered CCDC6 portion of the\nchimera (aa 53-101, see also figure 6 main text), so another hypothesis is that nuclear\nsubstrates may be affected by the signalling of this oncogenic fusion product. We have\nmade this point clear in the discussion of the revised version of the manuscript (see page\n36).\n5. Another point that the authors make is that ADP, as well as ATP, can serve as the\ndonor for phosphorylation. To make this claim I think it is important that the authors show\ndirect transfer of the beta phosphate using 32P-labeled ATP. An old set of papers by Ora\nRosen, indicated that ATP could be generated directly from a phosphorylated residue at\nthe active site of one of the PKA regulatory subunits. While such an observation to my\nknowledge has not been pursued for other kinases, it is still\npossible that the phosphates from ATP could somehow be “scrambled”. While ADP may stabilize one of the protomers,\nto claim that the phosphate is also transferred from ADP requires the radio-labelled ATP.\nAnswer 5: We agree with the reviewer#6 that in order to prove that the CCDC6-RET\nfusion uses ADP to phosphorylate itself we had to perform an auto-phosphorylation\nassay with radioactive ADP labelled on the beta phosphate. These data are now part of\nthe main text of the revised version of the manuscript and can be found on Fig. 4e.\nIn addition, we have performed a series of additional experiments to support the ADP\ndependency by the CCDC6-RET fusion product and the regulatory role of the di-\nphospho-nucleotide (see new Fig.4), in particular:\n1. We performed a quality control check of the purity of the nucleotide stocks to prove\nthat they were highly pure with no traces of contaminations (see Fig.1 rebuttal letter).\n2. We checked by spectroscopy that the protein come out of the cells already after the\nsecond affinity purification step free on DNA or nucleotide contamination as indicated by\nthe optimal (260/280 nm) ratio (see Fig. 1c).\n22\n3. We have data from other oncogenic RET fusions products that we work with in the lab\n(i.e. PTC2, RI-RET). The PTC2 fusion is capable of binding ATP and ADP, however\nand contrary to PTC1, it cannot use ADP efficiently as phosphoryl donor during the\nautophosphorylation process, contrary to PTC1.\n4. We have used ATPS as a phosphoryl donor, based on the premise that CCDC6-RET\ncan hydrolyse the beta-bond of the nucleotide derivative, as it does with ADP. In fact, we\nobserved CCDC6-RET autophosphorylation in the presence of ATPS, although (but as\nexpected) with slower kinetics than ATP, mirroring to some extent the\nautophosphorylation kinetics observed with ADP. These data are now provided in\nsupplementary Fig. 4 of the revised version of the manuscript.\n5. We have performed ADP titration experiments to prove a dose-dependent effect\n(inhibition) in autophosphorylation assays at 2.5- and 5-mM concentrations; and in\ncompetition assays we demonstrate a regulatory function (inhibition) of the ADP at high\nconcentrations (1 and 5 mM). See data on the revised version of the main manuscript\non Fig. 4.\n6. We have preliminary results using cell extracts of dormant (serum deprived) cells as\na substrate source where CCDC6-RET activated by ADP induce the phosphorylation of\na specific/different set of substrates compared with ATP, see figure 1 below. This work\nis ongoing and beyond the scope of this manuscript, but it is expected to be a follow-up\nstory (see Fig. 2 rebuttal, Martín-Hurtado et al. in preparation I).\n6. Heterodimers. Do the authors know if heterodimers are formed between the fusion\nprotein and wt CCDC6?\nAnswer 6: We have tried to assemble heterodimers of the CCDC6-RET fusion product\nwith a CCDC6 aa 1-101 fragment (the part of CCDC6 that forms the fusion product)\nwithout clear evidence of complex formation. Further, we have done biochemical and\nbiophysical characterization and found no effect when titrating increasing amount of the\nCCDC6 aa 1-101 fragment onto the catalytic core of the RET fusion product (RET aa\n713-1072) that would have been otherwise indicative of a direct interaction.\n23\n7. Where is the fusion protein localized in cells? Presumably targeting is mediated\nimproperly by the coiled:coil domains of CCDC6.\nAnswer 7: The fusion product is localized mainly in the cytoplasm and also in the nuclei.