The clinical missense variant E282K in PPP3CA/calcineurin shifts substrate dephosphorylation by altering active site recruitment

Domain: neuroscience
Published: 2026-02-16
Editorial outcome: major_revision

Abstract
Recently, de novo heterozygous variants of Calcineurin (CN) were reported as the cause of a neurodevelopmental disorder that presents with epileptic encephalopathy and dysmorphism (DEE91), with the largest group of patients harboring the CN missense mutation E282K (glutamate → lysine). Here, we use molecular and cellular techniques to define how this mutation alters CN activity. We discover that basophilic substrates use an arginine residue to bind to CN via an acidic substrate recruitment pocket adjacent to the CN active site, the E282 pocket. Furthermore, we show that basic residues in the i-1 position of the substrate relative to the substrate phosphosite enhance CN-mediated dephosphorylation. While the CN E282K structure shows that the overall conformation is unchanged, the E282 pocket transforms from acidic to basic, with pocket access blocked by the formation of a E282K-E237 salt bridge. Finally, in vitro assays and in cell phosphoproteomics show that CN E282K shifts CN substrate dephosphorylation profiles from basic to acidic, thereby altering CN-mediated dephosphorylation signaling. Together, these data define the molecular impact of the CN E282K variant in cells and development, providing a key step for developing strategies to treat this disorder and its accompanying complications.

Introduction
Phosphorylation is one of the most ubiquitous, reversible post-translational modifications (PTM) in cells1. Thus, defining the finely tuned specific and reciprocal relationship between kinases and phosphatases is essential for understanding their control and modulation of signaling pathways. Calcineurin (CN, Protein Phosphatase 2B [PP2B], PP3)2–4 is a ubiquitously expressed phosphoprotein phosphatase (PPP) that is especially abundant in the brain. Upon activation by Ca2+ (hence calcineurin)5, CN dephosphorylates an estimated 100–1000s of substrates to regulate development, synaptic plasticity, learning and memory as well as many other functions3,6,7. As a consequence, disruption of CN-regulated signaling in the brain has, for decades, been associated with disease, including Alzheimer’s (AD)8 and Parkinson’s disease9. Large-scale whole exome sequencing (WES) has greatly advanced the identification of genetic variants that underlie disease, especially rare diseases. During the last decade, WES studies have identified multiple de novo variants of CN (PPP3CA) that are associated with a distinct neurodevelopmental disorder that presents with epileptic encephalopathy, intellectual disabilities, dysmorphism, hypotonia and autism spectrum disorder (PPP3CA-related DEE91 [developmental and epileptic encephalopathy]; OMIM #617711)10–14, with the missense mutation E282K being the most frequently observed variant. Critically, the molecular mechanisms by which these de novo CN variants, including E282K, disrupt CN-mediated signaling are major open questions.

CN is a constitutive heterodimer with two subunits (Fig. 1a)5,15. The catalytic subunit A (CNA) contains the active site, the calmodulin binding region and an auto-inhibitory domain (AID). The regulatory subunit B (CNB) binds four Ca2+ ions, making CN responsive to calcium signaling. Substrates and regulators are recruited to CN via CN-specific short linear motifs (SLiMs), short conserved sequences that are present within intrinsically disordered regions (IDRs) of proteins that mediate protein:protein interactions16–24. The PxIxIT SLiM binds the CNA subunit16,20,21,23,24 while the LxVP SLiM binds the interface between the CNA and Ca2+-sensing CNB domain21; the latter is accessible only upon Ca2+-activation18,25,26. CN substrates, such as the NFATs17,18,22, NHE120,27, RCAN123, among others, use both PxIxIT and LxVP motifs to bind CN in a 1-to-1 manner. Mutating these SLiMs in substrates abolishes dephosphorylation by CN, demonstrating the essential role of recruitment for substrate dephosphorylation18–21. As a consequence, simply blocking substrate recruitment is sufficient to inhibit CN-mediated dephosphorylation in cells, a mechanism exploited by viruses (i.e., A238L of the African swine fever virus) and immunosuppressants such as FK50621,28,29.

Recent work has shown that the efficacy of CN catalysis can be modulated by additional mechanisms. For example, a recently discovered CN active site recognition sequence (TxxP) enhances recruitment to the active site, accelerating CN-mediated dephosphorylation20. However, many CN substrates do not have a phosphosite TxxP sequence, suggesting there may be additional mechanisms for enhancing phosphosite dephosphorylation. One such possibility is the enhanced binding of kinase-specific phosphosite sequences that are targeted by CN for dephosphorylation. For example, CN counteracts the activities of basophilic kinases, especially PKA, which requires an R-R-x-S/T motif for productive phosphorylation30. How and if this basophilic kinase motif is locally recruited to the CN active site for dephosphorylation processing remains an open question.

By leveraging two model PKA/CN substrate pairs, here we show an additional recruitment pocket adjacent to the CN active site that is critical for CN substrate recruitment, specificity and regulation. The two substrates include the canonical PKA regulatory subunit II (RII)31 that via a phosphorylation switch, controls PKA activity proceeding cAMP-binding, and the transforming growth factor beta (TGFβ) activated kinase (MAP Kinase Kinase Kinase 7 or Transforming growth factor β-activated kinase 1; TAK1)32. Leveraging both an in vitro peptide and cell-based proteomics approach, we tested how this pocket globally affects local CN active site recruitment. Our comprehensive structural, biochemical and proteomics data reveals that an arginine residue at the i-1 position relative to the phosphosite in CN substrates is critical for enhanced recruitment and dephosphorylation of these substrates. This enabled us to define an additional CN substrate recruitment pocket that facilitates recruitment of basophilic substrates. Furthermore, we show that this CN substrate recruitment pocket includes the major clinical CN variant residue E282, highlighting that the E282K variant likely results in a global shift of signaling outcomes resulting in neurodevelopment deficits. Defining how CN recruits and especially dephosphorylates distinct CN-specific substrates will not only advance our understanding of the role of PPPs in modulating signaling pathways, but also provide the data needed to understand how the de novo CN variants alter this signaling and, in turn, lead to the development of routes for treating PPP3CA-related DEE91.

Results
TAK1 uses an LxVP SLiM and a RRRS motif to bind CN
TAK1 is a mitogen activated protein kinase (MAPK) that contains an N-terminal kinase domain (Fig. 1a; aa 1–306) and a C-terminal IDR (Fig. 1a; aa 306–606) that is phosphorylated by PKA at Ser439 (436RRRS439). CN counteracts the activities of many basophilic kinases, including PKA, and its sequence contains a putative LxVP motif (422LDVP425) near the PKA phosphorylation site22. Therefore we tested if the TAK1 IDR binds CN using isothermal titration calorimetry (ITC). The data showed that both the extended C-terminal IDR (TAK1 306–590) and a shorter version including the target phosphorylation site (TAK1 384–474; hereafter, TAK1) bind CN and do so with nearly identical affinities (Fig. 1b, Supplementary Fig. 1a, b; Supplementary Table 1). Consistent with these data the 2D [1H,15N] heteronuclear single quantum coherence (HSQC) spectra of TAK1 306–590 and TAK1 show an early perfect overlap of all HN/N cross-peaks, confirming there were no local or long-range changes between the ensemble of conformations adopted in both TAK1 constructs (Supplementary Fig. 1c). We did not test if CN binds to the TAK1 catalytic domain (likely not as no CN SLiM was identified) nor if the TAK1 catalytic domain makes an intramolecular interaction with the TAK1 C-terminal IDR.

To identify the TAK1 residues that bind CN, we completed the sequence-specific backbone assignment of TAK1 (Supplementary Fig. 2a) and overlaid the 2D [1H,15N] HSQC spectra of 15N-labeled TAK1 in the absence and presence of CN (Fig. 1c, d). Many HN/N cross-peaks were significantly broadened with CN (Fig. 1d, e; Supplementary Fig. 2b–e), with most reduced intensities corresponding to TAK1 residues 415–441 (Fig. 1e; includes the putative LxVP motif and residues surrounding the Ser439 PKA phosphosite). We confirmed the importance of the LxVP motif for CN recruitment using ITC, as a variant lacking the LxVP sequence (LxVP dead: 422LDVP425 → 422AAAA425) failed to bind CN (Fig. 1f). TAK1 bound CN with an atypically strong affinity compared to other LxVP:CN interactions (TAK1 KD, 823 ± 8 nM; Fig. 1b; other reported LxVP KD’s, ~1800–500,000 nM; Supplementary Table 1)21,23,33, suggesting that residues outside the LxVP motif contribute to CN binding. Consistent with this, in the presence of CN, the peak intensities of the consensus site for PKA phosphorylation (436RRRS439) were reduced to the same extent as those observed for the LxVP residues (Fig. 1e). To test if these residues facilitate CN binding, we mutated the PKA phosphorylation consensus sequence in TAK1 (RRR dead: 436RRRS439 → 436AAAS439; Fig. 1a) and measured its affinity for CN. The affinity decreased ~4.5-fold (Fig. 1g, Supplementary Table 1), demonstrating that 436RRR438 enhances CN binding. Together, these data show that TAK1 strictly requires a functional LxVP motif to bind CN and that this is strengthened by the RRRS phosphosite motif.

The RRRS phosphosite motif binds CN near the active site
To define how the RRRS motif binds CN, we determined the crystal structure of a substrate-trapped CN complex. Trapping was achieved by using a catalytically inactive CN variant (D90A; lacks the M1 active site metal and thus is unable to dephosphorylate substrates) and a thiophosphorylated TAK1 substrate (tp, thiophosphorylated; hereafter, CN D90A:tpTAK1, Fig. 2a, b; tethered to the PxIxIT site using CN-specific the A238L viral inhibitor peptide, Supplementary Fig. 3a, b, Supplementary Table 2). Two copies of the CN D90A:tpTAK1 complex were present in the asymmetric unit, with clear electron density for TAK1 residues bound to the CN LxVP binding pocket and the CN active site (Fig. 2b–d; Supplementary Fig. 3c–g; aa 422–428, 435–442; no density was observed for aa 429–434, which form a dynamic linker between the LxVP binding pocket and CN active site). The TAK1 LxVP motif (422LDVP425) binds the CN LxVP binding pocket in a manner identical to those observed previously (Fig. 2c). TAK1 Leu422 and Val424 bind two deep hydrophobic pockets at the CNA/B interface. Electron density was also observed at the CN active site, corresponding to TAK1 residues 436RRRtpS439 (Fig. 2c, d). While the conformations of the Leu422 and Val424 side chains were nearly identical between the two copies of TAK1 in the asymmetric unit, as is typical for buried hydrophobic interactions, the arginine side chains of the RRRS motif (436RRR438) adopted slightly different conformations between the two copies, highlighting that these charge:charge interactions are more dynamic (i.e., fuzzy; Supplementary Figs. 3c–g). TAK1 arginine residues bind an extended acidic pocket on CN (Fig. 2d), making polar and/or ionic interactions (<4 Å) with CN residues Glu282 and Gln284 (100% conserved in CN from yeast to humans; Fig. 2e, Supplementary Fig. 3h). Arg438 (R i-1 relative to tpS), which is immediately adjacent to the active site, makes the most extensive interactions with CN, binding a deep, strongly acidic pocket defined by Glu237 and Glu282 (Fig. 2e).

Arginine i-1 phosphosite recruitment facilitates efficient TAK1 dephosphorylation
To quantify the contribution of these CN residues for TAK1 binding, we performed ITC measurements of TAK1 vs CN WT, CN E282K, CN Q284K and CN D285K. The data showed that affinity of TAK1 for CN E282K is 3-fold weaker than CN WT (akin to TAK1 RRR dead; Supplementary Fig. 4a, Supplementary Table 1), while binding is only minimally impacted for TAK1 vs CN Q284K or CN D285K (Supplementary Fig. 4b, c Supplementary Table 1). These data highlight the key role of the R i-1 residue for binding. Next, we performed CN dephosphorylation assays. We mutated these residues in the CN substrate binding pocket and measured their ability to dephosphorylate TAK1 pSer439 (uniformly PKA-phosphorylated Ser439, pS439, was achieved using the TAK1 S389A/S417A variant, as NMR experiments showed that in vitro Ser389 and Ser417 are partially, non-specifically phosphorylated by PKA). The data showed that CN variants with mutations in residues near the i-2 and i-3 arginine residues (Q284K, D285K, Y315A), had an increase in activity (Fig. 2f). In contrast, mutating the residues that define the CN i-1 pocket—E237K and E282K—significantly reduced activity, by ~50% and ~70%, respectively (Fig. 2f, Table 1). To further confirm that the arginine in the i-1 position facilitates TAK1 binding at the active site, we generated the TAK1 R438V variant (436RRRS439 → 436RRVS439) and measured its affinity for CN and CN E282K using ITC (Supplementary Fig. 4d, Supplementary Table 1). The data showed that the lack of an arginine in the i-1 position weakens the affinity ~3-fold, nearly to the same extent observed for the TAK1 RRR dead variant, but, as reasoned, has very minimal effect on CN binding (Supplementary Fig. 4e, Supplementary Table 1). Together, these data identify a negatively charged pocket, the E282 pocket, that facilitates the recruitment and dephosphorylation of substrates containing an arginine residue in the i-1 position (R i-1).

Arginine in the i-1 position is a general mechanism for enhanced CN substrate dephosphorylation
We next tested the generality of an arginine in the i-1 position for enhancing CN-mediated recruitment and dephosphorylation of CN substrates by measuring the dephosphorylation of a canonical CN substrate that lacks an i-1 arginine: the PKA regulatory subunit RII (RII Ser99 is phosphorylated and dephosphorylated by PKA and CN, respectively, and lacks an i-1 arginine, 96RRVS99; RII 44–103 construct used for these studies; hereafter RII). We also created an RII variant where the i-1 residue was mutated to arginine (Fig. 3a; i-1.e., contains an R i-1). Like TAK1, RII also uses an LxVP motif for CN recruitment, which we confirmed using NMR spectroscopy and X-ray crystallography (Fig. 2c, Supplementary Fig. 5a–d, Supplementary Table 2). To assay CN-mediated dephosphorylation, we phosphorylated Ser99 in wt RII and the RII V98R variant (RII V98R; 96RRVS99 → 96RRRS99) using PKA (phosphorylation assays performed using the RII S58A/S78A construct to ensure phosphorylation of only Ser99). The data showed that replacing Val98 with an arginine in the RII i-1 position enhanced CN-mediated dephosphorylation by ~50% compared to WT, confirming the enhanced dephosphorylation of substrates with an arginine in the i-1 position (Fig. 3b; Table 2). Second, while the i-1 pocket charge reversal variants CN E237K and CN E282K reduced the dephosphorylation of wt RII, the decrease in activity was significantly more pronounced with the RII V98R variant, by ~70% and ~85%, respectively (Fig. 3b; Table 2). Together, these data confirm the role of the acidic binding pocket adjacent to the CN active site, the E282 binding pocket, for CN substrate recruitment of R i-1 containing substrates.

CN E282K fails to effectively recruit R i-1-dependent CN substrates
These results show that the CN E282 binding pocket has a critical role in recruiting R i-1-containing substrates to the CN active site to facilitate their dephosphorylation. The most frequently observed variant in PPP3CA-related DEE91 is E282K (Fig. 4a). To test if the altered dephosphorylation of R i-1-containing substrates in CN E282K is due to a global or local change in CN conformation, we determined the crystal structure of CN E282K (Fig. 4b; bound to NHE1, a peptide that binds CN using a PxIxIT and LxVP motif20; Fig. 2c). The structures of CN WT and CN E282K (both bound to NHE1) are nearly identical (RMSD, 0.16 Å; Fig. 4b, c), demonstrating that there are no global changes in the CN conformation. The conformation of bound NHE1 is also identical, confirming that the CN PxIxIT and LxVP recruitment pockets are fully structured and functional (Fig. 4b); i.e., they are not impacted by the E282K mutation. The most significant change is observed in the E282 pocket itself, where E282K forms a salt bridge with E237, which likely sterically hinders substrate access to the E282 pocket (Fig. 4c). Furthermore, an analysis of the electrostatic surface potential of CN E282K shows that the negative charge of the R i-1 binding pocket is significantly attenuated compared to the substrate-trapped complex (Fig. 4d–f).