\nWe have performed some preliminary experiments in PTC1 cells where we observed\nnuclei staining when using a RET-specific antibody. This is something we did not look\ninto more detail, but we have now a working hypothesis that the fusion product may be\ninteracting with DNA by the leucine-zipper motif of the coiled-coil in the nucleus (work\nin progress, see also next point).\n8. Is the CCDC6 homodimer also expressed in cells? Presumably there is one good copy\nof both RET and CCDC6. Is this homodimer more stable so that neither free RET nor\nthe CCDC6 dimer are present in these cells that express the fusion protein? If this is so,\nthen this mutation not only creates an oncogene but also abolishes the normal function\nof both proteins.\nAnswer 8: This is another interesting point made by the reviewer. In the case of CCDC6-\nRET, the fusion product is caused by an intra-chromosomal translocation, hence a good\nviable copy of each of the genes are present in the cells. The working hypothesis made\nby the referee is very interesting. It also opens the possibility that the oncogenic fusion\nmay change the activating and signalling properties of the native protein by direct\nphosphorylation and/or aberrant complex assembly. This is something we would like to\nexplore in the near future together with the already mentioned ADP-dependency in a\ncellular context and the nuclear function of CCDC6-RET interacting with the DNA.\nThe authors have made important improvements in the revised manuscript. In\nparticular the new data with radiolabeled ATP and ADP makes a convincing case for\nthe ability of the fusion protein to transfer the b-phosphate of ADP in an\nautophosphorylation reaction (although the Figure provided for review is not helpful in\nthat all traces are identified as ATP in the legend). However, a number of issues\nremain.\n1. It is unclear that there is any physiological relevance to the transfer of ADP.\nExperiments performed here are at concentrations that are ~100 times greater than\ncellular concentrations, and given the more efficient phosphorylation with ATP, unclear\nthat there could ever be a cellular condition in which limiting ATP led to phosphorylation\nwith ADP. As the authors acknowledge, ATP concentrations are ~1000 times that of\nADP. The authors suggest that this might differ in certain subcellular environments, but\nwildly speculative to suggest that it could differ enough to make this relevant to the\nactivity of this RET fusion. That said, utilization of ADP by a kinase is an interesting\nbiochemical result.\nResponse reviewer #1point 1:\nWe appreciate this is an important point and we agree that the physiological relevance\nof ADP as a phosphate donor requires careful contextualization. We have expanded\nthe discussion in the revised manuscript to address this explicitly (on page 35-36).\n“In normal cells, ATP concentrations are maintained between 1–10 mM, while ADP levels\nare lower (20-100 µM), yielding an ATP/ADP ratio of ~100-500 59, 60. In contrast, cancer\ncells often exhibit suppressed mitochondrial metabolism and enhanced glycolysis (i.e.\nthe Warburg effect), leading to reduced ATP levels and ATP/ADP ratios as low as 5–20,\nwith ADP reaching 100–200 µM or more 61. Thus, tumor cells display ADP/ATP ratios of\n0.05–0.2, much higher than in normal cells. Under significant ATP-limiting conditions,\nADP can accumulate to comparable concentrations to those of ATP (~500 M)\n(Coccimiglio and Clarke, 2020) and under these conditions it is plausible that ADP can\nbe utilized as an alternative phosphate donor by certain protein kinases.\nWe further hypothesize that local concentration effects, such as the formation of\nCCDC6–RET condensates or lipid phase separation, could locally enrich ADP and\nthereby enable ADP-dependent phosphorylation. Similar mechanisms have been\nreported for oncogenic kinase fusions like EML4–ALK, which forms condensates that\ncompartmentalize signaling 62, and for ATP-driven phase separation of disordered basic\nproteins (Kota et al., bioRxiv 2023.08.20.554035). To test this working hypothesis, we\nare developing cellular systems that allow direct manipulation of the ATP/ADP ratio using\ngenetically encoded tools such as ATPGobble (Ekvik et al., bioRxiv 2025.08.12.670003).