To test if these changes impact TAK1 binding at the E282 binding pocket, we repeated the NMR analysis of TAK1 with CN E282K (Supplementary Fig. 6a–f). As expected, an overlay of the 2D [1H,15N] HSQC spectrum of 15N-labeled TAK1 384–474 in the absence and presence of CN E282K showed that many HN/N cross-peaks were significantly broadened partially beyond detection upon complex formation; however, the broadening observed for the TAK1 RRRS motif was less than that observed with CN WT, demonstrating that CN E282K recruits R i-1 containing substrates less effectively, consistent with their lower KD in ITC measurements and their inability to be efficiently dephosphorylated (Supplementary Fig. 6e, f). These data suggest that the observed changes in substrate recruitment and dephosphorylation by CN E282K are due to local changes at the E282 binding pocket.

To further confirm our findings, we generated additional TAK1 variants and compared their dephosphorylation efficiency with CN WT vs CN E282K. As expected, TAK1 R438K (436RRRS439 → 436RRKS439) showed no differences, while reversing the charge by creating an TAK1 R438D or R438E variant (436RRRS439 → 436RRDS439 or 436RRRS439 → 436RRES439) showed, as expected, increased dephosphorylation by CN E282K when compared to wt-TAK1 (Fig. 4g). We also tested the effect of deleting the i-2 or i-3 arginine side chains. TAK1 R436A (436RRRS439 → 436ARRS439) showed an additional small decrease in activity (highlighting that the ensemble of structures is less influenced by the likely interactions between the positive charged arginine and the negative charged phosphor-moiety), while TAK1 R437A (436RRRS439 → 436RARS439) showed a small increase in dephosphorylation. Lastly, we also tested the TAK1 R438V (436RRRS439 → 436RRVS439) and showed that its activity is slightly increased for CN E282K. Thus, both local electrostatic and substrate accessibility changes at the E282 pocket in the CN E282K variant result in the weakened recruitment and dephosphorylation of R i-1-containing substrates, and, therefore, is likely the molecular basis of the altered signaling that gives rise to PPP3CA-related DEE91.

The PPP3CA-related DEE91 CN missense mutation E282K globally alters CN substrate dephosphorylation
To determine the general importance of CN E282 for substrate recruitment and dephosphorylation, we performed an in vitro phosphoproteomics assay. Here, we quantified the differences between CN WT- and CN E282K-mediated dephosphorylation of substrates derived from a HeLa cell phosphopeptide library. Asynchronous HeLa cells were treated with the pan-PPP inhibitor Calyculin A for 30 minutes to induce a hyperphosphorylated state. Proteins extracted from these cells were digested with Lys-C to preserve arginine residues that are part of dephosphorylation motifs surrounding the phosphosites. These peptides were incubated with CN WT and CN E282K in vitro for 0, 15, 30, 60 and 120 min to determine CN sequence preferences in a time-dependent manner (Fig. 5a, Supplementary Fig. 7a; Supplementary Data 1). This resulted in 1274 substrate peptides for CN WT and 1267 substrate peptides for CN E282K, respectively. Using hierarchical clustering, we identified four clusters with fast, intermediate, or slow dephosphorylation kinetics or sites that were not dephosphorylated (Supplementary Fig. 7b). Next, we extracted peptides with either arginine (R), lysine (K), aspartate (D) or glutamate (E) amino acids in the i-1 position and compared their dephosphorylation rates (Fig. 5b).

The data show that CN WT readily dephosphorylates peptides with R-pS/T and K-pS/T motifs, while CN E282K dephosphorylates these peptides more slowly (Fig. 5c). Conversely, CN E282K dephosphorylates peptides with D/E-pS/T motifs faster than CN WT (Fig. 5c). These data confirm the importance of the negatively charged E282 active site recruitment pocket for CN activity and, furthermore, highlight how a single point variant, CN E282K, globally alters the CN substrate recruitment and dephosphorylation (Fig. 5c, Supplementary Fig. 7b). We also determined the amino acid enrichment of all phosphorylation sites in the fast dephosphorylation cluster of CN WT and CN E282K, which confirmed the preference of CN WT for basic and CN E282K for acidic residues in the i-1 position (Fig. 5d)34.

CN substrate preference in cells
To determine if CN WT and CN E282K display differential substrate preferences in cells, we stably expressed them in HEK293T cells and performed MS-IP and quantitative phosphoproteomics (Supplementary Fig. 8a, b). The MS-IP CN WT vs control data identified many established CN interactors, including NFATs, RCANs, constituents of the PI4K complex35, NUP proteins19, GSK3 kinases, SIK kinases and RANB3, all of which have been previously identified in CN MS data19,33. Comparing the MS-IP data between CN WT vs CN E282K showed that RCAN1/3 are enriched in CN E282K vs CN WT (as RCAN1 has been shown to bind at the CN active site23, it is possible that a similar active site interaction in RCAN1/3 may influence CN binding). Interestingly, most other known interactors showed depletion with CN E282K, including PI4K35,36. Next, we analyzed the HEK293T-based quantitative phosphoproteomics data. Here, we compared sequences surrounding single, localized phosphosites dephosphorylated by CN WT or CN E282K (Supplementary Fig. 8c, d, Supplementary Data 2). Consistently, as in our in vitro results, we observed an enrichment of arginines and lysines in the −1 position for CN WT and glutamate and aspartate for CN E282K.

To determine if the HEK293T MS data is mirrored in a cell line more physiologically relevant for DEE91, we repeated the MS-IP and quantitative phosphoproteomics measurements of CN WT and CN E282K in N2A cells, a neuronal mouse neuroblastoma cell line that has been widely used to study neuronal functions and disorders. The MS-IP CN WT vs control data identified again many established CN interactors, confirming our ability to perform these experiments in N2A cells and highlighting the consistency between the HEK293T and N2A CN interactomes (Fig. 6a). We then assessed if known CN interactors exhibit altered recruitment with CN E282K. While the RCANs and NFATC3 were enriched with CN E282K, many interactors were depleted, including the PI4K complex (Fig. 6b). Thus, the N2A MS-IP data largely mirrored that from HEK293T cells. We then analyzed the N2A-based quantitative phosphoproteomics data (Fig. 6c), which, like the HEK293T data, showed an enrichment of positively charged residues in the −1 position for CN WT and glutamate for CN E282K (Fig. 6d; Supplementary Data 2). We also analyzed which proteins experienced the largest increase or decrease in (de)phosphorylation, which, surprisingly, included many proteins not previously shown to be regulated or modulated, directly or indirectly, by CN (Fig. 6c). Because direct substrates of CN are recruited via CN-specific SLiMs, we used a bioinformatic pipeline to identify if CN-specific SLiMs (PxIxIT and LxVP) are present in these potential CN substrates (Supplementary Fig. 9a, b). The data show that all but one of the putative substrates contain 1 or more PxIxIT, LxVP or both SLiMs, highlighting the most differently affected phosphosites in N2A cells are putative direct CN substrates. Together, our comprehensive in vitro and cellular data confirm that, as observed for TAK1 and RII, CN has a preference at the active site for substrates with an arginine residue the i-1 position relative to the phosphosite in CN substrates. This residue is recruited by a negatively charged pocket consisting of E282 and E237. Together, with the previously defined TxxP motif, our data shows the importance of active site interactions for CN dephosphorylation and how a single residue substitution, E282K, can shift the overall substrate preference of CN.

Methods
Cloning
TAK1 384–474 /TAK1 384–474 LxVP dead (AAAA)/TAK1 384–474 RRR dead (AAAS) were subcloned into pTHMT containing an N-terminal His6-MBP for enhanced solubility followed by tobacco etch virus (TEV) protease cleavage site tag. Additionally, TAK1 306–590 and TAK1 384–474 were subcloned into pRP1b containing an N-terminal His6-tag followed by a TEV protease cleavage site. A TAK1 384–474 variant (S389A, S417A; S389 and S417 are non-specifically phosphorylated by PKA as detected by NMR spectroscopy) was created by site-directed mutagenesis and exclusively used for CN dephosphorylation assays. Site-directed mutagenesis was performed in TAK1 384–474 (S389A, S417A) to generate R436A (436ARR438), R437A (436RAR438), R438K (436RRK438), R438V (436RRV438), R438E (436RRE438) and R438D (436RRD438) variants. RII 44–103 was subcloned into pTHMT and site-directed mutagenesis was performed to generate RII 44–103 (S58A, S78A; S58 and S78 are non-specifically phosphorylated by PKA as detected by NMR spectroscopy) and RII 44–103 V98R (S58A, S78A) for dephosphorylation assays. CN 391 (CNA; 1–391, CNB; 1–169) was subcloned into pRP1b as a single bicistronic cassette containing an N-terminal His6-tag followed by a TEV protease cleavage site. CN 391 variants; E237K, E282K, Q284K, D285K, and Y315A variants (CNA subunit variants) were generated by site-directed mutagenesis from CN 391 wild type construct. The A238L-TAK1 peptide was designed as a G-Block containing residues of the African Swine Viral protein A238L PxIxIT motif (PKIIIT) and linker (aa 200–228) and the LxVP motif and RRRS motif from TAK1 (aa 422–440) (TAK1 chimera, motifs underlined: GHMRRFKKKPKIIITGCEDNVYEKLPEQNSNF422LDVPEIVISGNGQPRRRSI440RP, RP was added to the C-terminus to enhance the binding to the active site and facilitate crystallization). The A238L-TAK1 peptide sequence was subcloned into the pRP1b vector containing a His6-tag followed by a TEV protease cleavage site. The NHE1 679–723 peptide was subcloned into the pTHMT vector and used for CN E282K crystallization studies. For crystallization experiments, CNA 1–370 (hereafter, CNA 370), CNB 1–170 or CNB 15–170 were subcloned into pCDF_Duet1 (CNA in MCS1 and CNB in MCS2). CNA contains an N-terminal His6-tag followed by a TEV protease cleavage site. Site-directed mutagenesis was performed on CNA 370 to generate the D90A and E282K variants. All variants were sequence verified.

Protein expression
TAK1 (WT and variants), RII (WT and variants) and the A238L-TAK1 chimera proteins were expressed in Escherichia coli BL21 (DE3) cells (Agilent). CN 391 plasmids (WT and variants) were transformed into E. coli BL21 (DE3) RIL cells (Agilent). Freshly transformed cells were grown at 37°C in LB medium containing appropriate antibiotics (kanamycin [50 µg mL−1] for TAK1, RII and A238L-TAK1 plasmids; kanamycin [50 µg mL−1] and chloramphenicol [34 µg mL−1] for CN 391 plasmids) until they reached an optical density (OD600) of ~0.8. Protein expression was induced by addition of 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) to the growth and incubated for 18 h at 18°C, 250 rpm. Cells were harvested by centrifugation (6000 × g, 20 min, 4°C) and stored at −80°C until purification. For NMR experiments, expression of uniformly 13C- and/or 15N-labeled protein was carried out by growing freshly transformed cells in M9 minimal media containing 4 g L−1 [13C]-D-glucose and/or 1 g L−1 15NH4Cl (Cambridge Isotopes Laboratories) as the sole carbon and nitrogen sources, respectively.

NHE1 679–723 was expressed in E. coli BL21 (DE3) RIL cells (Agilent). Freshly transformed cells were grown at 37°C in LB medium containing the appropriate antibiotics (kanamycin [50 µg mL−1] and chloramphenicol [34 µg mL−1]) until an OD600 of ~0.8 was reached. Protein expression was induced by addition of 1 mM IPTG and cultures were allowed to grow overnight (18–20 h, 250 rpm shaking) at 18°C. Cells were collected by centrifugation (6000 × g, 15 min, 4°C) and stored at −80°C until purification.

CNA 370/ CNB 1–170 (WT) and CNA 370/ CNB 15–170 (WT, E282K, D90A) were expressed in E. coli BL21 (DE3) RIL cells (Agilent). Freshly transformed cells were grown at 37°C in LB medium containing the antibiotics (spectinomycin [120 µg mL−1] and chloramphenicol [34 µg mL−1]) until they reached an OD600 of ~0.8. Protein expression was induced by addition of 1 mM IPTG to the growth and incubated for 18 h at 18°C, 250 rpm. Cells were harvested by centrifugation (6000 × g, 20 min, 4°C) and stored at −80°C until purification.

PKA (human Cα1 in pet15b) was transformed into E. coli BL21 (DE3) RIL cells (Agilent). Freshly transformed cells were grown at 37°C in LB medium containing the appropriate antibiotics (carbenicillin [100 µg mL−1] and chloramphenicol [34 µg mL−1]) until an OD600 of ~0.8 was reached. Protein expression was induced by addition of 1 mM IPTG to the culture medium, and cultures were allowed to grow overnight (18–20 h, 250 rpm shaking) at 18°C. Cells were collected by centrifugation (6000 × g, 15 min, 4°C) and stored at −80°C until purification.

Protein purification
For all constructs, cell pellets were resuspended and homogenized in ice-cold lysis buffer (50 mM Tris pH 8, 500 mM NaCl, 5 mM Imidazole, 0.1% Triton-X-100) or CN lysis buffer (20 mM Tris pH 7.5, 300 mM NaCl, 50 µM CaCl2, 0.1% Triton X-100) with EDTA-free protease inhibitor tablet (Thermo Fisher), lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Cell debris was pelleted by centrifugation (45,000 × g, 45 min, 4°C) and the supernatant was filtered using 0.22 μm syringe filters (Millipore) before purification.

TAK1 and RII. His6-MBP-tagged TAK1 384–474/306–590 or RII 44–103 (S58A, S78A) (WT and variants) and His6-tagged TAK1 384–474 (S389A, S417A) (WT and variants) was loaded onto a HisTrap HP column (Cytiva) pre-equilibrated with Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM Imidazole). Proteins were eluted using a linear gradient of 0–80% Buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM Imidazole). Fractions containing the protein were pooled and dialyzed in buffer ([TAK1, 20 mM Tris pH 8.0, 500 mM NaCl], [RII, 20 mM CHES pH 9, 50 mM NaCl, 0.5 mM TCEP]) overnight at 4°C with TEV protease (in-house, His6-tagged) to cleave the His6-MBP-tag or His6-tag. Following cleavage, the sample was loaded under gravity onto Ni2+-NTA beads (Prometheus) pre-equilibrated with Buffer A. Proteins were eluted using a stepwise-elution of Buffer B (5–100%). The flow-through and wash A fractions were collected and then heat purified by incubating the samples at 80°C/TAK1 and 75°C/RII, for 15 min. Samples were centrifuged at 4000 × g for 15 min to remove precipitated proteins. TAK1 384–474 (S389A, S417A) was aliquoted, flash frozen and stored at −80°C for phosphorylation reactions. TAK1 384–474 or RII 44–103 (S58A, S78A) supernatant was concentrated and purified using size exclusion chromatography (SEC; HiLoad 26/600 Superdex 75 [Cytiva]) in TAK1 NMR Buffer (20 mM HEPES pH 6.5, 50 mM NaCl, 0.5 mM TCEP) or RII NMR buffer (20 mM HEPES pH 6.5, 50 mM NaCl, 0.5 mM TCEP) or SEC buffer, TAK1 (20 mM Tris pH 8.0, 50 mM NaCl, 0.5 mM TCEP) or RII (10 mM HEPES pH 7.0, 50 mM NaCl, 0.5 mM TCEP). Purified samples were heat purified for a second time at 80°C for TAK1 and 75°C for RII for 15 min and were either used directly for NMR data collection or flash frozen and stored at −80°C for ITC (TAK1) or dephosphorylation assays (RII).