\nThese models will enable us to reduce the ATP/ADP ratio even further and assess ADP-\ndependent phosphorylation under controlled, physiological conditions (work in progress).\nFinally, recent studies describe similar ADP-driven phosphorylation in MAP2Ks and IKK\n63, 64, supporting the emerging concept that ADP-dependent activity, though secondary\nunder normal conditions, may act as an adaptive mechanism during energy limitation.\nThis could have important implications for how (cancerous) cells maintain signaling\nfunctions under stressful conditions such as low oxygen, or ischemia, which are common\nin diseases like cancer and atherosclerosis.”\nRegarding the commentary “although the Figure provided for review is not helpful in that\nall traces are identified as ATP in the legend”. The referee should refer and see Fig. 4j\ninstead.\n2. Along the same lines, seems wrong to suggest that high concentrations of ADP \"regulate\"\nthe RET fusion, when the concentrations required are ~100x that found in cells (cells would\nnot survive such a concentration of ADP).\nResponse reviewer #1point 2:\nWe agree that the term “regulate” could be misleading outside an in vitro context. Our\ntitration data (Fig. 4a) show that CCDC6–RET activity increases with ATP starting at ~10\nµM, whereas activation by ADP becomes apparent at 25–50 µM, peaks near 1 mM, and\ndeclines at >2.5 mM. Furthermore, ADP at 1 mM (1:1 with ATP) slows ATP-driven\nautophosphorylation, while 5 mM ADP (5:1 ratio) completely abolishes it (Fig. 4c). These\nfindings demonstrate a clear in vitro modulatory effect of ADP on CCDC6–RET catalytic\nactivity. Nevertheless, since these experiments use purified protein at supraphysiological\nconcentrations, we have revised the section title to “ADP regulates CCDC6–RET\nphospho-tyrosine activity in vitro” to clearly delimit the scope of this conclusion.\n3. the authors us of \"apo\" to refer to the unphosphorylated kinase is confusing. \"Apo\"\ntypically means without a binding partner or co-factor. Clearer to refer the the\nunphosphorylated state as the \"unphosphorylated state\" or perhaps inactive state.\nResponse reviewer #1 point 3:\nWe use the term “apo” for the protein that is not stimulated with ATP/ADP nor MgCl (i.e.\n2\ninactive or unstimulated). To make this point clearer we will use the term inactive and/or\nunstimulated accordingly throughout the text.\n4. Authors at least imply that utilization of ADP for autophosphorylation is unique to the\nRET fusion. But not clear that is does not also happen with the isolated kinase domain\n(which has lower catalytic activity overall) but is at a level that is difficult to detect.\nResponse reviewer #1 to point 4:\nIn Fig. 3f we performed side-by-side experiments with the RET catalytic core (aa 713-\n1072) and demonstrated neglected/residual ADP-dependent activity by the native\nkinase. Further, we showed in the previous response to the referees, that PTC2 (RI-\nRET) lacked ADP dependent activity. We have performed additional experiments to\ndemonstrate that another RET fusion product, KIF5B-RET lacks ADP dependent activity\nand it is not able to autoactivate in the presence of ADP (see Fig. 2 and response to\nreferee 7 on this document). Together, these data confirm that ADP-dependent activity\nappears to be a mechanistic feature specific to CCDC6–RET and not a general feature\nof RET or other fusion variants.\nPlease note that the RI-RET, CCDC6-RET iso51 (see figure 3-5 on this letter) and\nKIF5B-RET data shown on the rebuttal letters are part of two current manuscripts that\nare in advance stages of preparation and must be kept strictly confidential.\n5. The authors appear to have misunderstood or at least incompletely understood my\nconcern about their conclusions regarding cis vs trans autophosphorylation. I think the\nauthors and I would both agree that their data argue against inter-dimer phosphorylation\nas the dominant mechanism. The question is how intra-dimer phosphorylation occurs, which\ncould either be in cis (each subunit of the dimer phosphorylating itself) or in trans (one\nsubmit phosphorylating the other). In the kinase field, cis-autophosphorylation would\nordinarily be taken to mean a given kinase domain phosphorylating itself, irrespective of\nits oligomeric state. For example in an RTK dimer, cis autophosphorylation would imply\nthat each subunit in a dimer transferred a phosphate to its own activation loop, while trans\nautophosphorylation would mean each transferred a phosphate to the activation loop of the\npartner subunit in the dimer. I still do not know for certain what the authors are concluding\nhere, but as best I can tell there are two possibilities: 1) they are using cis-autophosphorylation\nto mean that CCDC6-RET dimers are autophosphorylating themselves, without regard to\nwhether this intra-dimer phosphorylation is occuring in cis or in trans. This is a correct\ninterpretation of their data, but a mis-use (or at least a misleading use) of \"cis\", for the\nreasons outlined above. 