A238L-TAK1 chimera. Supernatant was loaded under gravity onto Ni2+-NTA beads pre-equilibrated with Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM Imidazole) and eluted using a step wise gradient 5–100% Buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM Imidazole). Fractions containing the protein were pooled and dialyzed (20 mM Tris pH 8.0, 500 mM NaCl) overnight at 4°C with TEV protease to cleave the His6-tag. Following dialysis and cleavage, the dialyzed sample was loaded under gravity onto Ni2+-NTA beads pre-equilibrated with Buffer A. The flow-through and wash A fractions were collected and heat purified at 70°C for 20 min, centrifuged at 4000 × g for 10 min to remove precipitation. The sample was concentrated and was either directly used or flash frozen and stored at −80°C.

NHE1 679–723. His6-MBP-tagged NHE1 679–723 was loaded onto a HisTrap HP column pre-equilibrated with Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM Imidazole). Proteins were eluted using a linear gradient of 0–80% Buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM Imidazole). Fractions containing the protein were pooled and dialyzed (50 mM Tris pH 7.5, 500 mM NaCl, 0.5 mM TCEP) overnight at 4°C with TEV protease to cleave the His6-MBP tag. Following cleavage, the sample was loaded under gravity onto Ni2+-NTA beads pre-equilibrated with Buffer A. The flow-through and wash A fractions were collected and then heat purified by incubating the samples at 75°C for 15 min. Samples were centrifuged at 4000 × g for 15 min to remove precipitated proteins. NHE1 679–723 supernatant was concentrated and purified using SEC (HiLoad 26/600 Superdex 75) in CN SEC Buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM CaCl2, 0.5 mM TCEP). Purified samples were a second time heat purified at 75°C for 15 min and was either used directly for co-purification with CN or flash frozen and stored at −80°C for future purifications.

CN wild type/variants (activity assays) and CN 370 (crystallization with RII). Supernatant was loaded onto a HisTrap HP column pre-equilibrated with Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM Imidazole) and eluted using a linear gradient of 0–80% Buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM Imidazole). Fractions containing the protein were pooled and dialyzed overnight (20 mM Tris pH 7.5, 25 mM NaCl, 1 mM CaCl2, 0.5 mM TCEP) at 4°C with TEV protease to cleave the His6-tag. Following cleavage, the sample was loaded under gravity onto Ni2+-NTA beads pre-equilibrated with low salt His Buffer A (50 mM Tris pH 8.0, 25 mM NaCl, 5 mM Imidazole) and eluted step wise with 5–100% low salt His Buffer B (50 mM Tris pH 8.0, 25 mM NaCl, 500 mM Imidazole). Flow-through and wash A fractions were collected and loaded directly onto HiTrap Q HP column for further purification. The proteins were loaded with IEX Binding Buffer (20 mM Tris pH 8, 25 mM NaCl, 1 mM CaCl2, 0.1 mM TCEP) and eluted with a linear gradient 0–80% of IEX Elution Buffer (20 mM Tris pH 8, 800 mM NaCl, 1 mM CaCl2, 0.1 mM TCEP). CN fractions were pooled, concentrated and further purified via SEC (HiLoad 26/600 Superdex 75; 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM CaCl2, 0.5 mM TCEP). Samples were either directly used or flash frozen and stored at −80°C.

CNA 370 (D90A)/CNB 15–170 :A238L-tpTAK1, CNA 370 (E282K)/CNB 15–170 :NHE1 679–723 co-purification (for crystallization). CN supernatants were loaded onto a HisTrap HP column pre-equilibrated with Buffer A (20 mM Tris pH 8, 500 mM NaCl, 5 mM Imidazole) and eluted using a linear gradient 0–80% with Buffer B (20 mM Tris pH 8, 500 mM NaCl, 500 mM Imidazole). Fractions containing CN variants were pooled; for CN 370 (D90A) a 1.5×-excess (molar concentration) A238L-tpTAK1 (thiophosphorylated) was added and for CN 370 (E282K) a 2×-excess (molar concentration) NHE1 679–723 was added. Both complexes were dialyzed (20 mM Tris pH 8.0, 25 mM NaCl, 1 mM CaCl2, 0.5 mM TCEP) overnight at 4°C with TEV protease to cleave the His6-tag. Following dialysis and cleavage, the complex sample was loaded under gravity onto Ni2+-NTA beads pre-equilibrated with Buffer A. The flow-through, and wash fractions were collected, concentrated, and centrifuged at 4000 × g for 10 min to remove precipitated proteins. The supernatant containing the CN complexes were further purified using SEC (HiLoad 26/600 Superdex 75; 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM CaCl2, 0.5 mM TCEP). Fractions from the peak containing the complex were pooled, concentrated, and either directly used for crystallography or flash frozen and stored at −80°C.

PKA. Supernatant was loaded onto a HisTrap HP column pre-equilibrated with Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole) and eluted using a linear gradient (0–80%) with Buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Fractions containing the protein were pooled and dialyzed overnight into PKA buffer (20 mM Tris pH 8, 50 mM NaCl, 1 mM EDTA, 2 mM DTT) at 4°C. Purified sample was centrifuged at 15,000 × g for 10 min to remove precipitated protein. Supernatant protein sample was mixed with 50% glycerol, flash frozen and stored at −80°C.

A238L-TAK1 thiophosphorylation for crystallization
Purified A238L-TAK1 chimera peptide was incubated with PKA kinase at 10:1 ratio (peptide:kinase) in phosphorylation buffer (100 mM Tris pH 7.5, 2 mM DTT, 10 mM MgCl2) with 10 µM ATP-γ-S (Sigma) for thiophosphorylation. The reaction was incubated at 37°C for 72 h. Thio-phosphorylated A238L-TAK1 (A238L-tpTAK1) was heat purified at 70°C for 30 min and centrifuged (4000 × g, 10 min) to remove precipitated PKA kinase. The final sample was concentrated, aliquoted in small volumes, snap frozen and stored at −80°C until used for crystallization.

Sequence-specific backbone assignment
For TAK1, all NMR measurements were collected on a Bruker Avance 500 MHz spectrometer with a HCN TCI z-gradient cryoprobe at 283 K. The sequence-specific backbone assignment was determined by collecting a suite of heteronuclear NMR spectra: 2D [1H,15N] HSQC, 3D HNCA, 3D HN(CO)CA, 3D HNCACB, 3D CBCA(CO)NH and 3D (H)CC(CO)NH (τm = 12 ms). For RII, all NMR data were collected on Bruker Advance Neo 600 MHz NMR spectrometer with HCN TCI z-gradient cryoprobe at 283 K. The sequence-specific backbone assignment was determined by collecting a suite of heteronuclear NMR spectra: 2D [1H,15N] HSQC, 3D HNCA, 3D HN(CO)CA, 3D HNCACB, 3D HNCO, and 3D HN(CA)CO. Both dataset spectra were processed in Topspin (Bruker Topspin v. 2.1/4.1.3/4.4) and referenced to internal DSS. Peak picking and sequence-specific assignment for TAK1 and RII were performed using the program CARA v. 1.9 (http://www.cara.nmr.ch).

NMR Interaction Studies of TAK1/RII with CN (WT) and CN 391 (E282K). All NMR interaction data of TAK1 384–474 /RII 44–103 with either CN 391 (WT) or CN 391 (E282K). was recorded using either a Bruker Neo 600 MHz or 800 MHz spectrometer equipped with a HCN TCI active z-gradient cryoprobe at 283 K. All NMR measurements of all TAK1 384–474 /RII 44–103 constructs were recorded using 15N-labeled protein in TAK1 384–474 /RII 44–103 NMR buffer and 90% H2O/10% D2O. For TAK1 384–474 interaction studies, an excess of unlabeled CN 391 (WT) or CN 391 (E282K) (3-fold molar excess) was added to 15N-TAK1 384–474 before the 2D [1H,15N] HSQC spectrum was collected. For RII 44–103 interaction studies, an excess of unlabeled CN 391 (WT) (2-fold molar excess) was added to 15N-labeled RII 44–103 before the 2D [1H,15N] HSQC spectrum was collected. NMR data were processed using nmrPipe43 and the intensity data were analyzed in Poky44 (Build: 20230213). For TAK1 384–474 /RII 44–103, the intensity data was normalized to the C-terminal residue. The difference in peak heights between the free 2D [1H,15N] HSQC spectrum of TAK1 384–474 /RII 44–103 was compared to its respective peak, if present, on the 2D [1H,15N] HSQC spectrum of TAK1 384–474 /RII 44–103 in complex with CN 391 (WT) or CN 391 (E282K) (Microsoft Excel, v2301, b16.0.16026.20002 and GraphPad Prism v. 10.2.2). Any overlapping peaks were omitted for analysis. Poky (Build: 20230213) was used to display spectra overlays.

CN complex crystallization
CNA 370 (D90A)/CNB 15–170 :A238L-tpTAK1 (hereafter CN D90A :tpTAK1). Crystals of the CN D90A :tpTAK1 complex were grown using the vapor diffusion hanging drop method, obtained after two rounds of micro-seeding and crystallization condition optimization. Purified CN D90A :tpTAK1 complex was crystallized in 0.2 M sodium sulfate decahydrate, pH 6.7, 20% (w/v) PEG 3350 or 0.2 M sodium phosphate dibasic dihydrate, pH 9.1, 20% (w/v) PEG 3350 at 4.5 mg/mL in drop ratios 1:1, 1:2 and 2:1 (protein:solution). Thin, long, ragged needles appeared after 5–8 days and were used to prepare seed stock A. For the initial round of micro-seeding, seed stock A was diluted at 1:20 ratio with 0.2 M sodium citrate tribasic pH 8.4, 20% (w/v) PEG 3350, and added to CN D90A :tpTAK1 at 5 mg/mL in 0.2 M sodium citrate tribasic pH 8.4, 22% (w/v) PEG 3350, 3% (v/v) ethanol in drop ratios 5:8:1 and 5:4:1 (protein:solution:micro-seeding; 1 µL: 1.6 or 0.8 µL: 0.2 µL). Crystals appeared after 7 days and were used to prepare seed stock B. For the second round of micro-seeding, seed stock B was diluted the same way as seed stock A, and added to the CN D90A :tpTAK1 complex at 6.3 mg/mL in 0.2 M sodium citrate tribasic pH 8.4, 22% (w/v) PEG 3350, 3% (v/v) ethanol in drop ratios 5:8:1 and 5:4:1. Long, thick plates appeared after 6 days. Crystals were grown using hanging drop vapor diffusion method at room temperature (22°C). The seed stock was prepared by mixing 10 µL of the reservoir solution in each drop five times and transferring to a 1.5 mL tube containing a seed bead (Hampton Research). The process was repeated until there was a total of 50 µL in the seed bead tube. The crystals were crushed by vortexing the tube with the bead for 1 min followed by 1 min on ice (repeated five times), and 5 µL aliquots were snap frozen and stored at −80°C for further use. Single crystals were cryoprotected with a mix of the reservoir solution and 15% glycerol before flash cooled in liquid nitrogen for data collection.

CN:RII. CN 370 was incubated with a 1.2-fold molar excess of RII and concentrated to 6 mg/ml. Crystals formed in 3 days in 100 mM sodium citrate, pH 5.0, 100 mM MgCl2 6H2O, 15% (w/v) PEG 4000. Crystals were cryoprotected using silicon oil and flash frozen in liquid nitrogen.

CN E282K :NHE1. CNA 370 (E282K)/CNB 15–170 :NHE1 (hereafter CN E282K :NHE1) was concentrated to 8 mg/mL. Crystallization was performed using Gryphon LCP (Art Robbins) into Intelli 3-well plates (Art Robbins). Initial crystallization trials were performed in the conditions used for crystallizing the CN:NHE1 complex, resulting in small needle clusters. Conditions optimized by testing varying high pH buffers (CHES/CAPS/TRIS/HEPES) and testing varying low MW PEGs (PEG 400 – PEG 6000). Small, moon shaped crystals were observed in 0.1 M CAPS pH 9, 0.2 M MgCl2 6H2O (v/v), 24% PEG 600. Crystals were cryoprotected with paraffin oil and flash frozen in liquid nitrogen.

Data collection, processing and structure solution and refinement
Data processing and refinement statistics for all complexes reported in Supplementary Table S2 with example electron density shown in Supplementary Figs. 3, 10.

CN D90A :tpTAK1 and CN E282K :NHE1. X-ray diffraction data were collected at the National Synchrotron Light Source II (NSLS-II) AMX 17-ID-1 beamline at Brookhaven National Laboratory (Upton, NY, US), at 100 K and a wavelength of 0.92 Å with 0.2-degree oscillations. Raw data were integrated and scaled using autoPROC (v.1.0.5)45 with XDS software package (Jun 2023, build 20230630)46, POINTLESS (v.1.12.10)47 and AIMLESS (v.0.7.7)48. The CN D90A :tpTAK1 structure was solved by molecular replacement (MR) using Phaser (v.2.8.3) in PHENIX (v.1.20.1-4487)49 with the structure of CN:A238L (PDB ID: 4F0Z21) as the search model. The CN E282K :NHE1 structure was solved by MR using Phaser in PHENIX with the structure of CN:NHE1 (PDB ID: 6NUC20) as a search model. The initial models of the complex were built using Phenix AutoBuild, followed by iterative rounds of refinement in PHENIX and manual building using Coot (v.0.9.8.7 EL)50. The A238L-TAK1 chimera peptide position in CN D90A :tpTAK1 was verified using PHENIX Polder maps51.

CN:RII. X-ray diffraction data were collected using a Bruker Metal Jet liquid gallium source (wavelength of 1.34 Å) with a photon III detector and the raw data were integrated and scaled using Bruker Proteum processing software, POINTLESS (v.1.11.16) and AIMLESS (0.7.3). The CN:RII structure was solved by MR using Phaser (v.2.8.3) in PHENIX (v.1.20.1-4487)49 with the structure of CN (PDB ID: 5SVE22) as the search model. The initial models of the complex were built using PHENIX AutoBuild, followed by iterative rounds of refinement in PHENIX and manual building using Coot (v.0.9.8.7 EL50).

ITC
ITC experiments were performed at 25°C using either a VP-ITC (Microcal), an Affinity SV ITC microcalorimeter (TA Instruments) or a Nano SV ITC microcalorimeter (TA instruments). All protein samples were dialyzed in ITC buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM CaCl2, 0.5 mM TCEP) prior to performing ITC. TAK1 variants were titrated into CN WT, CN E282K, CN Q284K, CN D285K, or CN 391 (for all ITC measurements CN 391 was used) Titrant (10 µL per injection) was injected into the sample cell over a period of 20 s with a 200–300 s interval between titrations to allow for complete equilibration and baseline recovery. 25 injections were delivered during each experiment, and the solution in the sample cell was stirred at 175 rpm or 350 rpm to ensure rapid mixing. Datasets were converted using NanoAnalyze (v.3.10.0) and the converted datasets were analyzed with a one set binding site model using NITPIC (v.1.2.252,53) and SedPhat (v.12.1b54). For each SedPhat evaluation the experimental parameters were set for the buffer used (HEPES buffer pH 7.5 at 25°C) and the cell concentration was corrected using the correction factor and fit local to account for small concentrations errors, as the less error-prone concentration is advised to be the one fixed55. The outlier data points were excluded from the analysis, and analysis was carried out using the model ‘A+B↔AB Hetero-Association’ and curve-fitting with Marquardt-Levenberg non-linear least-squares. Graphs were plotted using GUSSI (v.1.2.1)56 and all thermodynamic parameters reported are based on the average of three or more replicates.