2) the authors are claiming that intra-dimer phosphorylation occurs\nin cis - that is, that each subunit in the dimer transfers a phosphate to itself. I think this is\nwhat the interested reader would likely take as the authors meaning, but as best I can tell,\nthere is not a single experiment presented to discriminate between cis and trans\nphosphorylation within a CCDC6-RET dimer. The \"gold standard\" experiment the authors\nnow present supports intra-dimer phosphorylation, but does not address whether intradimer\nphosphorylation is occurring in cis or trans. The authors could clear all this up by simply\navoiding the \"cis\" designation, and concluding that autophosphorylation occurs in an\nintra-dimer manner, but acknowledging that they have not addressed whether this occurs\nin cis vs trans.\nResponse reviewer#1 to point 5:\nWe thank the reviewer for clarifying this important point. Our biochemical data exclude\ninter-dimer phosphorylation during CCDC6–RET activation (Supplementary Fig. 10), but\nwe acknowledge that these data do not definitively distinguish cis from trans events\nwithin the dimer.\nNevertheless, several lines of evidence favor a cis- mechanism:\n(i) Structural modeling shows that, in the inactive state, the kinase domains are\npositioned face-to-face, while upon nucleotide binding, they rotate/swing apart, orienting\ntheir active sites away from each other-compatible with each kinase phosphorylating its\nown activation loop.\n(ii) XL-MS data reveal that crosslinks between activation loops present in the inactive\ndimer disappear in the phosphorylated state, consistent with this conformational\ntransition (Fig. 8). Molecular-dynamics simulations support this rearrangement (Fig. 9).\nDespite attempts (serial dilution, mild denaturation, coiled-coil mutations), we could not\nisolate a monomeric form suitable for direct testing. Accordingly, we have tempered our\nclaims:\ni) “cis-autophosphorylation” has been removed from the title.\nii) the data are now presented in Supplementary Fig. 10, retitled “A concentration-\nindependent mechanism drives CCDC6–RET autophosphorylation”\niii) the abstract now states that “fast activation-loop phosphorylation could be driven by\na mechanism in cis”;\niv) and the Discussion clarifies that, while interdimer phosphorylation is excluded, the\nexact cis/trans relationship within the dimer remains unresolved (page 37-38). This\nrevised phrasing accurately reflects the evidence and addresses the reviewer’s concern.\n6. to my understanding, phosphotransfer with ATPgammaS (or more precisely, transfer\nof thiophosphate) typically involves transfer of the gamma thiophosphate and not transfer\nof the beta or beta-gamma phosphate unit as the authors suggest here. I think this\nexperiment detracts, rather than adds to the story.\nResponse to reviewer#1point 6:\nWe agree with the reviewer’s assessment and have removed the ATPγS experiment\nfrom the revised manuscript to maintain clarity and focus.\n7.How do the authors reconcile the high-molecular weight species seen in prior SDS-\nPAGE gel with crosslinked samples with the clean, dimeric peak seen in the SEC\nexperiment provided in the revision? Presumably this was carried out on a parallel\nsample, and not the actual sample used for crosslinking mass spec studies? Was the\nSDS-PAGE gel of the actual sample?\nResponse to reviewer#1point 7:\nIn all our crosslinking experiments, the samples eluted as a clear dimeric peak by SEC,\nconfirming a stable dimer in solution, and that the sample was not overcrosslinked. The\nsame samples migrate as high-molecular-weight smears on SDS–PAGE, a possible\nartifact of covalent crosslinking under denaturing conditions that alters mobility but not\noligomeric state. We have clarified this point in the revised Methods (page 16).\nReviewer #3 (Remarks to the Author):\nThe authors present a much improved version of the manuscript that broadly addresses\nour concerns, specifically on the phosphoproteomics and the crosslinking MS\nquantitation aspects.\nThere are still 2 areas that present major issues:\n- we requested to have all crosslinks of the datasets mapped on the structure. The\nauthors reply to this by saying that only few crosslinks arise from the site of interest. The\nissue is that in order to interpret those proposed crosslinks in a structural context, one\nwould need to understand if the proposed model is validated/consistent with the\ncrosslinking dataset as a whole. What is the percentage of crosslinks within distance? is\nthe sample overcrosslinked (as another reviewer was wondering)? Normally this is\naddressed also with a distogram over the whole dataset. Without this, we have no way\nof assessing what is the relevance and reliability of the specific crosslinks highlighted by\nthe authors\nResponse to reviewer#3:\nWe agree with the reviewer and considering his/her comment, we are now showing on\nthe two 3D-structural assemblies all the specific crosslinked peptides that define the two-\nfunctional and structural (inactive-unphosphorylated- and active-phosphorylated) states,\nand the percentage of crosslinked peptides within distance for each of them. These data\ncomplement the data already shown and synthesized in the circular diagrams where the\nspecific crosslinked peptides for both states were depicted (see Fig. 8c and d). These\ndata are now of Fig. 8g. As we can see, the crosslinked peptides that define the topology\nof the two functional-conformational states are distinct. There is one main central node\nof crosslinked peptides arising from activation loop K907 in the inactive (apo) state. In\nthe case of the specific crosslinked peptides that define the active (auto-phosphorylated)\nstate there are two main nodes, one at K965 just below the susbtrate binding pocket and\nanother at a K747 on the 2 of the G-loop. This information has been now included on\nthe result section corresponding to Fig. 8g, on page 31. These data address the\nreviewer’s request for model validation and confirm the internal consistency of the XL-\nMS data.\n- Again, alphafold models are presented without PAE plots or coloring by pLDDT. This is\ndiscouraged also by DeepMind themselves. Indeed, looking at the ModelArchive\ndeposition, one can see that the confidence of the inter-dimer arrangement of the\ncatalytic lobes is low, as well as the pLDDT scores around the active site and in the ADP\nitself. A model with ATP can also be predicted and the difference in pocket conformation\n(again, with judgment gauged by inspection of pLDDT) can support or refute some of the\narguments being made. Similarly, the fact that this \"fits well\" with the negative stain data\nis presented with no cross-correlation or percentage inside metrics.\nResponse to reviewer#3:\nThe reviewer is correct. The ModelArchive entry has been updated (DOI 10.5452/ma-\nb7la0) with a newer model with even higher confidence (mean pLDDT = 87.7). Both\nactive and inactive assemblies are now displayed with pLDDT-colored confidence maps\n(active = 87.7; inactive = 79.1) in Supplementary Fig. 7e-f. These visualizations now\nclearly indicate confidence levels across the model. The Data Availability section and\nfigures have been updated accordingly.\nWhile we acknowledge that the available EM and SAXS data cannot resolve atomic\ndetails, the combined metrics confirm a reliable overall architecture consistent with\nexperimental data. We also note this limitation explicitly in the Discussion on page 37-\n38\n.\npLDDT 87.74 pLDDT 79.08 Fig.1. AlphaFold\nmodels (pLDDT color\ncoded) for the active\n(phosphorylated) and\ninactive\n(unphosphorylated)\nstates of CCDC6-RET\nwith the overall\npLDDT values for\neach state and table\nwith reference quality\ncontrol figures.\nIn general, while the manuscript has greatly improved in transparency and in overall\nreadability, our objections were that the model uncertainty is not quantified, the model\nprecision is not quantified, the fit to the data (save for SAXS chi squared) is not really\nquantified. These remain problematic. Ultimately, this structure does not have the\nresolution or interpretability to explain why this kinase should use ADP as substrate,\nfor example with mutation of R912 that supposedly plays a key role in the proposed\nmechanism.