Phosphorylation of substrates for dephosphorylation assays
TAK1 384–474 (S389A, S417A; S389 and S417 are non-specifically phosphorylated by PKA as detected by NMR spectroscopy) and variants were phosphorylated by PKA for 18 h at 37°C at a 1:50 ratio (PKA:TAK1) in phosphorylation buffer (100 mM Tris pH 7.5, 2 mM DTT, 10 mM MgCl2) and 2 mM ATP (Sigma). Phosphorylated TAK1 384–474 (S389A, S417A) was heat purified at 70°C for 15 min, and the soluble fraction was further purified using anion exchange chromatography (MonoQ 5/50 GL column [Cytiva]) column pre-equilibrated with TAK1 IEX buffer A1 (TAK1 WT: 20 mM Tris pH 8, 0.1 mM TCEP) or A2 (TAK1 variants: 20 mM HEPES pH 7, 0.1 mM TCEP) and eluted with a linear gradient of increasing concentrations of 0–80% TAK1 IEX buffer B1 (TAK1 WT: 20 mM Tris pH 8.0, 800 mM NaCl, 0.1 mM TCEP) or B2 (TAK1 variants: 20 mM HEPES pH 7, 800 mM NaCl, 0,1 mM TCEP). The fractions containing pTAK1 384–474 (S389A, S417A) WT and variants were pooled and dialyzed (20 mM Tris pH 8.0, 50 mM NaCl, 0.1 mM TCEP) at 4°C for 16 h to remove trace phosphate species. The final sample was concentrated, flash frozen and stored at −80°C to be used for dephosphorylation assays.

RII 44–103 (S58A, S78A; S58 and S78 are non-specifically phosphorylated by PKA as detected by NMR spectroscopy) and RII 44–103 V98R (S58A, S78A) were phosphorylated by PKA for 18 h at 37°C in a 1:10 ratio (PKA:RII) in phosphorylation buffer (100 mM Tris pH 7.5, 2 mM DTT, 10 mM MgCl2) with 500 µM ATP (Sigma). Phosphorylated RII (pRII) was concentrated and purified using SEC (HiLoad 26/600 Superdex 75) using RII SEC buffer (20 mM HEPES pH 7.0, 50 mM NaCl, 1 mM DTT). Fractions containing pRII were further dialyzed for 16–18 h at 4°C (20 mM HEPES pH 7.0, 50 mM NaCl, 1 mM DTT) to remove trace phosphate species. pRII was concentrated, flash frozen and stored at −80°C to be used for dephosphorylation assays.

Dephosphorylation assays
Assays were designed using PiColorLock 600 kit (Abcam) and performed in 96-well flat-bottom plates with low evaporation lids (Costar). Control wells were included to ensure absence of trace phosphate species, 3 negative control reactions were implemented: without enzyme/substrate, enzyme only (3 concentrations) and substrate only (blank). Buffer and enzymes were added to appropriate wells, and dephosphorylation reactions were initiated by the addition of phosphorylated substrate (pTAK1 or pRII). All reactions were carried out at 37°C for 30 min and quenched with Gold Mix mixed with Accelerator at 1:100 ratio, incubated for 5 min and mixed, followed by addition of Stabilizer. Measurements were carried out on a CLARIOStar plate reader (BMG Labtech) at 635 nm. A minimum of three replicates were run for each substrate/enzyme. Data was blanked against wells containing substrate only and normalized against wild type to calculate % activity. Graphs were plotted using GraphPad Prism (v. 10.2.2).

In vitro dephosphorylation assay
Asynchronous HeLa was lysed in lysis buffer (8 M urea, 50 mM Tris pH 8.1, 150 mM NaCl, 2 mM sodium beta-glycerophosphate, 2 mM sodium fluoride, 2 mM sodium molybdate, 1 mM sodium orthovanadate, mini-Complete EDTA-free protease inhibitor cocktail tablet). Protein concentration was determined by BCA assay (Pierce/Thermo Fisher Scientific). Proteins were then reduced with 5 mM DTT at 55°C for 30 min, cooled to room temperature, and alkylated with 15 mM iodoacetamide at room temperature for 45 min in the dark. Alkylation reactions were quenched with an additional 5 mM of DTT. The lysates were then diluted 6-fold with 25 mM Tris pH 8.1 before tryptic digestion with 1:100 (w/w) trypsin (Sigma) at 37°C overnight. The next day, digests were quenched by acidification with triofluoroacetic acid (TFA), centrifuged to remove precipitation, desalted, and peptides were lyophilized.

Phosphopeptide enrichment was carried out using a High Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fischer Scientific) following the manufacturer’s instruction and desalted. All phosphopeptides were combined and split into eleven equal portions. Phosphopeptides were resuspended in 166 mM EPPS pH 8.5 and the respective TMT reagent of the TMT-11 plex (Thermo Fischer Scientific), followed by incubation at room temperature for 1 h. A sample was removed to determine labeling efficiency, while the remainder was stored at −80°C. Labeling was confirmed to be at least 95% efficient before quenching with hydroxylamine. TMT-labeled phosphopeptides were separately desalted and stored in 60% methanol at −80°C. TMT-labeled phosphopeptides were aliquoted and speed vacuum to dryness before dephosphorylation. Phosphatases were added to in vitro phosphatase dephosphorylation buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM MnCl2, 1 mM MgCl2) before adding to dried phosphopeptides for dephosphorylation. All reactions were quenched at their respective time point with 1% TFA and stored at −20°C. Once all reactions were completed, they were combined before desalted and analyzed by LC-MS3. Triplicate dephosphorylation time series were performed for each phosphatase. TMT-labeled peptides were analyzed on an Orbitrap Lumos mass spectrometer (Thermo Fisher Scientific) equipped with an Easy-nLC 1200 (Thermo Fisher Scientific), and raw data was searched and processed as previously described41. All phosphopeptides detected were filtered for single phosphorylated peptides with a false localization rate <0.1%. Furthermore, only phosphopeptides that were detected and quantified in 2 out of 3 replicates were picked to proceed with further analysis. Pair-wise correlation coefficient values were calculated using the Excel formula CORREL. Subsequently, p-values were calculated using the Excel formula T.DIST.2T with t-score derived from correlation coefficient values. Duplicated quantifications with the lowest p-values were selected for further analysis. The phosphorylation ratio at each time point for each phosphopeptide was calculated by dividing the TMT quantification at that time point by the maximum TMT quantification of all time points for that peptide. Data were input into Perseus57 (v2.0.11.0) for further analysis. Unsupervised hierarchical clustering of phosphopeptides was performed using Euclidean distance. For each phosphatase, four clusters were defined based on their dephosphorylation kinetics, from fastest to non-regulated. For motif generation, the web based application IceLogo34 was used.

Phosphoproteomics analysis
Flag-tagged CN WT and CN E282K were stably expressed in HEK293T (ATCC CRL-3216) and N2A cells (ATCC CCL-131). After selection with G418, equal expression was confirmed by western blotting. Control, CN WT, and CN E282K HEK293T cells were lysed in lysis buffer, digested, phosphopeptide enriched, and TMT labeled as described above. After off-line separation58, peptide fractions were analyzed by LC-MS3 and raw data were searched and processed as previously described41. All phosphopeptides detected were filtered for single phosphorylated peptides with a false localization rate <0.25% and data were input into Perseus57 (v2.0.11.0) for T-test statistical analysis. For motif generation, the web based application IceLogo34 was used.

To identify CN SLiMs sites in proteins identified in the N2A phosphoproteomics data, we used the ScanProsite tool from the Prosite database59. For the search, the full-length protein sequence, and specific definitions of the PxIxIT and LxVP SLiM motifs were used. For the PxIxIT site search19, P-x-[FILV]-x-[FILV]-x was used as query; for the LxVP site33, [NQDESRTH]-[YRTDFILV]-[LIVF]-x-[VPLIHA]-x was used as query. Disorder prediction via IUPred360 was used to calculate the disordered score for the identified SLiMs to ensure that putative SLiM sites are in an IDR (IUPred score ≥ 0.4) based on a previous CN SLiM analysis22.