\nResponse to reviewer#3:\nWe agree with the referee that the resolution of the electron microscopy and SAXS data\ndoes not allow the visualization and full depiction of the molecular determinants that\ncontrol/regulate ADP- versus ATP- dependent activities. We did mention and\nacknowledged this in the manuscript discussion on page 38. We are actively working on\nthe crystallization and in the cryoEM characterization of CCDC6-RET in the apo and\nactive states bound to both ATP and ADP, and in complex with novel RET kinase\ninhibitors to get high resolution structural and mechanistic information (work in progress)\nabout the proposed mechanism of CCDC6-RET autoactivation.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #6 (Remarks to the Author):\nThe authors did an extensive amount of work to address the various specific concerns\nthat were raised by the reviewers. My major concern was with regard to phosphorylation\nby ADP. The authors did the critical experiment of using radio-labeled ADP to show that\nthe bets-phosphate was transferred to the protein. While this may not be physiological,\nthe data nevertheless shows that some transfer is possible with this hyperactive\noncoprotein although it is not observed in the wt RET. If Reviewers 1 2, and 3, in\nparticular, are satisfied with the authors revised manuscript, then I would concur with\npublication.\nResponse to reviewer#6:\nWe thank reviewer#6 for their insightful and positive evaluation.\nReviewer #7 (Remarks to the Author):\nDear Authors,\nThank you for submitting the revised manuscript and for addressing some of the previous\ncomments. While the study has been improved, several important concerns remain that\nshould be clarified to strengthen the significance and rigor of your work.\n1. In your response, you indicate that other RET fusion proteins (e.g., PTC2 and RIα-\nRET) were used to support the claim that ADP-dependent phosphorylation is a distinctive\nfeature of CCDC6-RET. However, it is important to note that fusion partners such as RIα-\nRET are rare in human cancers, especially when compared to more common and\nclinically relevant fusions such as NCOA4-RET (PTC3) or KIF5B-RET. The use of such\nuncommon variants as primary comparators limits the generalizability and biological\nrelevance of your conclusions. To more convincingly demonstrate the uniqueness of\nCCDC6-RET’s biochemical behavior, direct comparative analyses with frequent RET\nfusions—particularly those with well-established roles in oncogenesis—are essential.\nFurthermore, it appears that these critical comparative data are not included in Figure 4,\nwhich diminishes support for this central claim.\nResponse to reviewer #7:\nWe have conducted experiments with another RET fusion product, KIF5B-RET, which is\na driver and therapeutic target in NSCLC. This RET-fusion product is more clinically\nrelevant due its high incidence and bad prognosis in human cancers. We have performed\nadditional experiments to demonstrate that KIF5B-RET has no apparent ADP dependent\nkinase activity as is unable to autophosphorylate (see Fig. 2). Please note that the\nCCDC6-RET, RI-RET and KIF5B-RET data shown on the rebuttal letters are part of\nseveral manuscripts (3) that are in advance stages of preparation and should be kept\nstrictly confidential.\nThe RET gene produces two isoforms, RET9 and RET51, through alternative splicing,\nwhich differ in their C-terminal sequences and signaling properties. There is\naccumulating evidence that the RET51 isoform may play a more prominent role in cancer\ndevelopment. However, your study is based primarily on the RET9 isoform. A key\nunresolved issue is which RET isoform is predominantly involved in genomic\nrearrangements with CCDC6 in actual human tumors. Is CCDC6-RET9 the predominant\nform, or does CCDC6-RET51 also occur?\nWe believe that addressing these issues will significantly enhance the impact and\ntranslational relevance of your study.\nResponse to reviewer #7:\nThis is a very interesting question and something we have considered and think about\ncarefully. RET fusions can result in different c-terminal splicing isoforms, including RET9\nand RET51(Grieco et al. Cell 1990, Ju et al. Genome Res 2012, Drilon et al Cancer\nDiscovery 2013 and Liam et al. 2017). There is accumulating evidence however about\nthe RET51 isoform may play a more prominent role in cellular transformation in cell-\nbased experiments/preclinical models (Liam et al. 2017). However, whether any specific\nc-terminal isoform is predominantly associated with any RET fusion product and with a\nmore aggressive phenotype is a critical question that requires further empiric evidence\nto be answered. In our case, we have convincing evidence that CCDC6-RET_iso9 and\nCCDC6-RET_iso51 have the same enzymatic/catalytic and phospho-tyrosine activities\nin vitro (Fig. 3 and 4 on this rebuttal letter). Note that CCDC6-RET_iso51 has also ADP\ndependent activity (work in progress, confidential data). Crucially when we\nperformed a whole-cell lysate kinase assay coupled with LC/MS-MS under saturating\nconcentrations of ATP we found changes in substrate specificity by both c-terminal\nsplicing isoforms that could account for their specific potential in tumorigenesis (Fig. 5,\nrebuttal letter, Hurtado and Contreras in preparation). Please note these data are part of\na paper in preparation to be submitted in the following weeks and should be kept strictly\nconfidential.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have adequately addressed my concerns in this revision.\nResponse to reviewer #1:\nWe thank the reviewer for the constructive comments and suggestions provided during\nthe review process, which helped us to improve the clarity and robustness of our study.\nReviewer #3 (Remarks to the Author):\nThe authors present a revised manuscript that partially addesses my 2 concerns, which\nhowever remain the same\n- include pLDDT AND pAE plots (only plddt is shown in the new supplementary figure).\nResponse to reviewer #3:\nIn the new version of the manuscript, we have now included both pLDDT scores and\npAE plots for each of the two structural models, see supplementary Fig. 7e and 7f. We\ndid not include the pAE plots in the last version of the manuscript (despite having them\nin the first version of the manuscript submitted) to avoid data redundancy, as we\nunderstood they come along in the same line as the pLDDT scores, which are indicative\nof high-quality data. Our apologies for any misunderstanding or inconvenience this may\nhave caused. Note that the pAE plot of the structural model for the inactive-unstimulated\nstate was generated in AlphaFold2 multimer with the complete CCDC6-RET sequence,\nincluding the N- and C-terminal IDR regions. This is clearly stated in the figure legend.\n- plot ALL detected crosslinks on the model with a distance distribution histogram, not\nonly the crosslinks arising from sites of interest. As I mentioned in my previous\ncomment: \"The authors reply to this by saying that only few crosslinks arise from the\nsite of interest. The issue is that in order to interpret those proposed crosslinks in a\nstructural context, one would need to understand if the proposed model is\nvalidated/consistent with the crosslinking dataset as a whole\". Unfortunately, the edited\nmanuscript still does not provide this information- the distance distribution of ALL the\ndetected crosslinks, not the subset picked by the authors as \"of interest\" or the ones\nspecific to a particular state. One can generate this easily using ProXL or xiview. The\nassertion that the data confirms the “internal consistency” of the crosslinking dataset is\nalso dependent on the whole dataset being assessed. I would surmise that %ge within\ndistance being so low (45% within distance for a stated FDR of 5% for supposedly\nconformation-specific restraints) is not a good sign of the model being consistent with\nthe data or the data being consistent with itself.\nResponse to reviewer #3:\nWe did not show all the detected crosslinks in the structural models for simplicity and for\na better visual representation. That is why we focused on the specific crosslinks that\ndefined each of the two functional states. However, we recognize that this approach did\nnot address completely the reviewer’s concern and that mapping all the detected\ncrosslinks in the structural models and showing their distance distribution will confirm the\ninternal consistency and robustness of the data.