Immunoprecipitations
Flag-tagged CN WT and CN E282K were stably expressed in HEK293T and N2A cells. After selection with G418, equal expression was confirmed by western blotting. For immunoprecipitation, cells were lysed in lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl and 0.5% (w/v) Triton X-100, with an EDTA-free protease inhibitor tablet (Roche) per 10 mL of lysis buffer). Flag-tagged proteins were purified using Flag M2 affinity gel (Sigma) rotated end over end for 3 h at 4°C. The beads were washed five times with lysis buffer and eluted with 3X Flag peptide. Proteins were precipitated using the SP3 methods61 and digested overnight in 50 mM ammonium bicarbonate with trypsin (Promega) for mass spectrometric analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors characterize a variant of calcineurin (CN) in which glutamic acid at position 282 is substituted with lysine (E282K). Heterozygous occurrence of this variant has been associated with neurodevelopmental disorder. Crystal structure of the variant shows significant remodeling of the substrate binding pocket transforming an acidic cavity into a basic one with additional steric restriction. This explains a global shift in dephosphorylation profile of the variant from basic to acidic substrates as assessed with phosphoproteomics. The data defines and mechanistically explains the molecular impact of the variant on cellular phosphorylation pattern.\nMajor comments\n1. How do affinities of fragments of TAK1 compare to full length affinity? Are there any sites involved in binding other than LxVP and RRRS? The same goes for RII\n2. The effect of mutating sites i-2 and -3 in CN (Q284K, D285K, Y315A) is unexpected given the interactions observed in the crystal structure (increased dephosophorylation). How would the authors explain the increased activity of Q284K, D285K and Y315A variants? The authors should use their system to try to explain this phenomenon (for example by testing if mutating relevant arginines in TAK1 would have similar effect).\n3. E237K and E282K reduce activity of CN in dephosphorylating TAK1. Would this effect be reversed by mutating relevant basic residues in TAK1 to acidic residues?\n4. The variant 436RRVS439 should be characterized in the functional dephosphorylation assay. This concerns all the other variants where ITC affinities and dehosphorylation potential should be determined.\n5. Chapter “Arginine in the i-1 position is a general mechanism for enhanced CN substrate dephosphorylation” – contrary to the title, the data presented in this chapter does not show the generality of the arginine mechanism. A single example is provided in support of the proposed mechanism, but single example does not allow to conclude on generality. Omic analysis of substrates could possibly allow to conclude on generality of the proposed mechanism, but even than authors should reconcile how substrates like 96RRVS99 get dephosphorylated when they show that -2 and -3 sites are less relevant (or irrelevant) for recognition.\n6. “crystal structure of a substrate trapped CN complex” – the manuscript text states “tethered to the PxIxIT site using the A238L viral inhibitor peptide”, Figure 2a and the further manuscript text discuss binding at LxVP site. Please, clarify. Also, please clarify what “A238L viral inhibitor peptide” is, and detail in the main text on the design of the thiphosphorylated TAK1 and CN constructs that were used for crystallization.\n7. RII dephosphorylation – the difference in dephosphorylation rate for WT enzyme and E237K and 282K mutants is significant already for WT substrate. How would the authors explain this effect? In addition, it would be interesting to test if similar is true when TAK1 RRVS is used.\n8. “we determined the crystal structure of CNE282K“ – to my understanding this is not a structure of CNE282K alone, but in complex with NHE1. If so, this should be indicated in the sentence “we determined the crystal structure of CNE282K in complex with HNE1”. The text should also address the question why catalytically dead CN variant was necessary to crystalize with TAK1 while it was possible to crystallize HNE1 with active (I assume) CN?\n9. Is the (i-1) arginine containing structure exceptional or had there been other structures earlier (if so should be discussed in the introduction)\n10. My major comment pertains to the phosphoproteome study. The study on phosphopeptides isolated form HeLa cells clearly shows a shift in substrate preference in E282K variant of CN. The particular experiment provides a clear indication of the shift, but only allows to conclude on the shift in a completely nonphysiologically model. The substrates, though diverse, are short peptides and not full proteins – therefore most of the complexity of substrate recognition is lost. Secondly, HeLa cell proteome is largely irrelevant for CN, especially in the context of neurodevelopmental pathologies related to E282K and treatment with pan-PPP inhibitor likely induces significant non-physiological phosphorylation. Further, digestion with Lys-C removes all potential Lys sites (it would also be interesting to test if R(i-1) can be substituted with K using the peptide substrates from the initial part of the study (TAK1); it should be explicitly stated on the panel d in figure 5 that the assay is not capable of detecting lysine containing substrates).\nTo evaluate the substrate preference at more physiological conditions, authors overexpress the CN in 293T cells (is it in CN negative background?). This allows to assess intact proteomes (rather than over-phosphorylated peptide mixture), but the choice of cell line is irrelevant to CN and PPP3CA related DEE.\nIt would be beneficial if the authors could confirm their findings analyzing the effect of CN mutation on intact proteome in a cell line relevant to the major pathology, for example by comparative analysis of heterozygous neuronal cell line with E282 mutation compared to homozygous wt cell line, or similar, physiologically relevant setup which could provide clues as to the major imbalances in phosphorylation leading to the pathogenesis of the disease. Authors are strongly encourage to try to address this comment, however it may be difficult experimentally and thus should not preclude publication of this interesting manuscript.\nAs it is, the data does not substantiate the conclusion that “These data (…), highlights how a single point variant, CNE282K, globally alters the CN substrate recruitment and dephosphorylation.” as the recruitment and dephosphorylation of full length substrates in a cellular context relevant to PPP3CA related DEE may be significantly different.\n11. As it is now, the authors should tone down the conclusion stated in the title regarding global shift of substrate dephosphorylation profile. They should also tone down the claims that activity of the mutant have been studied in cells – it indeed was, but not in cells relevant to the pathology of interest.\nMinor comments;\n1. Abstract – if calcineurin is abbreviated CN in the text, this abbreviation should be the first one in the list of abbreviations given in the first sentence “(PP3CA; CN, Protein Phosphatase 2B [PP2B], PP3”).\n2. “To quantify the contribution of the CN residues for TAK1 recruitment and dephosphorylation, we mutated these residues and measured their ability to dephosphorylate TAK1 pSer439”. First part of the sentence – which residues?; CN has many residues and it is unclear what is meant here. Second part of the sentence – residues with “ability to dephosphorylate TAK1” implies active site (catalytic) residues. It is unlikely that this was meant in the sentence. The entire sentence should be corrected referring to specific residues and their functions.\n3. “The regulatory subunit B (CNB) binds four Ca2+ ions, making CN responsive to calcium signaling.” – A sentence above mentions that CNA has a calmodulin binding site which is likely to already “make CN responsive to calcium”. The role of calmodulin binding site mediated regulation and CNB regulation should be described in few sentences.\n4. It is mentioned that E282K is the most common mutation. It would be useful to map other known mutations resulting in similar clinical outcomes at the structure of CN and speculate as to their influence on the phosphoproteome. For example, is E237K a clinically relevant variant – one would expect so given the data shown in Figure 3b.\nIf excessive number of mutations is known, such discussion could be presented in detail in the supporting information.\n5. The functional sites within CN discussed in the text should all be depicted in figure 1a (ex. SLiM binding site, calmodulin binding region, auto inhibitory domain, calcium binding sites, etc.), either in 2D or 3D representation.\n6. The introduction states “Mutating either of these SLiMs abolishes their ability to be dephosphorylated by CN…”. But the tested substrate (TAK1 fragment) contains only the LxVP SLiM. This apparent discrepancy should be clarified (and Figure 1a updated accordingly if necessary)\n7. The authors mention several times that their results “lead to the development of novel routes for treating PPP3CA-related DEE”. The results are interesting and explain the molecular pathology in PPP3CA-related DEE, but I do not see how this knowledge might lead to a treatment. With the described functional mechanism it is difficult to imagine small molecular weight compounds restoring the specivicity of CN vairant while genetic correction does not require the knowledge gained through this study. Authors are kindly requested to elaborate on their view or remove the statements pertaining to influence of their results on treatment development.\n8. Figure 1a; TAK1; it is unclear what CN SLiM refers to (ie. which part of the protein)\n9. “CN counteracts the activities of many basophilic kinases, including PKA, and its sequence contains a putative LxVP motif (422LDVP425) near the PKA phosphorylation site,2” – “its sequence” points at CN in this sentence while it refers to TAK1. Please, rephrase.\n10. “TAK1 bound CN with an atypically strong affinity compared to other LxVPpeptide:CN interactions (TAK1 KD, 860 ± 23 nM; Fig. 1b; other LxVP KD’s, ~1800-500000 nM; Table S1),21,23,33” – please, clarify the nomenclature – LxVPpeptide seems to signify a peptide consisting only of LxVP. Additionally, summarize the data on LxVP KD’s in a supporting figure giving exactly the constructs and affinity determination methods used to determine the affinity and construct alignment if significant differences between the constructs are present.\n11. Figure 2f – please, indicate which residues constitute the -3, -2 sites and which build the -1 site.\n12. Use consistent labeling of residues (ie. Glu282 or E282) through the manuscript\n13. Please, use a common nomenclature throughout the manuscript. Why is CND90A:tpTAK1-tpS439 not simply called CND90A:tpTAK1 in Figure 2 legend? Especially that it has a different description in the figure title and panel legends.\n14. Figure 2, panel d – label E282 and other important residues at the figure\n15. Figure 2e – please suggest a more comprehensive depiction of ionic interactions. An ionic interaction is not an interaction of a particular atom in a COO- moiety as currently suggested by the figure. (ex. Asp285-Arg(-2) or Glu237-Arg (-1)).\n16. Figure 4b legend – make clear that both the structure of wt and mutant were determined in complex with HNE1. As currently is, it is not clear if CN(E282K) apo is not compared to WT with HNE1.\n17. In the final part of the “results” section, which concludes the findings, I am missing an important sentence, that is that E282K shifts the overall substrate preference of CN (the major conclusion of the study).\n18. “Because PPPs are metalloenzymes, with the residues that coordinate the metal binding being 100% conserved between the family members, this enzyme family, unlike many kinase families, mostly lacks specificity at the active site.35,36” – many enzyme classes will have identical catalytic residues (ie. ion coordination in this particular case) while at the same time particular representatives of the class may largely differ in substrate preference because they differ in residues directly adjacent to catalytic residues which adjacent residues will modulate specificity. Please, rephrase this fragment – active site is broader term than the catalytic residues.\n19. “This CN active site recruitment is sufficiently enhanced that it enabled the structure of CN bound to a PKA phosphorylated substrate to be determined.” – please rephrase. The substrate contained additional tether.\n20. “by creating a peptide library from PPP-inhibited HeLa cells and HEK293T cells expressing CNWT and CNE282K“ – please, be more exact. The peptide library in HeLa experiment was prepared at a very different stage of experiment and served a different purpose than in 239T so should not be combined in a single sentence.\n21. “active site recruitment is necessary for a broad range of peptide sequences” – meaning unclear – please, rephrase.\n22. “…but, as we show, globally alters substrate preference from basophilic to acidophilic both in vitro and in cells.” – indicate that the cells used in the experiment are not relevant to physiological role of CN in the clinical pathology.\n23. “These results not only provide a molecular basis for altered signaling in patients with the E282K variant” – tone down. The results do not provide any data on the influence of E282K on signaling in a setup relevant to pathology.", "reviewer_2": "Overall summary of manuscript: This manuscript provides a comprehensive study of the calcineurin E282K variant, a mutation most frequently associated with PPP3CA-related developmental and epileptic encephalopathy. The authors identify a novel acidic substrate recruitment pocket (termed the E282 pocket) adjacent to the calcineurin active site that preferentially interacts with basophilic substrate motifs, particularly those with an arginine at the i-1 position. Using a combination of X-ray crystallography, NMR spectroscopy, biophysical assays, in vitro dephosphorylation, and phosphoproteomics, the authors demonstrate that the E282K mutation impairs the ability of calcineurin to recruit and dephosphorylate these substrates, resulting in a shift in substrate preference from basic to acidic motifs. This mechanistic insight provides a plausible molecular explanation for how the E282K mutation contributes to altered calcineurin signaling.\nStrength of manuscript: The manuscript leverages structural (X-ray, NMR), biochemical (ITC, activity assays), and global phosphoproteomics approaches, which collectively provide strong and converging evidence for the discovery and functional significance of the E282 substrate recruitment pocket. Also, the identification of the E282 pocket as a determinant of substrate specificity, particularly the preference for Ri-1 containing basophilic substrates, is novel and will advance the understanding of calcineurin substrate recognition. Finally, the work has implications for understanding the pathophysiology of PPP3CA-related disorders and provides a molecular rationale for the consequences of the E282K mutation, offering a clear foundation for translational follow-up studies focused on therapeutic targeting.\nWeaknesses and Major Concerns:\n1) Lack of Functional Cellular/Phenotypic Assays to Link Biochemistry to Disease Phenotypes\nIssue - While the phosphoproteomics and structural biology support the biochemical consequences of the E282K mutation, the manuscript lacks a functional readout that links these molecular changes to cellular phenotypes relevant to neurodevelopment or epilepsy (e.g., NFAT signaling, calcium flux, neuronal morphology, or activity, etc). This would help establish a causal link between altered substrate specificity and disease-relevant cellular dysfunction.\n2) Limited Exploration of the Broader Substrate Repertoire Affected by E282K - The manuscript demonstrates that Ri-1-containing substrates are affected, but the scope of calcineurin substrates modulated by this mechanism remains incompletely characterized. A deeper analysis of the phosphoproteomics data is needed, such as emphasizing which peptide substrates correspond to known, well characterized calcineurin substrates whose dephosphorylation is impaired in the E282K context, would strengthen the manuscript and validate the peptide phosphatase assay approach.", "reviewer_3": "The manuscript by Shirakawa et al. discusses how calcineurin (CN) recognizes its substrates. CN is known to use short recognition motifs to anchor to substrates. In this original study, the authors discovered a new binding pocket adjacent to the phosphatase active site that binds the phosphorylated motif. Besides the fundamental importance of this finding, it enables the authors to explain the effect of the E282K clinical mutation, which leads to rare neurodevelopmental disorders. Using X-ray structures of CN and CNE282K with different substrates, the authors demonstrate how an arginine residue at the -1 position of the phosphorylation site enables fast dephosphorylation by facilitating substrate recruitment. CN mutations coupled with activity assays and nuclear magnetic resonance (NMR) spectroscopy validated the structure-based hypothesis. Based on this, the authors performed a proteomic analysis to define a consensus sequence for substrate recognition at this new pocket. Interestingly, the consensus sequence shifts with the E282K mutation.\nThis is a very thorough and elegant study. The manuscript is well illustrated, clearly written, and the experiments appear to have been performed to the highest standards.\nI still have a few concerns and suggestions.\nI agree that the NMR experiments suggest CN E282K binds to TAK1 RRRS less efficiently. However, it would be interesting to quantify this effect by using ITC, as presented in Table S1, with the mutated kinase. Similarly, it is important to validate the RII V to R mutation with an additional Kd measurement. In the current version, we only have activity data in Figure 3.\nI would also like to know if the enhanced activity of mutants Q284K and D285K in Figure 2F is linked to increased binding, i.e., by using ITC.\nThe data on enhanced activity for mutants Q284K and D285K are barely discussed. They seem to indicate that residues R-2 and R-3 are not favorable for enzymatic activity. However, the authors discuss the PKA consensus RRxS/T as matching the CN dephosphorylation of basic substrates. Additionally, the consensus sequence in Fig. 5f shows a proline residue at the +1 position, indicating a preference for phosphorylation catalyzed by proline-directed kinases. This should be at least addressed in the discussion.\nConsensus sequences are proposed in Figures 5f and g. It would be interesting to see if the peptide sequences with the highest probability are indeed dephosphorylated faster or slower in an enzymatic assay with CN and CNE282K.\nMinors:\nFigure 2C: The colors in the legend for RIIa/NFATc1 do not seem to match the colors in the figure (red, blue, yellow, and red).\nA reference to Fig. 2C could be made in the paragraph on the structure of CNE282K (together with NHE1).\nThis paragraph does not present the phosphorylation motif of NHE1. I guess it is not an R, but I would like it to be stated.\nIn Fig. 4 E, F, and G, the E282/E237 sticks between the two compared structures are difficult to distinguish. Perhaps using different colors would help?"}, "author_response": "We thank the reviewer for her/his careful review and the strong support for our presented data.\nMajor comments\n1. How do affinities of fragments of TAK1 compare to full length affinity? Are there any sites involved in binding other than LxVP and RRRS? The same goes for RII\nWe thank the reviewer for her/his question. The reviewer is correct that it is important to ensure that when shorter protein constructs are used in a study, they include all interactions of interest of the longer (full) construct. This is exactly what we have done. Namely, we confirmed that the TAK1 construct that we use throughout the manuscript includes all interactions of interest of the full-length TAK1 C-terminus. The data are described in Supplementary Figure 1, which shows both NMR and ITC data (see also Supplementary Table 1 for details on the ITC data). The ITC data show that the long and short C-terminal fragment of TAK1 bind with similar K s. The NMR data shows a nearly perfect overlap, demonstrating that the shorter TAK1 construct has the same conformation in solution as the longer TAK1 construct. Thus, based on the combined data, the residues described for the TAK1:CN interaction in the manuscript are the only ones identified to interact with CN and no residues outside of this region make significant contributions to binding.\nRII is the dimerization and docking domain of the PKA regulatory subunit. It has an N-terminal helical domain (residues 1-43), an intrinsically disordered domain (residues 44-145) and two C-terminal cAMP binding domains (residues 145-379). Dimerization occurs via the N-terminal helical domain, which forms two parallel helices that bind and recruit the C-terminal helix from the scaffolding protein AKAP5/79. There is no evidence RII (residues 1-43) bind CN. Residues 44-103 form an extended ‘RII peptide’ that has been used for years as a substrate for testing CN activity (e.g. a ~15 aa shorter version is commercially available from Enzo BML-AK816-0001 or other companies). We tested different RII constructs to design the most reliable CN substrate, including RII 1-103 and RII 44-103. The protein stability for RII 1-103 is much lower than RII 44-103, resulting in lower quality (less reproducible) CN activity assays. It has been previously shown that the folded RII dimerization domain is in intermediate exchange on the NMR timescale, and the quality of the resulting NMR data is very low (this has also been previously reported; see Embo J. 2001;20:1651–1662). After careful optimization, we selected RII 44-103 as the optimal construct, as it allows for rigorous and reproducible data in our interaction studies and in CN assays.\n2. The effect of mutating sites i-2 and -3 in CN (Q284K, D285K, Y315A) is unexpected given the interactions observed in the crystal structure (increased dephosophorylation). How would the authors explain the increased activity of Q284K, D285K and Y315A variants? The authors should use their system to try to explain this phenomenon (for example by testing if mutating relevant arginines in TAK1 would have similar effect).\nWe thank the reviewer for this comment. We realized that some of these concerns may have arisen from our imperfect display of our structural results. We did not accurately distinguish the interactions between TAK1 and CN between those with strong (<4 Å) and weak (>4 Å) interactions. Thus, we updated Figure 2 to only highlight polar/ionic interactions that are <4 Å (i.e. the most significant interactions); we also added the distances to the figure to further improve clarity. These criteria show that the key interactions are made between the TAK1 Ri-1 and CN E282 and the TAK1 Ri-2 and CN Q284 (the latter includes a backbone hydrogen-bond, which will be present independent of any mutation made to Q284). While electrostatic/polar interactions can also be longer than 4 Å, their impact on binding is much lower, as electrostatic and polar interactions weaken as the distance increases.\nAs requested by the reviewer, we also performed additional experiments. First, we performed ITC measurements between TAK1 and CN or CN , which showed little change to the values reported for CN . This contrasts with the data for TAK1 with CN which showed a 3-fold weaker K by ITC. This supports the central importance of the E282 binding pocket for CN substrate recruitment. Second, we generated new TAK1 variants and measured their dephosphorylation by CN. TAK1 variants with either the i-2 or i-3 arginine residue changed to an alanine were generated (we cannot change both at the same time, as at least one arginine is required for PKA phosphorylation). The dephosphorylation assays showed that the Ri-3 TAK1 variant (RRRS → ARRS) behaves essentially identical to wt-TAK1 (same reduction in dephosphorylation with CN vs CN ), with the Ri-2 variant (RRRS → RARS) slightly more effectively dephosphorylated with CN vs CN (likely due to the observed polar interactions with Ri-2 and the Q284 sidechain/backbone). Together, these data further confirm the key role of the R-1 position residue for TAK1 binding and dephosphorylation.\nThese data have been added to the revised manuscript (Figure 2, Supplementary Figure 4, Figure 4). We thank the reviewer for these suggestions, which further strengthened the results of our work.\n3. E237K and E282K reduce activity of CN in dephosphorylating TAK1. Would this effect be reversed by mutating relevant basic residues in TAK1 to acidic residues?\nAs requested, we created TAK1 RR-D/E-S variants and tested the dephosphorylation efficiency for CN . The data show that both variants are more efficiently dephosphorylated by CN than wt-TAK1.\nThese data have been added to the revised manuscript (Figure 4g). We thank the reviewer for this suggestion, which further strengthens the results of our work.\n4. The variant 436RRVS439 should be characterized in the functional dephosphorylation assay. This concerns all the other variants where ITC affinities and dehosphorylation potential should be determined.\nAs requested, we have performed dephosphorylation assays of the TAK1 RR-V-S variant vs CN , which showed the expected outcome.\nThese data have been added to the revised manuscript (Figure 4g). We thank the reviewer for this suggestion, which further strengthens the results of our work.\n5. Chapter “Arginine in the i-1 position is a general mechanism for enhanced CN substrate dephosphorylation” – contrary to the title, the data presented in this chapter does not show the generality of the arginine mechanism. A single example is provided in support of the proposed mechanism, but single example does not allow to conclude on generality. Omic analysis of substrates could possibly allow to conclude on generality of the proposed mechanism, but even than authors should reconcile how substrates like 96RRVS99 get dephosphorylated when they show that -2 and -3 sites are less relevant (or irrelevant) for recognition.\nWe agree with the reviewer that a single example is not enough to test (or claim) generality. It is for this reason that we expanded from TAK1 to RII. RII is a canonical CN substrate (likely the substrate used most often to confirm CN activity). We tested if substituting the RII Vi-1 residue to an Ri-1 residue would enhance dephosphorylation efficiency. It did, and this is what we reported in the submitted manuscript (Figure 3). Next, as suggested by the reviewer, we also performed Omic analysis, first leveraging an in vitro peptide analysis/assay and second a cellular analysis using HEK293T and now also N2A cells (please see below).\nThe reviewer is concerned (asking) why wt RII, which has a RRVS dephosphorylation sequence, is still dephosphorylated by CN. Clearly, RRVS sequences are still viable CN dephosphorylation sites, they are just not as effectively/efficiently dephosphorylated as RRRS sites (even in the title, we highlight that is an enhancement and not an on/off switch). A key aspect of CN dephosphorylation is that CN substrates must first be recruited to CN via SLiM motifs (PxIxIT and/or LxVP). It is only after recruitment that active site recognition sequences can enhance dephosphorylation. For RII:CN, RII is recruited to CN via its LxVP motif leading to wt-RII dephosphorylation (shown in Figure 3). However, while wt-RII is clearly dephosphorylated by CN, this dephosphorylation is enhanced when the Vi-1 is mutated to Ri-1.\nThus, in a scenario where a CN substrate with a single LxVP motif yet multiple phosphorylated residues (the most studied example is the NFATs), our data shows that the RRRS phosphosites, versus RRVS phosphosites, will be more rapidly dephosphorylated. Using similar experiments, we previously discovered that S/TxxP motifs are also preferentially dephosphorylated by CN (see Nature Communication. 2019 Aug 2;10(1):3489).\nRegarding the reduced importance of the i-2/i-3 sites: the reviewer is correct these sites are not important for enhanced substrate recruitment to CN (shown by additional ITC and activity assays, Figures 4 and S4). However, these arginine residues do have an essential biological role as they are critical for the preceding event: phosphorylation. That is, PKA is an Arg-directed kinase, with a consensus sequence of R-R-x-S/T; i.e., the i-2/i-3 arginines ensure these TAK1/RII sequences are phosphorylated by PKA.\nWe hope this explanation and data clarify the issues at hand – we have added a sentence to the discussion to make this clearer. Thank you for pointing out that additional information will be helpful for the general reader.\n6. “crystal structure of a substrate trapped CN complex” – the manuscript text states “tethered to the PxIxIT site using the A238L viral inhibitor peptide”, Figure 2a and the further manuscript text discuss binding at LxVP site. Please, clarify. Also, please clarify what “A238L viral inhibitor peptide” is, and detail in the main text on the design of the thiphosphorylated TAK1 and CN constructs that were used for crystallization.\nWe thank the reviewer for highlighting that additional information will be helpful for clarity. First, we want to point out that immediately after the statement “tethered to the PxIxIT site using the A238L viral inhibitor peptide” is a reference to Supplementary Figs. 3a,b, which provide additional information. In addition, reference 21 refers to our manuscript that describes the structure of CN in complex with the potent (K = 4 nM) protein regulator A238L, which is a protein from the African swine fever virus.\nDuring the last 20 years, it has become apparent that CN crystallizes much more readily when one or both of its SLiM sites is/are occupied (i.e. bound to a regulator/substrate). By design, substrates typically engage weakly at enzyme active sites (otherwise they would be inhibitors). Thus, to ‘trap’ substrates at active sites, different strategies are used. One of the most common is to artificially tether the substrate, i.e. via their C/N-terminus, to the enzyme to increase their ‘local concentration’. We did not want to use an artificial tethering approach but rather leveraged a ‘biologically existing tether’: a modified A238L peptide which contains the TAK1 sequence starting from the LxVP site (as described in detail in the figure caption of Supplementary Figure 3 as well as in the methods section – see A238L-TAK1 chimera in cloning [where we also provide its full aa sequence] and expression and purification). This tethering has the key advantage that we know how A238L binds to CN and thus fitting the new electron density due to the RRRS motif is straightforward and is much less likely to be misinterpreted. We hope this explanation clarifies the rational as well as highlights all the details provided in the methods and figure legends of the manuscript.\n7. RII dephosphorylation – the difference in dephosphorylation rate for WT enzyme and E237K and 282K mutants is significant already for WT substrate. How would the authors explain this effect? In addition, it would be interesting to test if similar is true when TAK1 RRVS is used.\nThe reviewer is correct pointing out that there is a dephosphorylation rate difference for RII between CN and CN and CN . This was not surprising for us as the different conformation of TAK1 in complex with CN in the two molecules in the unit cell already highlighted that the interaction is dynamic (fuzzy; we also highlight this explicitly in the manuscript). Thus, the substrate can ‘move’ and adapt to the optimal position for dephosphorylation, as observed in the presented data. While we were able to visualize a subset of positions that TAK1 can adopt when bound to CN – there are likely additional conformations that are not stable enough (populated enough) to be seen in the crystal structure. Furthermore, there are likely even more degrees of freedom for the interaction in the variants that also show reduced binding to TAK1. Finally, as already shown in response to comment 4, we have now also performed a dephosphorylation assay for the TAK1 RR-V-S variant vs CN . These data have been added to the revised manuscript (Figure 4).\n8. “we determined the crystal structure of CNE282K“ – to my understanding this is not a structure of CNE282K alone, but in complex with NHE1. If so, this should be indicated in the sentence “we determined the crystal structure of CNE282K in complex with HNE1”. The text should also address the question why catalytically dead CN variant was necessary to crystalize with TAK1 while it was possible to crystallize HNE1 with active (I assume) CN?\nThe reviewer is correct that the crystal structure of CN was determined in complex with NHE1. As highlighted in our response to point 6 – CN crystallizes more readily when its SLiM binding pockets are populated. We crystallized CN bound to NHE1 not only to facilitate its crystallization but to also confirm that its SLiM binding pockets are not negatively impacted by the E282K mutation. The sentence in the manuscript highlighted by the reviewer reads as follows: “we determined the crystal structure of CN (Fig. 4b; bound to NHE1, a substrate that binds CN using a PxIxIT and LxVP motif).” This directly reports that we determined the structure of CN in complex with NHE1 – we try to be as transparent as possible with all of our work. All experimental details, including the full sequences, are also provided in the methods section.\nThe reviewer wants to know why we had to use a catalytically inactive variant of CN to get the structure of TAK1 – a substrate – bound to CN, while it was not necessary for NHE1. For TAK1 we trapped the substrate bound to the CN active site using a (thio)phosphorylated substrate; if we had formed the complex with active CN, the substrate would have been dephosphorylated, and we would not have been able to observe the interactions made at the CN active site between a phosphorylated substrate and CN. Indeed, to prevent dephosphorylation – we used multiple ‘tricks’, including using the inactive CN variant and thiophosphorylating the substrate (even thiophosphorylated substrates are slowly dephosphorylated).\nWhile NHE1 is a CN substrate, the construct we used for crystallization lacks the dephosphorylation residues (these are 50+aa beyond the CN SLiM interaction motifs) and only includes the SLiM interaction motifs. Here, NHE1 was solely used to stabilize CN for crystallization and thus there was no need to use a catalytically inactive variant of CN.\n9. Is the (i-1) arginine containing structure exceptional or had there been other structures earlier (if so should be discussed in the introduction)\nTo the best of our knowledge the (i-1) arginine containing structure is exceptional. To the best of our knowledge, this is also the first CN structure with any substrate bound to the active site. We have refrained from using ‘first’ throughout the manuscript, as this is generally not accepted per most journals’ policies.\n10. My major comment pertains to the phosphoproteome study. The study on phosphopeptides isolated form HeLa cells clearly shows a shift in substrate preference in E282K variant of CN. The particular experiment provides a clear indication of the shift, but only allows to conclude on the shift in a completely nonphysiologically model. The substrates, though diverse, are short peptides and not full proteins – therefore most of the complexity of substrate recognition is lost. Secondly, HeLa cell proteome is largely irrelevant for CN, especially in the context of neurodevelopmental pathologies related to E282K and treatment with pan-PPP inhibitor likely induces significant non-physiological phosphorylation. Further, digestion with Lys-C removes all potential Lys sites (it would also be interesting to test if R(i-1) can be substituted with K using the peptide substrates from the initial part of the study (TAK1); it should be explicitly stated on the panel d in figure 5 that the assay is not capable of detecting lysine containing substrates).\nTo evaluate the substrate preference at more physiological conditions, authors overexpress the CN in 293T cells (is it in CN negative background?). This allows to assess intact proteomes (rather than over-phosphorylated peptide mixture), but the choice of cell line is irrelevant to CN and PPP3CA related DEE.\nThe reviewer is correct with her/his statements and summarizes effectively our intended design. We used HeLa cells to generate a phosphorylated library of peptides to assay CN vs CN active site dephosphorylation preferences. Our use of short peptides minimizes the chance that any CN-specific SLiMs are present in the peptides – this is by design, as the presence of SLiMs would reduce the significance of the observed active site motifs and thus would be counterproductive. We selected HeLa cells because they are easy to work with, allowing the ‘general’ phosphopeptide library to be readily generated. In short, the goal of this study was to use the HeLa-generated phosphorylated library of peptides to determine if/how the active site substrate preference shifts because of the E282K mutation (i.e., CN to CN ).\nThe author is also correct that next we tested dephosphorylation changes in HEK293T cells. While we agree that HEK293Ts are not perfect cell lines, ≥90% of all reported MS data is from HEK293T experiments, enabling us to compare our data with existing, comprehensive databases.\nBut we also agree with the reviewer that one should always try to provide data from cell lines relevant to the system under investigation. Thus, in response to the reviewer, we now also performed the MS experiments using N2A cells. N2A cells are a commonly used mouse neuronal cell line. The N2A results show a similar trend as the HeLa phosphorylated peptide library and the HEK293T MS data. All these data, including those from HEK293T and N2A cells, are included in this revised manuscript (Figure 6 and Supplemental Figures).\nLastly, the reviewer comments that we should also test if Lys can substitute for Arg in the -1 position. To answer this question, we generated a TAK1 RR-K-S variant and measured its dephosphorylation. As expected, dephosphorylation is similar to wt-TAK1 (RR-R-S) and further confirms our results (Figure 4).\nThese data have been added to the revised manuscript. We thank the reviewer for these suggestions, which further strengthened the conclusions of our work.\nIt would be beneficial if the authors could confirm their findings analyzing the effect of CN mutation on intact proteome in a cell line relevant to the major pathology, for example by comparative analysis of heterozygous neuronal cell line with E282 mutation compared to homozygous wt cell line, or similar, physiologically relevant setup which could provide clues as to the major imbalances in phosphorylation leading to the pathogenesis of the disease. Authors are strongly encourage to try to address this comment, however it may be difficult experimentally and thus should not preclude publication of this interesting manuscript.\nWe fully agree with the reviewer and indeed we are working on such experiments. CN iPSC E282K cells are currently being created by the Simmons foundation, and we are also creating a heterozygous CN mouse. Clearly, we plan to use these tools in additional experiments in the future (2026-2030), including the reviewer’s requested experiments. These tools will be made fully available to the research community for further testing in future experiments. However, as the reviewer highlights, these experiments are difficult, very time-consuming and, in our case, ongoing. We very much appreciate that the reviewer highlights that these efforts should ‘not preclude publication of this interesting manuscript’.\nWhat we have been able to do, and as discussed above, is perform the MS experiments using a mouse neuronal cell line (N2A); these results have been added to the revised manuscript.\nAs it is, the data does not substantiate the conclusion that “These data (…), highlights how a single point variant, CNE282K, globally alters the CN substrate recruitment and dephosphorylation.” as the recruitment and dephosphorylation of full length substrates in a cellular context relevant to PPP3CA related DEE may be significantly different.\nWe are not certain that we fully understand these concerns of the reviewer and, in particular, why the reviewer would think that the recruitment of full-length substrates might be different in cells. Data from many groups (including ours) have shown that SLiM-based substrate recruitment is both necessary and sufficient for the cellular fidelity observed in cells. This has been shown not only for CN, but also for a plethora of other signaling enzymes, including kinases and phosphatases. If the reviewer refers to the fact that SLiM residues can be modified by post-translational modification (PTM) themselves, and thus modify signaling outcomes, that is certainly correct and another layer of the complex signaling network. However, at this point, a PTM that modifies a CN SLiM has not been reported.\nTaken together, our data highlights that there is a different active site preference for substrate dephosphorylation between CN and CN . If recruitment of a CN substrate via CN SLiMs to CN is identical (as we show in our crystal structures), dephosphorylation of Ri-1 containing substrates will be slower in CN than for wt-CN; these differences in dephosphorylation rates/preferences can/do alter CN signaling networks.\nIf the reviewer highlights that, at this point, we have not identified all relevant substrates that are changed – that is 100% correct. This is indeed a very important and complex task that we will pursue in the next (likely 10) years. What makes this even harder is the fact that we simply do not have a very good understanding of the CN interactome/proteome (for that matter this is true for all ser/thr protein phosphatases). As already highlighted, we are producing the CN mouse to test for signaling differences – however, because of this study, we will initially focus on Ri-1 substrates. We hope that these future studies will indeed identify substrates that have a changed phosphorylation profile in CN vs wt-CN that might lead to new treatments for one or more secondary conditions (pain/epilepsy/others) of this disease. Clearly this will take years to come to full fruition.\nOverall, we present work that spans molecular biology to biochemistry to structural biology to cellular MS work. This comprehensive picture allows us, and many others, to leverage these data and provide the necessary next steps for concrete strategies and hypotheses for helping PPP3CA DEE91 patients. This is an open problem and an opportunity to be tackled in the next 5-10 years.\n11. As it is now, the authors should tone down the conclusion stated in the title regarding global shift of substrate dephosphorylation profile. They should also tone down the claims that activity of the mutant have been studied in cells – it indeed was, but not in cells relevant to the pathology of interest.\nWe thank the reviewer for her/his input. Indeed, our MS studies have been done in HEK293T and now also neuronal N2A cells; thus, we have studied the activity of the CN variant in a biologically relevant cell line. Many published MS studies have been performed in HEK293T cells. N2A is a mouse neuronal cell line, and we have now repeated the MS analysis and report on the outcome.\nWe agree with the reviewer that the next logical step will be a (or multiple) mouse model(s) to study the effects of this CN variant further. While this is beyond our current scope, it clearly does not change the impact of the conclusions of our existing, comprehensive data. Indeed, I would argue that all future studies will dramatically gain from these detailed molecular biochemical and MS analyses, which are missing from many studies that start with mouse models. We have edited the manuscript to ensure the tone is appropriate for the results.\nMinor comments;\n1. Abstract – if calcineurin is abbreviated CN in the text, this abbreviation should be the first one in the list of abbreviations given in the first sentence “(PP3CA; CN, Protein Phosphatase 2B [PP2B], PP3”).\nThank you – changed as requested.\n2. “To quantify the contribution of the CN residues for TAK1 recruitment and dephosphorylation, we mutated these residues and measured their ability to dephosphorylate TAK1 pSer439”. First part of the sentence – which residues?; CN has many residues and it is unclear what is meant here. Second part of the sentence – residues with “ability to dephosphorylate TAK1” implies active site (catalytic) residues. It is unlikely that this was meant in the sentence. The entire sentence should be corrected referring to specific residues and their functions.\nThank you – we have added new data and changed this sentence/paragraph to clearly highlight which residue is referred both on TAK1 and CN.\n3. “The regulatory subunit B (CNB) binds four Ca2+ ions, making CN responsive to calcium signaling.” – A sentence above mentions that CNA has a calmodulin binding site which is likely to already “make CN responsive to calcium”. The role of calmodulin binding site mediated regulation and CNB regulation should be described in few sentences.\nMuch of this basic knowledge about CN has been described in reviews, which are cited throughout the manuscript. However, some of the details regarding CN activation by Ca2+ have not yet been explored at molecular detail. In particular, the role of Calmodulin binding to CN and the mechanism of its subsequent activation has been not well studied nor well-defined. It is assumed that Calmodulin binding releases the autoinhibitory domain (AID); however, to the best of our knowledge, how this is achieved has not been experimentally shown. Ca2+-binding to CNB ensures that LxVP SLiMs can bind to CN. We would prefer to leave the introduction as is and focus on the new insights gained in the manuscript (multiple foundational manuscripts and reviews are referenced for further information in the introduction). But the reviewer is correct we should indeed revisit Ca2+-activation of CN and develop a detailed model in vitro.\n4. It is mentioned that E282K is the most common mutation. It would be useful to map other known mutations resulting in similar clinical outcomes at the structure of CN and speculate as to their influence on the phosphoproteome. For example, is E237K a clinically relevant variant – one would expect so given the data shown in Figure 3b.\nIf excessive number of mutations is known, such discussion could be presented in detail in the supporting information.\nThere are several published manuscripts that report on clinical CN variants – these are carefully cited in the manuscript. However, beyond these published reports, we are in contact with the group of families that founded the PPP3CA Hope Foundation, which aims to collect clinical data from families around the world. This has been (partially) successful, but that data has not been made public. As said, we are working with them and are urging them to make the data available – even if only posted on their webpage. Nevertheless, the literature described variant data is very accurate and cited throughout our manuscript.\nTo the best of our knowledge, E237K is not a clinical variant; alternatively, if it is a clinical variant, it has not yet been identified or reported.\n5. The functional sites within CN discussed in the text should all be depicted in figure 1a (ex. SLiM binding site, calmodulin binding region, auto inhibitory domain, calcium binding sites, etc.), either in 2D or 3D representation.\nThe reviewer is correct to point this out – but indeed this is much harder to achieve than a linear SLiM sequence on an IDP. Binding surfaces are 3-dimensional in a folded protein (e.g. the LxVP site includes residues from CNA and CNB etc.). Thus, this is hard to show in the depiction in Figure 1a. But the PxIxIT and LxVP sites are highlighted on the 3D structure of CN both in Figure 4 and in Supplemental Figures.\n6. The introduction states “Mutating either of these SLiMs abolishes their ability to be dephosphorylated by CN…”. But the tested substrate (TAK1 fragment) contains only the LxVP SLiM. This apparent discrepancy should be clarified (and Figure 1a updated accordingly if necessary)\nThe introduction provides a general concept for CN. Most regulators and substrates indeed have both SLiM sequences and the statement is correct. The reviewer is correct that TAK1 only has a LxVP site. We hope this clarifies the statement.\n7. The authors mention several times that their results “lead to the development of novel routes for treating PPP3CA-related DEE”. The results are interesting and explain the molecular pathology in PPP3CA-related DEE, but I do not see how this knowledge might lead to a treatment. With the described functional mechanism it is difficult to imagine small molecular weight compounds restoring the specivicity of CN vairant while genetic correction does not require the knowledge gained through this study. Authors are kindly requested to elaborate on their view or remove the statements pertaining to influence of their results on treatment development.\nThe reviewer is correct that it is unlikely that a small molecular weight compound will be effective in treating all the complications associated with a heterozygous CN . However, it is important to know that treatments can also mean simply treating one or more of the underlying associated complications (epilepsy, pain, others). Our work sets the much-needed stage for developing such strategies. We will perform additional MS experiments using iPSC patient derived cells (hopefully by mid-2026) – such as the ones now reported for N2A cells to specifically start to understand proteins that are the target of CN and differentially regulated in CN . Over the next years we will then test these findings/substrate changes in vitro and in cells. Furthermore, and as already highlighted, we are creating the heterozygous CN mouse (hopefully available also in mid-2026 for experiments). Based on our data we will focus on CN substrates with R-1 signatures (and expanded signatures that are summarized in Fig. 5 and 6). As done by many others working on rare diseases, we hope to identify specific signaling pathways that are influenced by the CN variants, which may be regulated by enzymes that already have FDA approved drugs, and their use in PPP3CA-related DEE91 patients might be possible.\nClearly this approach is not unique to CN but is also actively being used for a variety of rare diseases that arise due to spontaneous mutations in signaling enzymes. The advantage for the CN community is that we defined how this mutation changes CN function and signaling, which our community can now leverage in future analyses.\n8. Figure 1a; TAK1; it is unclear what CN SLiM refers to (ie. which part of the protein)\nWe thank the reviewer for this question: Indeed, CN SLiM does not refer to any part of the protein, but rather CN SLiM indicates the two SLiMs highlighted next to it – the LxVP and RRRS site in TAK1. Based on the reviewers’ feedback, we can see that this might be easily misunderstood and deleted CN SLiM from the figure to improve clarity – thank you.\n9. “CN counteracts the activities of many basophilic kinases, including PKA, and its sequence contains a putative LxVP motif (422LDVP425) near the PKA phosphorylation site,2” – “its sequence” points at CN in this sentence while it refers to TAK1. Please, rephrase.\nThank you – we changed the sentence to:\nThe amino acid sequence of TAK1 contains a putative LxVP motif (422LDVP425) near the PKA phosphorylation site,22 therefore we tested if TAK1 binds CN using isothermal titration calorimetry (ITC).\n10. “TAK1 bound CN with an atypically strong affinity compared to other LxVPpeptide:CN interactions (TAK1 KD, 860 ± 23 nM; Fig. 1b; other LxVP KD’s, ~1800-500000 nM; Table S1),21,23,33” – please, clarify the nomenclature – LxVPpeptide seems to signify a peptide consisting only of LxVP. Additionally, summarize the data on LxVP KD’s in a supporting figure giving exactly the constructs and affinity determination methods used to determine the affinity and construct alignment if significant differences between the constructs are present.\nThank you: to clarify the issue, we deleted the word other and replaced it with reported. The sentence now reads:\nTAK1 bound CN with an atypically strong affinity compared to reported LxVPpeptide:CN interactions (TAK1 KD, 860 ± 23 nM; Fig. 1b; other LxVP KD’s, ~1800-500000 nM; Table S1),21,23,33 suggesting that residues outside the LxVP motif also contribute to binding.\nThe highlighted references provide ITC or SPR measurements for LxVP SLiMs in proteins and peptides. In particular, reference 33 reports on several LxVP :CN interactions measured by SPR and highlights the range of affinities measured. There, we show that the binding strength of intrinsically disordered proteins with LxVP motifs is sequence independent; rather, it depends on the k rate, i.e. it depends on the ensemble of conformations of the IDP and how that ensemble ‘presents’ the LxVP motif to CN for binding (accessibility and conformation). Since paper 33 provides such comprehensive data, we refer to this primary literature.\n11. Figure 2f – please, indicate which residues constitute the -3, -2 sites and which build the -1 site.\nWe thank the reviewer for this suggestion. A correlation between the TAK1 sequence and the Arg residues as well as how Arg sites correspond to the CN residues are established in Figure 2d/e just adjacent to Figure 2f. In particular, we used the same color code between Figure 2e and Figure 2f to ensure this can be readily understood. We hope these correlations clarify the issue. Thank you.\n12. Use consistent labeling of residues (ie. Glu282 or E282) through the manuscript\nThank you – done. We have decided to keep Glu where we describe the structure and the molecular interaction but use E when the variant is described. This is meant to facilitate readability.\n13. Please, use a common nomenclature throughout the manuscript. Why is CND90A:tpTAK1-tpS439 not simply called CND90A:tpTAK1 in Figure 2 legend? Especially that it has a different description in the figure title and panel legends.\nThank you – updated.\n14. Figure 2, panel d – label E282 and other important residues at the figure\nWe thank the reviewer for this suggestion, but as highlighted in our response to point 11, these residues are all labeled in Figure 2e, which is directly next to Figure 2d. Indeed, the design of the figure was as follows: In Figure 2d we focus on the identification of all TAK1 residues, in Figure 2e we correlate these with CN residues and finally in Figure 2f show the effect of changes in the CN pockets that are displayed in Figure 2e.\nWe would feel that adding too much information into a single figure make the figures even harder to follow and would appreciate the possibility of keeping all three figures. Thank you.\n15. Figure 2e – please suggest a more comprehensive depiction of ionic interactions. An ionic interaction is not an interaction of a particular atom in a COO- moiety as currently suggested by the figure. (ex. Asp285-Arg(-2) or Glu237-Arg (-1)).\nWe have changed the figure as requested and now highlight only a small number of interactions that are shorter than 4 Å and thus clearly contribute to the interaction. Thank you.\n16. Figure 4b legend – make clear that both the structure of wt and mutant were determined in complex with HNE1. As currently is, it is not clear if CN(E282K) apo is not compared to WT with HNE1.\nWe thank the reviewer to pointing this out – but we are indeed comparing the structure of CN and wt CN, both bound to NHE1. We agree completely that an ‘apple to orange’ comparison would need to be highlighted – but this is a true ‘apple to apple’ analysis.\n17. In the final part of the “results” section, which concludes the findings, I am missing an important sentence, that is that E282K shifts the overall substrate preference of CN (the major conclusion of the study).\nPer the reviewer we have added a sentence summarizing the major conclusion of the structure to the final part of the results section.\n18. “Because PPPs are metalloenzymes, with the residues that coordinate the metal binding being 100% conserved between the family members, this enzyme family, unlike many kinase families, mostly lacks specificity at the active site.35,36” – many enzyme classes will have identical catalytic residues (ie. ion coordination in this particular case) while at the same time particular representatives of the class may largely differ in substrate preference because they differ in residues directly adjacent to catalytic residues which adjacent residues will modulate specificity. Please, rephrase this fragment – active site is broader term than the catalytic residues.\nThe reviewer is correct that the active site is broader than the core catalytic residues. For the PPP family, this is a key issue; indeed, PPP conservation extends much beyond the core catalytic residues explaining, in part, why no specific inhibitors for individual members of this family have been identified. Natural compounds bind with nearly identical K s to the family of enzymes. The only slight outlier is indeed CN; one reason for this is the identified i-1 binding pocket that we report here.\n19. “This CN active site recruitment is sufficiently enhanced that it enabled the structure of CN bound to a PKA phosphorylated substrate to be determined.” – please rephrase. The substrate contained additional tether.\nWe are not certain how to rephrase the sentence, as the active site interaction is really driven by the active site i-1 binding event. We do want to highlight that we have tried to get substrate-bound structures of a PPP for many years (since 2010) and have continuously failed (so have indeed several groups). Even when substrates were tethered in a variety of ways (many unpublished experiments) there was never significant electron density identified around the active site, but rather only at some tethered sites (i.e. SLiM sites etc.). Thus, while the tether is overall important for binding (rationale for doing it), we can assure the reviewer that only additional binding at the active site – such as the i-1 site here at CN – allows for enough interaction strength to see electron density in the structures, i.e. to be able to trace a model of the structure.\n20. “by creating a peptide library from PPP-inhibited HeLa cells and HEK293T cells expressing CNWT and CNE282K“ – please, be more exact. The peptide library in HeLa experiment was prepared at a very different stage of experiment and served a different purpose than in 239T so should not be combined in a single sentence.\nChanged as requested:\nIndeed, by creating a peptide library from PPP-inhibited HeLa cells and by testing sequence specificity in HEK293T and N2A cells expressing CNWT and CNE282K, we showed that an arginine residue at the i-1 site allows for enhanced CN active site recruitment.\n21. “active site recruitment is necessary for a broad range of peptide sequences” – meaning unclear – please, rephrase.\nPlease see response to point 20. Thank you.\n22. “…but, as we show, globally alters substrate preference from basophilic to acidophilic both in vitro and in cells.” – indicate that the cells used in the experiment are not relevant to physiological role of CN in the clinical pathology.\nAs requested, we defined the cell lines now in detail.\nThus, its mutation to a lysine reverses its charge from acidic to basic and weakens not only the recruitment and dephosphorylation of basophilic substrates like TAK1 but, as we show, globally alters substrate preference from basophilic to acidophilic both in vitro and in cells (here tested in HEK293T and N2A cell lines).\n23. “These results not only provide a molecular basis for altered signaling in patients with the E282K variant” – tone down. The results do not provide any data on the influence of E282K on signaling in a setup relevant to pathology.\nThe sentence reads as follows:\nThese results not only provide a molecular basis for altered signaling in patients with the E282K variant but will enable future detailed CN proteomics studies to understand the consequences on specific CN substrates.\nWe agree with the reviewer that at this point we do not show data on the influence of CN on signaling in a setup relevant to pathology, but neither do we claim this here. Rather we only highlight that we have identified the molecular basis for the altered signaling that is performed by CN , which now can be leveraged in future studies to hopefully start to understand the specific changes in signaling, which is really highlighted already in the second part of the sentence."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have provided additional data which in my opinion strengthens the original conclusions. At the same time, the changes to the manuscript text do not entirely reflect the new insight gained with the additional data provided (especially regarding structural biology / biochemistry part). Also, some additional, minor experiments are still missing. I therefore suggest further modifications to the manuscript text and accepting the communication after those changes are introduces.\nMajor reservations\n1. Comment: „How do affinities of fragments of TAK1…” – the reviewer appreciates the similar behavior of TAK1306-590 and TAK1384-474 in the performed assays. My original question however was if TAK11-606 behaves similarly to TAK1306-590 (and TAK1384-474), and this question has not been addressed. While using truncated variants of proteins in biophysical studies is a common practice and it is generally assumed that functional domains of a protein (in this case the kinase domain and IDR domain) act independently, this is rarely tested and not always true. One cannot exclude that the kinase domain of TAK1 influences the affinity of the C-terminal IDR.\nThe reviewer appreciates the difficulties in expressing and handling full length proteins, but at least a comment towards the untested possibility of kinase domain influencing the binding affinity of C-terminal IDR should be made in the manuscript. Also, the authors should avoid referring to TAK1384-474 as TAK1 which suggests a full length protein.\nEven stronger reservation goes towards the rebuttal provided for RII. The fact that everybody uses the same truncated variant is not a proof that it fully reflects the physiological situation. Again, appreciating the experimental difficulties, a relevant comment should be made in the manuscript following the information provided in the rebuttal.\nFrankly pointing out the limitations of the model allow the readers to take them into account while interpreting the data.\n2. Comment: “The effect of mutating sites i-2 and i-3 in CN…”. Asp285 and Tyr315 are now arbitrarily removed from the description of CN binding pocket which is poorly justified since Asp285 seems to form the side of i-3 site while Tyr315 seems to form a stacking interaction with Arg(i-3) (though difficult to judge analyzing only 2D images). The description of the pocket at the end of part “The RRRS phosphosite motif binds CN near the active site” should be based on solid structural criteria and not tied to later findings pertaining to the importance of particular residues. These structural criteria are more than hydrogen bonds as multiple other interactions (including stacking and vdW constraints) are involved. In analyzing the pocket authors should use an unbiased methodology characteristic for structural biology and refrain from describing the pocket through the lens of further functional findings (ie. removing residues Asp285 and Tyr315 from the description, while the authors themselves considered those residues of importance in the prior version of the manuscript, to the extent they tested their effects in functional assays).\nFurther text in chapter “Arginine i-1 phosphosite recruitment is critical for efficient TAK1 dephosphorylation” refers to E282, Q284 and D285 and some “additional” residues, mysteriously selected in the absence of systematic description of the binding site in the proceeding chapter. Clearly defining the interactions at i-1, i-2 and i-3 sites in the prior chapter would justify the selection of residues for functional assays in the following chapter.\nThe activity and affinity data should be discussed together, instead of separately, to allow a holistic picture.\nThe new data provided in review consistently show the importance of i-1 site and more difficult to explain role of i-2 and i-3 sites. Mutations at i-1 site (E282K, E237K) decrease activity (Figure 2f) and affinity (Table S1; only shown for E282K; E237K should be included to complete the description of the i-1 site). Effects for sites i-2 and i-3 are also consistent. Q284K, D285K, Y315A increase activity (Figure 2f) while having little effect on affinity (Table S1; Y315A should be included to complete the description of the site).\nDespite such consistent data, the description in the revised manuscript is inconsistent. Tyr315 has been omitted form the description of the binding pocket, while the residue is tested among the mutants evaluating the contribution of particular interactions in the pocket (Figure 2f). The description in the text focuses solely on i-1 effects while neglecting effects of equal strength, but opposite quality (increased vs. decreased activity) for sites i-2 and i-3. The text should consistently describe i-1 pocket (E282, E237), i-2 interaction (E284) and i-3 site (Y315, D285), and include the effects of mutations at site i-1 (decreased affinity and activity) and i-2 and i-3 (little effect on affinity and increased activity), even though the reason for the increased activity of mutants at i-2 and i-3 sites is not entirely clear (which should also be directly mentioned; Reviewer #3 also expressed this reservation about the same thing and yet it has not been addressed in the revised version neither as mine. The reason for the increased activity is simply unclear – it should be explicitly stated in the revised version).\nAuthors also mention testing Ri-3 and Ri-2 variants, but I can’t find the results demonstrating the effect of Ri-3 and Ri-2 on dephosphorylation rate by WT CN (only on E282K; Figure 4).\n3. Comment: “E237K and E282K reduce activity of CN in dephosphorylating TAK1.”\nConstructively addressed with data summarized at Figure 4g.\n4. Comment: “The variant 436RRVS439…”\nThe variant has been tested with E282K, but I can’t find the results demonstrating the influence on dephosphorylation rate by WT CN.\n5. Comment: “Chapter “Arginine in the i-1 position…”\nI still fail to recognize that two examples justify calling a mechanism “general”, but the rest of the explanation clarifies my reservations.\n6. Comment: “crystal structure of a substrate trapped CN complex”\nThank you for the clarification. It was clearly my omission. All the data were indeed present in the original manuscript.\n7. Comment: “RII dephosphorylation…”\nConstructively addressed.\n8. Comment: “we determined the crystal structure of CNE282K“\nThank you for a clear clarification. In such a case, please modify the sentence “bound to NHE1, a substrate that binds CN using a PxIxIT and LxVP motif” by changing the world “substrate” into a “peptide”. A peptide lacking the dephosphorylation residues is not a substrate and the statement is misleading.\n9. Comment “Is the (i-1) arginine containing structure exceptional…”\nConstructively addressed.\n10. Comment: “My major comment pertains to the phosphoproteome study…”\nThe authors have constructively addressed the reviewers reservation for the most part. It is useful to see that experiments in less mechanistic and more physiological setting are ongoing, but due to their length, complexity and scope beyond the mechanistic interest of this manuscript delaying its publication while waiting for their completion would not have been justified.\nHowever, I still ask to address the following point:\na. It should be clearly stated that because Lys-C is used for digestion all the potential sites having K(i-1) are not detected – unless relevant provisions were made in data analysis which should also be mentioned.\n11. Comment: “As it is now,…”\nConstructively addressed\nMinor comments\n1. Please, rephrase: “Mutating either of these SLiMs abolishes their ability to be dephosphorylated by CN, …”. It is not SLiMs that get dephosphorylated.\n2. “Recent work has also shown that the efficacy of CN catalysis can be modulated by…” – remove “also”", "reviewer_2": "It is well apparent that the authors have considered the concerns of the reviewers and have done a thorough effort to either address these concerns through new experimentation, edits to the manuscript text or have provided acceptable rationale in their response to the reviewers explaining the limits to the scope of the current study. The revisions have strengthened the manuscript and I have no major remaining concerns. I recommend acceptance.", "reviewer_3": "The comments have been addressed appropriately, including the addition of new experiments (ITC to determine Kd) and improvements to the representation of structures in some of the figures."}, "author_response": "We thank the reviewer for her/his support of our work. It is great to read that our additional data strengthens the original conclusions.\nMajor reservations\n1. Comment: „How do affinities of fragments of TAK1…” – the reviewer appreciates the similar behavior of TAK1306-590 and TAK1384-474 in the performed assays. My original question however was if TAK11-606 behaves similarly to TAK1306-590 (and TAK1384-474), and this question has not been addressed. While using truncated variants of proteins in biophysical studies is a common practice and it is generally assumed that functional domains of a protein (in this case the kinase domain and IDR domain) act independently, this is rarely tested and not always true. One cannot exclude that the kinase domain of TAK1 influences the affinity of the C-terminal IDR.\nThe reviewer is correct that the potential influence(s) of intradomain interactions on specific biochemical assays are not commonly tested, especially in the absence of published cellular data that would indicate that such interactions might have functional importance. We have tested for such interdomain interactions in other biological systems, such as those highlighted here. Our data have not detected effects in the cases tested. Rather, our work and that of others have shown that long IDRs like that present in TAK1 almost exclusively function independently from the folded domains present within the same protein. This is indeed not a surprise. While the kinase domain (residues ~26-300) has dimensions of ~35x35x50 Å, the dimensions of the IDR tested here (residues 306-590) can reach a length of ~850 Å when fully extended; thus, at any point in time, the vast majority of the IDR residues are very far from the kinase domain. For these reasons, it is highly unlikely that the kinase domain influences the affinity of the C-terminal IDR.\nFinally, it is important to emphasize that, by definition, SLiMs are only identified in IDRs; thus, there is a clear physiological reason to focus on the IDR of TAK1 for mediating CN binding. Indeed, and as the reviewer knows, TAK1 activation requires that TAK1 binds another protein, namely TAB1 (TAB1 binds TAK1 via a structured helical interaction to the TAK1 kinase C-lobe). Here, we solely focus on the key interaction of TAK1 with CN. Clearly, it might be of interest for others to further investigate if CN also interacts with TAB1/2/3 as well (that said, thus far no SLiMs have been identified in TAB1). Furthermore, we have not seen TABs in our MS data and thus we have not followed up. We hope that this further strengthens the rationale why we chose to focus on the TAK1 IDR region in this manuscript.\nThe reviewer appreciates the difficulties in expressing and handling full length proteins, but at least a comment towards the untested possibility of kinase domain influencing the binding affinity of C-terminal IDR should be made in the manuscript. Also, the authors should avoid referring to TAK1384-474 as TAK1 which suggests a full length protein.\nEven stronger reservation goes towards the rebuttal provided for RII. The fact that everybody uses the same truncated variant is not a proof that it fully reflects the physiological situation. Again, appreciating the experimental difficulties, a relevant comment should be made in the manuscript following the information provided in the rebuttal.\nFrankly pointing out the limitations of the model allow the readers to take them into account while interpreting the data.\nAs requested, we added a statement that we did not test for the influence of other domains on the TAK1 IDR domain.\nFirst, we modified the sentence:\nTherefore, we tested if the TAK1 IDR binds CN using isothermal titration calorimetry (ITC).\nIDR was added to highlight that we only tested binding of CN to the IDR.\nSecond, we added the following sentence:\nWe did not test if CN binds to the TAK1 catalytic domain (likely not as no CN SLiM was identified) nor if the TAK1 catalytic domain makes an intramolecular interaction with the TAK1 C-terminal IDR.\nWe would like to keep the nomenclature as is – simply because the manuscript would gain significant length with the additional annotations, making it much harder to read. We are confident that the readers of Nature Communications will be able to distinguish the different domains as is – especially as they are detailed in the methods as well as clearly illustrated in the figures (including Figure 1).\n2. Comment: “The effect of mutating sites i-2 and i-3 in CN…”. Asp285 and Tyr315 are now arbitrarily removed from the description of CN binding pocket which is poorly justified since Asp285 seems to form the side of i-3 site while Tyr315 seems to form a stacking interaction with Arg(i-3) (though difficult to judge analyzing only 2D images).\nWhile we appreciate the careful review of reviewer 1, we disagree that anything we do or represent in the data has been done ‘arbitrarily’. As explained in our original rebuttal letter, we ‘updated Figure 2 to only highlight polar/ionic interactions that are <4 Å (i.e. to focus on only the most significant interactions); we also added the distances to the figure to further improve clarity.’ Clearly, using a number cutoff is not arbitrary but rather 100% reproducible.\nWe have requested all PDB files to be released (they will be released on 12/17/2025) – so the reviewer can see them in 3D. We are always happy to provide all data to the reviewer.\nThe description of the pocket at the end of part “The RRRS phosphosite motif binds CN near the active site” should be based on solid structural criteria and not tied to later findings pertaining to the importance of particular residues. These structural criteria are more than hydrogen bonds as multiple other interactions (including stacking and vdW constraints) are involved. In analyzing the pocket authors should use an unbiased methodology characteristic for structural biology and refrain from describing the pocket through the lens of further functional findings (ie. removing residues Asp285 and Tyr315 from the description, while the authors themselves considered those residues of importance in the prior version of the manuscript, to the extent they tested their effects in functional assays).\nAs highlighted above, and in response to the previous review, our revision implemented a rigorous and reproducible criteria for defining interactions: using distances for the structural analysis. As stated before, the original Figure 2 was suboptimal as it did not highlight widely differing distances. Indeed, the response highlights this issue. The distances highlighted in the original figure for Asp285 and Tyr315 are ≥ 5 Å (some even 6-7 Å) and thus have significantly less or no impact on binding (it was my oversight that these were included in the original submission). Indeed, we have now proven their lack of importance for TAK1 binding with the additional ITC experiments performed in the revised manuscript. Taken together the combined data ensures correct, rigorous and unbiased interpretation of the structural and functional data.\nAs stated, the original figure was poorly prepared in the sense that the differences in the distances, which typically correlate with interaction strength, were not illustrated, giving the incorrect impression that all interactions were equally important. I apologize for this oversight during the original submission – my mistake. In its current form, this paper includes 3 crystal structures, multiple NMR data sets, ITC experiments, biochemical and cellular studies, including multiple MS experiments carried out in 2 different cell lines. We always aim to describe our data to the highest standards and accuracy, and I did not achieve the highest level of quality on the original figure 2 – not because we did not aim for it – but rather because it simply ‘slipped through the cracks’. But the careful review ensured that this ‘crack’ is successfully closed – thank you for your careful review and help.\nWe have always followed (and will always follow) all structural biology standards (see Supplementary Table 2 and PDB file) for our data. However, the reviewer might also have not fully appreciated that we described in the manuscript (and highlighted in Supplemental Figure 3) that the interaction at the active site is dynamic (fuzzy) and indeed adopts at least 2 conformations that we captured in our structure. Thus, additional experiments will always be necessary to fully understand the complete picture of such a fuzzy interaction – this is what we have done here.\nWe have recently summarized some of the aspects of fuzzy interactions in catalysis in a review article – PMID: 40580813. We are sure many more of these interactions are identified that are not locked down into a single conformation and will require complementary techniques for their full descriptions.\nFurther text in chapter “Arginine i-1 phosphosite recruitment is critical for efficient TAK1 dephosphorylation” refers to E282, Q284 and D285 and some “additional” residues, mysteriously selected in the absence of systematic description of the binding site in the proceeding chapter. Clearly defining the interactions at i-1, i-2 and i-3 sites in the prior chapter would justify the selection of residues for functional assays in the following chapter.\nWe deleted the word ‘additional’.\nThe activity and affinity data should be discussed together, instead of separately, to allow a holistic picture.\nThe new data provided in review consistently show the importance of i-1 site and more difficult to explain role of i-2 and i-3 sites. Mutations at i-1 site (E282K, E237K) decrease activity (Figure 2f) and affinity (Table S1; only shown for E282K; E237K should be included to complete the description of the i-1 site). Effects for sites i-2 and i-3 are also consistent. Q284K, D285K, Y315A increase activity (Figure 2f) while having little effect on affinity (Table S1; Y315A should be included to complete the description of the site).\nDespite such consistent data, the description in the revised manuscript is inconsistent.\nWe thank the reviewer for highlighting the excellent consistency of our data from different sources (X-ray, NMR, ITC, activity assays). This orthogonal approach is critical for describing complex data as presented in our work. We are certain that the current presentation allows for the best presentation of the complex data for a broad audience of Nature Communications and thank Reviewer 2 and 3, who clearly support the current presentation.\nTyr315 has been omitted form the description of the binding pocket, while the residue is tested among the mutants evaluating the contribution of particular interactions in the pocket (Figure 2f). The description in the text focuses solely on i-1 effects while neglecting effects of equal strength, but opposite quality (increased vs. decreased activity) for sites i-2 and i-3. The text should consistently describe i-1 pocket (E282, E237), i-2 interaction (E284) and i-3 site (Y315, D285), and include the effects of mutations at site i-1 (decreased affinity and activity) and i-2 and i-3 (little effect on affinity and increased activity), even though the reason for the increased activity of mutants at i-2 and i-3 sites is not entirely clear (which should also be directly mentioned; Reviewer #3 also expressed this reservation about the same thing and yet it has not been addressed in the revised version neither as mine. The reason for the increased activity is simply unclear – it should be explicitly stated in the revised version).\nWe want to highlight one more time that no data has been omitted from the manuscript, rather we display the data in the most coherent and clear way possible for the readers. Indeed, we are focusing on all the residues that have interactions that are 4 Å or less, as these are the most critical interactions (it is well established that ionic, polar and van der Waals interactions correlate with distance). We prove that this assumption is correct using ITC – where the data shows that only residues within the i-1 pocket have significant impacts on binding affinity with TAK1.\nThe reviewer is correct – we do not know the effect of increased activity for CN Y315A. We could speculate that it is most likely the change from a bulky tyrosine sidechain to a small alanine sidechain is the main driver, which allows, due to the reduced size, additional degrees of freedom of the substrate to dock for dephosphorylation, likely leading to increased dephosphorylation. We prefer not to speculate.\nIn the current manuscript, we solely focus on the i-1 interaction pocket, as it is completely novel.\nAuthors also mention testing Ri-3 and Ri-2 variants, but I can’t find the results demonstrating the effect of Ri-3 and Ri-2 on dephosphorylation rate by WT CN (only on E282K; Figure 4).\nThe reviewer is correct – we describe the data for CN E282K but compare it to the WT CN dephosphorylation in Figure 4. This is the only possible way to accurately describe the data as the ensemble of substrate structures (TAK1 ensemble) that is presented for CN dephosphorylation will change in each substrate variant, i.e. deletion of N-terminal arginine residues will change the interaction with the phosphorylated pS residue and thus change the ensemble of structure in the IDP for binding and subsequent dephosphorylation presentation. This does not happen for a small molecule substrate (DiFMUP or pNPP) or folded proteins, where such comparisons are possible. It is for this reason we did all experiments in a manner that changed CN and kept the substrate constant as otherwise we would have introduced a broad range of unknown factors, which would not lead to a rigorous comparison of the data.\n3. Comment: “E237K and E282K reduce activity of CN in dephosphorylating TAK1.”\nConstructively addressed with data summarized at Figure 4g.\nWe thank the reviewer for her/his support of our data and work.\n4. Comment: “The variant 436RRVS439…”\nThe variant has been tested with E282K, but I can’t find the results demonstrating the influence on dephosphorylation rate by WT CN.\nWe thank the reviewer for her/his support of our data and work. We want to refer to our detailed response above.\n5. Comment: “Chapter “Arginine in the i-1 position…”\nI still fail to recognize that two examples justify calling a mechanism “general”, but the rest of the explanation clarifies my reservations.\nWe thank the reviewer for her/his support of our data and work.\n6. Comment: “crystal structure of a substrate trapped CN complex”\nThank you for the clarification. It was clearly my omission. All the data were indeed present in the original manuscript.\nWe thank the reviewer for her/his support of our data and work.\n7. Comment: “RII dephosphorylation…”\nConstructively addressed.\nWe thank the reviewer for her/his support of our data and work.\n8. Comment: “we determined the crystal structure of CNE282K“\nThank you for a clear clarification. In such a case, please modify the sentence “bound to NHE1, a substrate that binds CN using a PxIxIT and LxVP motif” by changing the world “substrate” into a “peptide”. A peptide lacking the dephosphorylation residues is not a substrate and the statement is misleading.\nWe thank the reviewer for her/his support of our data and work. We are not fully convinced that using the word peptide make this easier to understand (especially, as a peptide has no meaning regarding a biological function of a protein, and NHE1 is not a random peptide that we created by design, but rather it has a specific function and way to interact with CN, which has been investigated in high detail in a previous manuscript that is cited in our work).\nNevertheless, as requested we changed the word to peptide.\n9. Comment “Is the (i-1) arginine containing structure exceptional…”\nConstructively addressed.\nWe thank the reviewer for her/his support of our data and work.\n10. Comment: “My major comment pertains to the phosphoproteome study…”\nThe authors have constructively addressed the reviewers reservation for the most part. It is useful to see that experiments in less mechanistic and more physiological setting are ongoing, but due to their length, complexity and scope beyond the mechanistic interest of this manuscript delaying its publication while waiting for their completion would not have been justified.\nWe thank the reviewer for her/his support of our data and work.\nHowever, I still ask to address the following point:\na. It should be clearly stated that because Lys-C is used for digestion all the potential sites having K(i-1) are not detected – unless relevant provisions were made in data analysis which should also be mentioned.\nIf the reviewer is referring to the dephosphorylation assays in original submitted Figure 5D then the reviewer is correct, the K in the i-1 position might be lost due to the Lys-C digest. However, because of the reviewers’ concerns in the last round of reviews, we already deleted this figure in the originally revised manuscript (R1).\nIf the reviewer refers to the in-cell data shown in Figures 5 and 6 (as well as Supplemental Figure 8), the dephosphorylation occurs before digest. So, in these data we can clearly assay K in the i-1 position. We hope this clarifies the concerns of the reviewer.\n11. Comment: “As it is now,…”\nConstructively addressed\nWe thank the reviewer for her/his support of our data and work.\nMinor comments\n1. Please, rephrase: “Mutating either of these SLiMs abolishes their ability to be dephosphorylated by CN, …”. It is not SLiMs that get dephosphorylated.\nThank you – done.\nrephrased to:\nMutating either of these SLiMs in substrates abolishes dephosphorylation by CN,\n2. “Recent work has also shown that the efficacy of CN catalysis can be modulated by…” – remove “also”\nThank you – done.\nReviewer #2 (Remarks to the Author):\nWe thank reviewer 2 for her/his support of our work.\nReviewer #3 (Remarks to the Author):\nWe thank reviewer 3 for her/his support of our work."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have made substantial improvements to the presented data in round R1 of the review and clarified the remaining issues in their detailed rebuttal letter in round R2. With the additional data and explanations provided I find the current manuscript an excellently presented and very interesting piece of data. I recommend publication of the manuscript in its current form."}, "author_response": ""}]

Decision letter