\nIn the revised manuscript we have now:\ni) Mapped all detected crosslinks in the CCDC6-RET structural models (coiled-coil plus\nkinase core) for both the apo and phosphorylated states.\nii) Displayed, the distance distribution histograms and the percentage of crosslinks within\nthe 27.5 Å cutoff for each structural state, using the full set of mappable crosslinks.\nThese data are shown in supplementary Fig. 11 d and e.\nCrosslinks that involve residues located in the N- and C-terminal intrinsically disordered\nregions were not included in the distance analysis due to a lack of structural confidence.\nAll experimentally observed crosslinks—including those that involve IDR residues—are\nfully reported in the XL-MS tables and deposited in the PRIDE databank.\nCrucially, the subset of distance-compatible crosslinks and their clustering on specific\nstructural elements (for example, around K907 in the apo state and around K965/K747\nin the phosphorylated state) is consistently observed across replicates and functional\nconditions, supporting further the internal consistency of the XL-MS dataset and its\nsuitability as a structural restraint for the proposed models. We have added a brief\nclarification of this point in the main text to make this explicit (see results section on page\n31).\n“Mapping all the detected crosslinks in both structural models revealed a percentage of\ncrosslinks within distance (27.5 Å cutoff) of 63.5 and 61.9 % for the unstimulated and\nphosphorylated states, respectively (supplementary Fig. 11 d and e). The subset of\ndistance-compatible crosslinks and their clustering on specific structural elements is\nconsistently observed across replicates and functional conditions, supporting further the\ninternal consistency of the dataset. These data together demonstrate significant\nconformational changes during the mechanisms of CCDC6-RET autoactivation, and at\nthe same time validate the internal consistency of the XL-MS dataset and its suitability\nas a structural restraint for the proposed models.”\nSo the authors have addressed one concern partially, and the other not at all- neither of\nmy objections asked for additional experiments, it is information the authors have\nreadily available and are not being forthcoming with. I was also not provided with the\nPRIDE reviewer details, as far as I can tell. So I could not check this myself either. I\ncan understand if the authors are frustrated with additional reviews and feel an unfair\nburden is placed upon them- at the same time, I feel they are choosing to not reply to\nthese simple requests.\nResponse to reviewer #3:\nWe are sorry for any confusion regarding the PRIDE access details. However, we have\nto make clear that the PRIDE accession codes and credentials were provided in the initial\nsubmission of the manuscript upon request by the editors. You can find the information\nagain below:\nProject accession: PXD053907\nToken: dQcrqzsuYnzx\nAlternatively, reviewer can access the dataset by logging in to the PRIDE website using\nthe following account details:\nUsername: reviewer_pxd053907@ebi.ac.uk\nPassword: KlpwHu4rkXby\nOverall, the manuscript remains improved and I leave it to the editor to make a\ndecision.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #7 (Remarks to the Author):\nI have reviewed the authors' responses to the reviewers' comments and the revised\nmanuscript. I am pleased to note that the authors have undertaken a comprehensive\nrevision to tackle the various specific issues raised. The explanations and changes\nmade regarding my key points are clear and have alleviated my initial concerns. If the\nother reviewers are content with the revisions pertaining to their comments, I am happy\nto support the acceptance of this work.\nResponse to reviewer #7:\nWe are very grateful for the reviewer’s careful reading and for the constructive\nsuggestions throughout the review process. We are pleased that the revisions and\nadditional clarifications satisfactorily address the reviewer’s concerns, and we thank for\nthe supportive recommendation."}, {"round": 3, "reviewer_comments": {"reviewer_3": "I thank the authors for their reply and understanding throughout the process. The authors have addressed all my concerns\nand I recommend the manuscript be publish.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter