The N-Myc MB0-MBI region interacts specifically and dynamically with the N-lobe of Aurora kinase A

Domain: chemistry
Published: 2026-02-24
Editorial outcome: minor_revision

Abstract
The intrinsically disordered MYC proteins are master regulators of cellular growth and function, but when deregulated they become cancer drivers. MYC-protein interactions are key to oncogenesis, and while disrupting such interactions would be of significant therapeutic benefit, the intrinsically disordered properties of MYC have dramatically hampered their characterization. Here, we apply an integrated structural biology approach to describe the structure and dynamics of the N-Myc–Aurora A complex, which is critical in neuroendocrine tumor progression. We reveal a functional interaction where multiple binding sites on N-Myc interact with the Aurora A N-lobe. The interaction is governed by aromatic clusters within the conserved MB0 and MBI motifs in N-Myc that interact with Aurora A in a dynamic binding mode that allosterically promotes kinase activation. We show that N-Myc binding to the Aurora A N-lobe can be inhibited by the small-molecule Aurkin A, providing opportunity for therapeutical strategies to disrupt this interaction.

Introduction
The MYC family of proteins (c-Myc, N-Myc, L-Myc) are master transcription factors whose activity is highly regulated as they play key roles in many cellular processes, such as cell differentiation, proliferation, and metabolism. In cancer, multiple mechanisms deregulate MYC expression and activity, leading to uncontrolled cell growth and proliferation. This turns MYC into a potent oncoprotein that significantly contributes to the development of both adult and pediatric human cancers 1–4. Within the MYC protein family, the N-Myc protein is recognized as a key cancer driver mainly within the central and peripheral nervous system, promoting high-risk cases most often manifested in children. Amplification of the MYCN gene has long been used as an indicator of poor prognosis within neuroblastoma 5,6, and characterizes one-in-six cases with dismal prognosis 7.

Directly inhibiting MYC with small molecule inhibitors would mark a key advance in cancer drug development, but this has proven difficult as the MYC proteins are intrinsically disordered in the absence of a protein binding partner 2,8,9. Intrinsically disordered proteins (IDPs) possess distinct conformational malleability and adaptability, enabling MYC proteins to be highly responsive to regulatory cues and to accommodate versatility in their protein interactions 10,11. The nature of complexes formed by IDPs spans a continuum, ranging from coupled folding upon binding, over multistate, or “fuzzy”, complexes with structured partners, to fully disordered complexes with no persistent structure in either partner 10,12,13. Within the MYC family, six highly conserved but unstructured regions known as MYC Boxes (MB0-MBIV) have been proposed to serve as focal points for the regulation of protein interactions 2 (Fig. 1a). Supporting this, an in situ proximity labeling approach revealed 336 high-confidence c-Myc interactors, of which more than half were interrupted by one or more MYC box deletion 14. Recent advances identifying which of these proteins directly interact with MYC has afforded us and others to reveal structural and dynamic features of protein interactions to MYC boxes and adjacent residues 15–22, several of which show characteristic IDP recognition modes 10,12,23. With these insights, alternative opportunities to inhibit MYC oncogenic activity could be made possible by disrupting MYC interactions with key partner proteins.

Within N-Myc driven neuroblastoma, the Aurora A (AurA) protein kinase is upregulated and stabilizes cellular N-Myc levels in a post-transcriptional manner, presumably as part of an oncogenic mechanism 24. Aurora kinases are widely overexpressed in tumors, with specific biochemical roles identified for AurA in melanoma, neuroblastoma and prostate cancer 25,26. AurA shares a common regulatory architecture with other eukaryotic protein kinases, comprising a bilobal kinase domain with the active site positioned between its N- and C-lobes 25,27 (Fig. 1b). A regulatory spine spans four layers of interacting side chains, including the catalytically critical DFG(W) motif at the active site. This network enables allosteric kinase activation by connecting the active site with the N-lobe αC-β4 loop and the activation loop, which contains an activating phosphorylation site (Fig. 1b) 27. By crystallographic studies, the spindle assembly factor TPX2 was shown to activate AurA by binding to hydrophobic pockets in the N-lobe, thereby affecting the adjacent αC helix 28. However, later studies suggest that Aurora kinases in solution comprise an ensemble of states, and that their activation is conveyed through the regulatory spine by induced population shifts 29. Furthermore, an expanding body of research highlights the role of correlated motions and dynamics-based allostery in regulating catalytic turnover in kinases 30,31.

Inhibiting the N-Myc–AurA interaction has been suggested as a therapeutic strategy for childhood cancers 9. Several AurA inhibitors promote proteasomal breakdown of N-Myc but were cytotoxic in subsequent clinical trials 32–34, prompting further investigation of the N-Myc–AurA complex. While it has been suggested that larger regions of the N-Myc transactivation domain (TAD) bind AurA 35, the crystal structure of AurA together with N-Myc 28-89 only resolved N-Myc 61-89, in a position supporting the AurA activation loop in its open state 18. This prompted the working model that the N-Myc–AurA interaction protects N-Myc from degradation via the E3 ligase SCF FbxW7 pathway 18 and subsequent work has thus been focused on this N-Myc 61-89 region 35–37. However, while N-Myc 28-89 was able to activate AurA, N-Myc 61-89 alone had no effect on AurA function 18 and constraining the conformational space of N-Myc 61-89 by shorter cyclic peptides did not significantly enhance inhibitory potency of the N-Myc–AurA interaction 36. Taken together, this suggests that the full molecular mechanism describing the N-Myc–AurA functional interplay has not yet been resolved.

In this work, we show that N-Myc binds AurA in a 1:1 complex via several contact regions on N-Myc. Among these, the N-terminal MB0 and MBI of N-Myc jointly comprise primary binding regions, with distinct hydrophobic patches that bind and stabilize the AurA N-lobe, thereby enhancing AurA activity. Using an experimentally integrated AlphaFold-based modeling approach, we derived a conformational ensemble model for the N-Myc-AurA interaction, consistent with experimental data from multiple structural and biophysical techniques in solution, including nuclear magnetic resonance spectroscopy (NMR), small angle X-ray scattering (SAXS), hydrogen deuterium exchange mass spectrometry (HDX-MS), nano differential scanning fluorimetry (nano-DSF), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), dynamic light scattering (DLS) and size-exclusion chromatography with multi-angle light scattering (SEC-MALS). In the context of an intact N-Myc segment encompassing both MB0 and MBI (N-Myc 1-100), the previously identified binding region N-Myc 61-89 is a minor contributor to AurA binding and activation and does not alter the conformational ensemble. We propose that N-Myc binding via MB0 and MBI to the AurA N-lobe can be targeted as an alternative therapeutic route.

Results
AurA forms 1:1 complexes with both N-Myc 1-69 and N-Myc 1-100
To understand how the N-terminal region of N-Myc binds AurA in solution we produced N-Myc 1-69 and N-Myc 1-100 from TEV-cleavable His-TRX fusion proteins, adding only one additional Ser at their N-termini. Both constructs contain the entire MB0 and MBI regions, which are well conserved in the MYC family, known hubs for protein interactions, and functionally important for MYC oncogenesis 14 (Fig. 2a, Supplementary Fig. 1a). The longer N-Myc 1-100 construct also includes the non-conserved region C-terminal to MBI (N-Myc 70-89) which co-crystallized with AurA 122-403 18. We produced AurA 122-403 including previously described mutations C290A and C393A 38 but with a cleavable SUMO tag, resulting in protein with no non-native N-terminal residues and insufficient yields for us to probe the N-Myc interaction with the kinase domain of AurA.

All constructs were monomeric as judged by SEC-MALS, each eluting as a single peak with respective average molecular masses in agreement with a monomeric state (Fig. 2b, Supplementary Fig. 1b). By DLS, the calculated hydrodynamic radii for N-Myc 1-69 and N-Myc 1-100 were significantly larger and had more variability than expected for a globular protein 39 (Supplementary Fig. 1c). This agrees with the longer-than-expected retention times observed in the SEC elution profiles and suggests a high degree of intrinsic disorder. This was further supported by SAXS, where smooth I(q) scattering profiles suggest an averaging of multiple conformational states and the plateau at large angles in the dimensionless Kratky plot indicates a natively unfolded state (Supplementary Fig. 2). Furthermore, the maximum dimensions (Dmax) of the two N-Myc constructs as derived from the pair-distance distribution function analysis (P(r)) are large, in agreement with the DLS results (Supplementary Table 1) 40.

To characterize stoichiometry and affinities of the N-Myc–AurA interaction, we used SEC-MALS and SAXS together with ITC. Judging by the clear shift in elution volume and SDS-PAGE analysis, both N-Myc 1-69 and N-Myc 1-100 eluted as complexes with AurA on a MALS coupled SEC column (Fig. 2c, Supplementary Fig. 3a). SEC-SAXS independently confirmed 1:1 stoichiometry of N-Myc–AurA complexes by Guinier analysis and molecular weight estimation. In addition, P(r) analysis indicated an increase in overall dimension upon complex formation compared to AurA alone, while Kratky analysis revealed predominantly globular conformation with some retained flexibility, as evidenced by the bell-shaped curve and upturn at higher q-values (Fig. 2d, Supplementary Fig. 3b, c, Supplementary Table 1). By ITC, the dissociation constants (KD) were 4.0 ± 0.45 µM and 0.90 ± 0.15 µM for N-Myc 1-69 and N-Myc 1-100 respectively (Fig. 2e, Supplementary Table 2), which is consistent with KD s obtained for N-Myc 28-89 by other methods 18, and for c-Myc TAD binding to regulatory partners 16,17,21. While ITC revealed a slightly higher affinity to AurA for the longer N-Myc 1-100 construct, the similar KD s indicate that the main binding determinants are contained within N-Myc 1-69, whereas additional residues in N-Myc 1-100 contribute to binding, but are not solely responsible. All ITC data were evaluated using a 1:1 binding model, in agreement with the stoichiometry identified by SAXS and SEC-MALS. With this model the N-value was consistently lower than 1, suggesting additional binding sites on the titrand that contributes to early saturation 41. This would be consistent with co-elution on a SEC column, as multiple interaction sites could maintain complex formation despite relatively low affinities. The fitted ITC data further revealed similar thermodynamic profiles, suggesting similar binding modes to AurA for the two N-Myc constructs (Supplementary Table 2).

Three distinct N-Myc regions interact with AurA
To learn more about the N-Myc–AurA interaction, we proceeded to NMR. 1H 15N-HSQC of N-Myc 1-69 and N-Myc 1-100 confirmed their intrinsically disordered nature, as evidenced by narrow linewidths and backbone amide proton chemical shifts confined to the 7.4–8.6 ppm range, consistent with corresponding NMR analysis of similar c-Myc and N-Myc fragments 16,20,21,35. The superposition of the N-Myc 1-100 and N-Myc 1-69 spectra is nearly perfect, suggesting only minor, if any, internal interactions between N-Myc residues 70-100 and regions within N-Myc 1-69 (Supplementary Fig. 4a, b). Chemical shift secondary structure population inference (CheSPI) analysis 42 further confirmed that the two N-Myc constructs are largely disordered, with only slight, possibly transient, helicity proposed within MBI and in residues 75-86 in N-Myc 1-100 (Supplementary Fig. 4c).

To identify specific residue motifs within N-Myc that interact with AurA in solution, we titrated unlabeled AurA into 13C 15N-labeled N-Myc 1-69 and N-Myc 1-100. The resulting NMR spectra revealed three distinct amino acid regions exhibiting pronounced line broadening (reduced I/I0) even at substoichiometric concentrations of AurA, accompanied by minor chemical shift perturbations (CSPs) (Fig. 3). This pattern is characteristic of interactions between IDPs and globular proteins 43–47 and has also been reported for several c-Myc interactions 16,17,21. The affected regions in N-Myc include the entire MB0 (residues 18-38), residues 48-55 within MBI (both N-Myc 1-69 and N-Myc 1-100), and residues 76-89 C-terminal to MBI (in N-Myc 1-100). The most prominent, but still small, CSPs were centered around aromatics in MB0 (F21, F28, Y29, F35, Y36, F37) and in MBI (W50, F53), including charged residues D20 and D22 which flank F21, and K51 and K52 located between W50 and F53. In N-Myc 1-100, the third region affected by AurA includes aromatic residues W77 and W88, as well as adjacent residues, and encompass the structured N-Myc 61-89 binding site previously identified by crystallography 18. In solution, the CSPs and I/I0 in this region closely resemble those observed in MB0 and MBI. Importantly, the similar NMR binding pattern for MB0 and MBI residues in both N-Myc 1-69 and N-Myc 1-100 suggests that AurA binds MB0 and MBI independent of the N-Myc C-terminal segment beyond MBI. This agrees well with our ITC, SEC-MALS and SAXS results suggesting that both N-Myc 1-69 and N-Myc 1-100 form 1:1 AurA complexes and bind AurA with comparable affinities.

The detected line broadening in N-Myc upon AurA addition may arise from an enhanced observed transverse relaxation (R2) due to one or more of the following factors: i) the increased molecular size of the complex resulting in a longer correlation time, ii) conformational exchange on the µs-ms timescale between free and bound states, and/or iii) exchange processes in the bound state due to transient intra- and intermolecular interactions 47. As peak intensities for N-Myc could not be recovered at >90% saturation (Supplementary Fig. 5), we speculate that transverse relaxation in the bound state is much larger than that of free N-Myc, indicating partial restriction of its intrinsically disordered nature. Furthermore, considering the expected high relaxation in the bound state and the extent of signal loss due to line broadening exceeding the proportion of added AurA in our substoichiometric titrations, we conclude that the system undergoes conformational exchange at a rate that is faster than the transverse relaxation of free N-Myc 48.

To further explore the interaction profile and dynamics of the N-Myc 1-69–AurA complex, we determined fast relaxation parameters; longitudinal relaxation rates (R1), transverse relaxation rates (R2) derived from rotating frame relaxation rates (R1ρ) and R1s, as well as heteronuclear NOEs (hetNOEs) of N-Myc 1-69, both in isolation and in the presence of increasing substoichiometric concentrations of AurA (Fig. 4). Consistent with the observed intensity changes, the 15N R2 rates increased significantly across the MB0 region and within a narrower segment within MBI, while remaining low for terminal residues and within the linker between MB0 and MBI, indicating that the MB0 and MBI regions are specifically involved in the interaction with distinct dynamic profiles. In agreement with the CSPs, the largest increases in 15N R2 rates were observed for F35, Y36 and F37 in MB0, and I49, W50 and K51 in MB1, where these resonances were broadened beyond detection at addition of only 10% AurA. In contrast to the observed changes in R2 rates, R1 remained largely unaffected by the addition of AurA. This observation is reasonable even if the free and bound states differ in correlation time or intrinsic dynamics, since R1 rates are in general small for 15N in biomolecular systems 48. Furthermore, hetNOE values remained largely unchanged upon addition of AurA, suggesting no significant increase in backbone rigidity upon complex formation. However, it is possible that the low concentration of AurA was insufficient to reveal subtle dynamic changes. Concluding our NMR analysis, three distinct regions in N-Myc are similarly perturbed by AurA binding, as shown by CSPs, line broadening, and increased R2 values. The loss of signal at substoichiometric AurA concentrations likely reflects conformational exchange on the μs-ms timescale and a system with substantial differences in transverse relaxation rates.

Hydrogen-deuterium exchange shows that N-Myc 1-69 and N-Myc 1-100 both bind to the AurA N-lobe
As poor NMR resolution of AurA has hitherto precluded direct NMR analysis 29, we turned to HDX-MS to investigate the effects in AurA upon N-Myc binding in solution. Amides in well-ordered and/or buried regions exchange more slowly compared to those in flexible and/or solvent-exposed regions, rendering this technique suitable both to explore changes in internal dynamics and monitor protection of exposed surface areas by an interacting protein or ligand 49. To probe the effects of N-Myc binding on AurA, we compared the HDX patterns of AurA alone to that of AurA in complex with N-Myc 1-69 and N-Myc 1-100 at four time points: 30, 300, 3000 and 9000 s (Supplementary Data 1-2). To enable comparative analysis of the N-Myc 1-69 and N-Myc 1-100 binding across the two distinct HDX-MS runs, we selected 96 AurA peptides with high significance and reproducibility in both experiments, resulting in a common peptide pool representative for all regions in AurA (Fig. 5a, b, Supplementary Data 3; see Methods).

The AurA HDX pattern revealed significantly reduced H/D exchange upon N-Myc binding. The most pronounced effects were localized in the AurA N-lobe, while the C-lobe remained comparatively unaffected (Fig. 5a). The AurA HDX patterns obtained from N-Myc 1-69 and N-Myc 1-100 binding were surprisingly similar and no additionally protected regions in AurA were observed for N-Myc 1-100 (Fig. 5b), even though our NMR and ITC data suggest that this longer construct holds a third AurA binding site. Rather, minor differences were observed only in the magnitude of the protection (Supplementary Fig. 6a), where the higher affinity of N-Myc 1-100 compared to N-Myc 1-69 enable a greater degree of saturation under the experimental conditions (87% compared to 69%, see Methods for details), potentially leading to an underestimation of the protection conferred by N-Myc binding. Within the AurA C-lobe, residues 334-351, encompassing αG which is contacted by N-Myc-W77 and -W88 in the N-Myc–AurA crystal structure 18, showed only a slight reduction in H/D exchange at the earliest time point for N-Myc 1-69 and N-Myc 1-100 (Fig. 5a–c, Supplementary Fig. 6a). While a highly transient interaction comprising residues within N-Myc 70-100 and αG in AurA might not have been resolved in the HDX-MS experiment, a direct comparison of the magnitude of the HDX effects across the AurA sequence supports that the AurA N-lobe is indeed the main N-Myc binding region (Supplementary Fig. 6a). Furthermore, the similar HDX protection pattern on AurA observed for both N-Myc 1-69 and N-Myc 1-100 suggests that N-Myc 1-69 houses the main AurA interaction site.

We further analyzed the time-dependence of the AurA HDX pattern and compared this with known dynamic features in kinases. The largest cumulative difference in H/D exchange overall time points was observed within residues 133-196 in the AurA N-lobe (Fig. 5b, Supplementary Fig. 6a). Within this region, residues 162-184 encompass helices αB and αC, which together with the tip of β3 line the so-called “Y-pocket”, a well-known regulatory binding groove in kinases 50. Here, the AurA HDX pattern reveals significantly reduced H/D exchange upon N-Myc binding at short D2O exposure times, but at longer time points the H/D exchange becomes similar for the bound and free state (Fig. 5b, c). Such early protection features are commonly observed at direct interaction sites by transient, dynamic, or fuzzy binders, where rapid on- and off-rates allow for continued H/D exchange in the bound state 51. To visualize the protected areas on the AurA structure (Fig. 5d), we classified significant protection at early time points as ‘strong” if it persisted over several of the time points measured, and ‘weak’ if it persisted over only one time point. Residues 184-208 include the AurA N-lobe αC-β4 loop, a functional and well-recognized element within protein kinases that structurally connects the N- and C-lobe 25,27,52, and the β4-β5 region. In contrast to the early protection observed around the Y-pocket, N-Myc binding conveys significant late protection within residues 184-208 at longer time points (Fig. 5b, c), as visualized in Fig. 5d. This suggests a reduction of conformational dynamics in this already well-protected region, which includes highly conserved residues such as Q185 and L196 of the regulatory spine, thereby connecting to the AurA active site. A weak initial HDX protection pattern was also observed at residues 236-262, comprising part of the C-lobe αE (Fig. 5b, c), which connects to the AurA active site via R-spine residue H254 and packs against the AurA αC-β4 loop via Y246-H190 stacking interactions 52.

To investigate the H/D exchange on N-Myc in the AurA-bound state, we performed an N-Myc optimized experiment at 1:1 molar ratio (77% saturation) using enhanced proteolytic resolution (Fig. 5e, Supplementary Data 4-5). This setup contrasts the AurA-optimized HDX-MS experiments, which were performed at 3-fold N-Myc 1-69 excess to maximize AurA saturation and could therefore not resolve N-Myc protection in the bound state. While the same pattern of large HDX perturbations were observed for AurA (ΔHDX 1-3 Da), indicating high occupancy of N-Myc at the AurA binding sites, only very weak perturbations were detected for N-Myc at the shortest time points (ΔHDX 0.2-0.6 Da), where the deuterium uptake was already high across the N-Myc sequence (Fig. 5f, Supplementary Fig. 6b). This suggests that N-Myc remains largely disordered upon binding and does not adopt any stable secondary structure. These results distinguish the N-Myc–AurA interaction from the INCENP-Aurora B interaction, where HDX-MS studies revealed folding-on-binding of a highly protected INCENP peptide 53. Furthermore, N-Myc binding at the Y-pocket reduces the conformational dynamics in the αC-β4 loop, a change which may propagate into the active site via the regulatory spine and thus affect AurA activity.

N-Myc binding is independent of AurA activation loop phosphorylation
In the HDX-MS experiment, AurA peptides encompassing the activation loop were equally poorly protected in the absence and presence of N-Myc (Fig. 5a–d), suggesting maintained activation loop dynamics of AurA in complex with the N-Myc constructs investigated here. The MS/MS evaluation of our E. coli expressed AurA construct performed within the HDX-MS-setup showed phosphorylation in at least one site in the AurA activation loop (Supplementary Fig. 7a), in agreement with previous observations 28, and to increase sequence coverage these modified peptides were therefore included in the HDX-MS analysis. Comparing the H/D exchange for phosphorylated and unphosphorylated peptides within the activation loop revealed identical patterns for both groups (see deposited HDX-MS data to the PRIDE repository). However, to further evaluate whether N-Myc binding to AurA is dependent on the phosphorylation status of the activation loop, we expressed AurA with mutations T287A:T288E to mimic the activating phosphorylation at T288 in the activation loop while prohibiting phosphorylation at the adjacent T287. We also co-expressed AurA with λ-phosphatase 54, resulting in AurA with no phosphorylations. Binding analysis monitored by BLI using these AurA variants revealed no significant changes in affinities for N-Myc binding based on the AurA phosphorylation status (Supplementary Fig. 7b). This suggests that N-Myc binding is not affected by phosphorylation in the AurA activation loop, once again in contrast to the INCENP interaction with Aurora B, which is highly stabilized by Aurora B phosphorylation 53.

N-Myc MB0 stabilizes and activates AurA
To investigate whether N-Myc binding modulates AurA thermal stability, we monitored the thermal melting point (Tm) by nanoDSF 55. The AurA tryptophan (Trp) residues in the N-lobe (W128), in the C-lobe (W313, W382), and within the DFGW motif at the active site (W277) are well positioned to monitor global unfolding. N-Myc has one Trp (W50) and N-Myc 1-100 another two (W77, W88). Neither N-Myc 1-69 nor N-Myc 1-100 showed any sign of cooperative melting (Fig. 6a), as expected for an IDP. Still, both N-Myc constructs significantly stabilized AurA to similar extents, and in a concentration dependent manner in line with their respective KD s as observed by ITC. In contrast, addition of peptides N-Myc 61-90 or N-Myc 69-90, corresponding to the third AurA interaction site in N-Myc 1-100, resulted in a slightly decreased Tm compared to free AurA (Fig. 6a, b). This effect could be due to an excess of unbound N-Myc, yielding an increase in the 350 nm signal and a masking of the unfolding effect (Supplementary Fig. 8). Indeed, a slight increase was observed in the onset (Ton) of the AurA melting curve, but this increase was still much lower than that observed for N-Myc 1-69 and N-Myc 1-100 (Fig. 6b). Taken together, these results support that the main N-Myc determinants for AurA interaction reside within N-Myc 1-69.

To assess possible N-Myc anchor points for binding AurA, we designed alanine mutations in N-Myc 1-69 guided by our NMR analysis. We focused on the aromatic residues and clusters affected by AurA binding and analyzed the effects on Tm in the respective N-Myc–AurA complexes. Mutation of either of the two aromatic clusters in MB0 (F28A:Y29A or F35A:Y36A:F37A) significantly reduced thermal stabilization of AurA, as judged from the onset and inflection points of the complex melting curve (Fig. 6b, Supplementary Fig. 8). Substitution of W50A within MBI had similar effects, however not to the same extent. The smallest effects were seen for mutants F53A and F21A, which only marginally affected the stability compared to wild type N-Myc.

To assess whether reduced stability of the N-Myc–AurA complex correlates with binding affinity, we performed ITC measurements using N-Myc anchor point mutants. While all mutants exhibited reduced affinity for AurA, the aromatic cluster mutations within MB0 showed the most pronounced effects (Fig. 6c, d, Supplementary Fig. 9, Supplementary Table 2). ITC measurements further indicated KD s exceeding 100 μM for the N-Myc 69-90 peptide, suggesting that this region alone cannot bind AurA efficiently. The magnitude of the affinity reduction seen by ITC correlates well with the diminished thermal stabilization observed by nanoDSF (Fig. 6e) supporting a correlation between binding strength and complex stability.

We continued to deconstruct the functional components of N-Myc binding by performing AurA activation assays in the presence and absence of the designed N-Myc variants using the ADP-Glo Kinase Assay (Promega). The results showed that binding of N-Myc 1-69 or N-Myc 1-100 increases the kinase activity of AurA (Fig. 6f). In contrast, N-Myc peptides 61–90 and 69–90 did not have any significant effect on AurA activity (Fig. 10a). When comparing the generated models, we noted a tendency of multimer_v1 to preferentially generate tightly packed complexes with smaller radius of gyration, thus disfavoring variability. Indeed, when selecting models that fit the experimental SAXS data (χ2 < 1.1) to structurally describe the conformational ensembles of N-Myc 1-69 and N-Myc 1-100 when bound to AurA, only models generated by multimer_v2 were obtained (Supplementary Fig. 10b). Further refinement by including additional structures with higher χ2 did not result in any lowering of the χ2 of the ensemble.

Overall, the N-Myc 1-69–AurA and N-Myc 1-100–AurA conformational ensembles both revealed N-Myc binding to several parts of the AurA N-lobe and indicated extensive variability within the complex (Fig. 7a, b). To further investigate the degree of consistency of the SAXS-filtered ensemble with our independently obtained NMR and HDX-MS data, we first calculated the per-residue buried surface area both in the selected conformational ensemble and in all generated complex models (Supplementary Fig. 10c, d) 58. The SAXS-filtered ensemble revealed a consistent subset of partially buried residues in both N-Myc and AurA, whereas the unfiltered ensemble revealed a broader set of buried residues. This suggests that the filtered ensemble describes a specific contact pattern despite the retained flexibility of the ensembles.

We continued to ask whether the SAXS-filtered ensemble could help identify potential residue-specific contacts between AurA and N-Myc, and therefore generated per-residue contact maps for both N-Myc 1-69–AurA and N-Myc 1-100–AurA complexes, and further calculated the fraction of contacts per residue in N-Myc and AurA across the selected ensembles (Fig. 7c, d). Partially protected residues were identified throughout MB0 and MBI in both N-Myc 1-69 and N-Myc 1-100, in line with our experimental NMR and biophysical data. Within the ensembles, and in agreement with HDX-MS results, several regions in the AurA N-lobe were significantly buried and showed a high fraction of contact in both complex ensembles. The highest degree of burial was observed within residues 171–189, which encompass the αC-helix and part of the β4-connecting loop. The AurA N-terminal residues 125–128, where peptides were not captured by HDX-MS, were also consistently protected throughout the ensembles. A smaller fraction of contacts were seen for residues 255–258, located within the protected αE-peptides in the HDX-MS experiments, and moderate burial was observed within the AurA activation loop (residues 270–293), where transient N-Myc–AurA interactions cannot be ruled out although this loop appeared highly dynamic both in the absence and presence of N-Myc in the HDX-MS experiment. Very few contacts were seen for residues 334-337 in the C-lobe αG helix, previously postulated as a major site for N-Myc binding 18, in agreement with the slight perturbations observed in the HDX-MS experiments (Fig. 5, Supplementary Fig. 6). The burial pattern on N-Myc agrees closely with the amino acid regions identified by NMR to be affected by AurA, both in N-Myc 1-69 and N-Myc 1-100. Reciprocally, the AurA N-lobe is partly buried in all models, in agreement with this being the main protected region observed by HDX-MS. Although only SAXS experimental data was used to confine the ensembles, their consistency with NMR and HDX-MS data independently confirms the validity of the computational approach.

Blocking the AurA N-lobe Y-pocket inhibits N-Myc 1-69 and N-Myc 1-100 binding
Based on our finding that N-Myc interacts with the AurA N-lobe, mediated by MB0 and MBI, and thereby both stabilizes and activates AurA, we asked whether this interaction could be specifically inhibited. TPX2 has been shown to compete with N-Myc binding to AurA 18, and we therefore asked if an inhibitor designed to block TPX2-AurA binding to the N-lobe could interfere with N-Myc binding. We chose the AurA inhibitor Aurkin A, which impairs TPX2 by binding to the AurA Y-pocket between αB and αC in the AurA N-lobe (Fig. 7e), as our work shows that this AurA site is also protected by N-Myc (Fig. 5). In competition assays monitored by BLI, we found that Aurkin A, which has comparable affinity for AurA to N-Myc (KD = 10.6 μM) 59, was able to efficiently compete with both N-Myc 1-69 and N-Myc 1-100 for binding to AurA (Fig. 7f). These results reveal the potential for targeting the AurA N-lobe to therapeutically disrupt the N-Myc–AurA complex.

Methods
Production of N-Myc proteins
Human N-Myc (residues 1–69 or 1–100) were subcloned into the pNH-Trxt vector (Addgene #26106). The protein was overexpressed in E. coli BL21(DE3) cells in LB medium supplemented with 50 µg/ml kanamycin by induction with 0.5 mM 1-thio-β-D-galactopyranoside (IPTG) at an OD 600 of 0.6 at 37 °C for 3 h. Harvested cells were resuspended in lysis buffer (50 mM NaPO 4, 10 mM Tris-HCl, 100 mM NaCl, 10 mM imidazole, 5% glycerol, 5 U/ml DNaseI (Roche), cOmplete EDTA free protease inhibitor (Roche), 10 mM β-mercaptoethanol, pH 8.0) and sonicated on ice. Following centrifugation, the supernatant was affinity purified at 4 °C using Ni 2+-NTA resin (Invitrogen™ R90110) and eluted with 300 mM Imidazole. Following dialysis into 40 mM NaCl, purification was done by anion exchange using a 5 ml XL High Trap column (Cytiva) and eluted by gradual increase in NaCl to 400 mM. The 6x-His-Trx-tag was cleaved with TEV at 4 °C during dialysis overnight and removed by Ni 2+ chromatography using 300 mM NaCl. Final polishing was done using a HiLoad 16/600 Superdex 75 column (Cytiva) equilibrated in GF buffer (50 mM NaPO 4, 10 mM Tris-HCl, 100 mM NaCl, 5% glycerol, 10 mM β-mercaptoethanol, pH 8.0).

For biotinylated proteins, human N-Myc fragments (residues 1–69 or 1–100) were subcloned into a modified pET15-MHL vector (Addgene #26092) such that they contained an N-terminal 6xHis-Thioredoxin tag cleavable by TEV protease, and a C-terminal AviTag (GLNDIFEAQKIEWHE). Proteins were co-expressed with biotin ligase BirA from the pCDF-BirA vector (GenBank ID #JF914075) from E. coli BL21(DE3) cells in TB medium supplemented with 100 µg/ml ampicillin, 50 µg/ml spectinomycin, and 20 µM D-biotin. Protein expression was induced overnight at 16 °C with 0.5 mM IPTG when the cells reached OD 600 ≈ 0.8. Proteins were purified according to the non-biotinylated N-Myc protocol, excluding the anion exchange step.

Peptide synthesis
N-Myc peptides corresponding to residues 61-90 and 69-90 of human N-Myc were synthesized using Fmoc-chemistry on an automated microwave peptide synthesizer (Liberty Blue, CEM, Matthews, North Carolina, USA) in 100 µM scale. ProTide Rink amide (LL) resin was used as solid support for synthesis yielding an amidated C-terminal. For synthesis, Fmoc-protected amino acids were sequentially coupled using a four-fold excess of amino acid, Oxyma as base and DIC as coupling reagent in DMF under microwave conditions. Fmoc-deprotection was achieved by treatment with 20% Piperidine in DMF under microwave conditions. After final Fmoc-deprotection N-terminal acetylation was done by acetic anhydride and DIEA (5 eq) in DMF for 1 hr. Global deprotection and cleavage of peptides from resin was achieved by treatment with TFA:H2O:Phenol:DTT (88/5/5/2, v/v/v/v) for 3 h before being concentrated using a stream of nitrogen. The crude peptides were precipitated in ice cold diethyl ether, twice, and the ether was discarded. The crude peptides were purified on a semi preparative HPLC system (Dionex) equipped with a RP-C18 column (ReproSil Gold) using a gradient of acetonitrile containing 0.1% TFA. Peptide identity and purity was confirmed using MALDI-ToF mass spectrometer (Bruker) and HPLC (Thermofischer) respectively (Supplementary Fig. 11).

Production of Aurora A protein kinase
Aurora A kinase residues 122-403, with the double Cys-mutations C290A:C393A described previously 38 and with or without the phosphorylation mimic mutation T287A:T288E, was subcloned into two different vectors: pNH-Trxt (Addgene #26106), which were transformed into E. coli BL21(DE3) cells containing the ROS-2 pRAR3 plasmid, and SUMO-6xHis-Lic1.10 (a gift from Patrick Celie at Netherland’s Cancer Institute 69), which was transformed into E. coli BL21(DE3) pLysS cells (Sigma Aldrich). Protein overexpression was done in LB medium supplemented with 50 µg/ml kanamycin and 34 µg/ml Chloramphenicol at 18 °C overnight following induction with 0.5 mM IPTG at an OD 600 of 0.6. Harvested cells were resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl 2, 10 mM imidazole, 10% glycerol, 5 U/ml DNaseI (Roche), cOmplete EDTA free protease inhibitor (Roche), 10 mM β-mercaptoethanol) and sonicated on ice. The protein was affinity purified at 4 °C using Ni-NTA resin (Invitrogen™ R90110) following centrifugation. The 6x-His and solubility tag was cleaved with SENP2 during dialysis at 4 °C overnight and the protein was purified through incubation with Ni-NTA resin before the final polishing step by a HiLoad 16/600 Superdex 75 column (Cytiva) using GF buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and 10 mM β-mercaptoethanol). Unphosphorylated AurA 122-403 with mutations C290A:C393A was produced by co-expression with λ-phosphatase 54 (a gift from John Chodera, Nicholas Levinson and Markus Seeliger (Addgene plasmid # 79748; http://n2t.net/addgene:79748; RRID: Addgene_79748) in BL21(DE3) cells. The co-transformation was carried on in a consecutive manner, where the λ-phosphatase was transformed first to BL21(DE3) and once the transformation was confirmed, it was followed by the transformation of AurA. The co-expressed unphosphorylated AurA 122-403 C290A:C393A was purified as mentioned above.

Size-exclusion chromatography with multi-angle light scattering (SEC-MALS)
SEC-MALS analysis was done by injecting samples onto a Superdex 75 Increase 10/300 column (Cytiva) equilibrated in the sample buffer (50 mM NaPO 4 (pH 8), 10 mM Tris, 10 mM BME, 5% v/v glycerol, 100 mM NaCl) with a flow rate of 0.5 mL/min. Complex formation was done prior to injection by mixing N-Myc constructs with AurA in a 2:1 molar ratio followed by incubation for 2 h on ice. The MALLS-RI data were measured using Mini-Dawn TREOS module coupled to an OptiLab T-Rex refractometer and a DynaPro Nanostar (Wyatt Technologies). The molecular weight distribution of species eluting from the SEC column was evaluated using ASTRA 6.1 (Wyatt Technologies).

Dynamic light scattering (DLS)
DLS of N-Myc 1-69 and N-Myc 1-100 was measured on a 100 μM sample in the sample buffer (50 mM NaPO 4, 10 mM Tris-HCl, 100 mM NaCl, 5% v/v Glycerol, 10 mM b-mercaptoethanol, pH 8.0), using a DynaPro Plate reader (Wyatt Technologies) at 20 °C using ten acquisitions at 5 s. Data was analyzed using the accompanied software DYNAMIC 7.10.1 (Wyatt Technologies).

Isothermal titration calorimetry (ITC)
ITC measurements were performed on a Microcal PEAQ-ITC (Malvern) at 25 °C, using a reference power of 10.0 µcal/s and a stir speed of 750 rpm. Samples were dialyzed into the ITC buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM MgCl 2, 5 % v/v glycerol, 2 mM TCEP), and assayed using N-Myc constructs at approximately 400 mM, which were titrated into a 45 mM AurA sample over 19 injections of 2 µl each. Data analysis was done using using a 1:1 binding model in the MicroCal PEAQ-ITC Analysis Software (Malvern), where the baseline was manually adjusted, and where the 1:1 binding model was validated by stoichiometry assayed by SAXS and SEC-MALS. As more complex binding models did not improve the fit to data for the strongest binders, and to allow direct comparisons between constructs with a different number of binding regions, we applied the 1:1 ITC model to all data.

Small-angle X-ray scattering (SAXS)
SAXS data collection was performed on the P12 bioSAXS beamline 70 at PETRA III (Hamburg, Germany) using in-line SEC-SAXS setup for AurA, N-Myc 1-69, N-Myc 1-100, and their respective complexes. Parallel MALLS-RI measurements 71 were recorded for AurA, N-Myc 1-100, and the respective complexes. The protein samples were injected onto Superdex 75 Increase 10/300 equilibrated in the sample buffer (20 mM HEPES 150 mM NaCl 5 mM MgCl 2 3% v/v glycerol 2 mM TCEP pH 7.5) with a flow rate of 0.70 mL/min. The 2400 individual frames for each run were processed using SASFLOW 72 and CHROMIXS 73 to obtain final buffer-subtracted scattering profiles, and the data analysis was performed using the ATSAS 3.2.1 suite 74. The radius of gyration, Rg, and forward scattering intensity, I(0), were obtained using Guinier approximation 75 and GNOM program 76 was used to evaluate a paired distance distribution function p(r) and to obtain maximum particle dimension, Dmax. Molecular weight estimates were calculated using concentration-independent methods including DatBayes 77, volume of correlation, Vc 78, SAXSMoW 79. Estimation of Δρ was done using MULCh 80. The MALLS-RI data were measured using Mini-Dawn TREOS module coupled to an OptiLab T-Rex refractometer (Wyatt Technologies). The molecular weight distribution of species eluting from the SEC column was evaluated using ASTRA 7 (Wyatt Technologies). The detailed information about samples, data collection and processing, and structural parameter analysis are outlined in Supplementary Table 1, in accordance with the publication guideline 81. Data and models are deposited in SASBDB 82.

NMR spectroscopy (NMR)
Isotopically labeled N-Myc (residues 1–69 and 1–100) was overexpressed in BL21(DE3) cells in M9 minimal media (6 g/l NaHPO 4, 3 g/L KH 2PO 4, 0.5 g/L NaCl, 50 μM CaCl 2, 1 mM MgSO 4) supplemented with 10 ml/L Gibco Vitamin solution, 2 g/L 13C-based glucose, 1.5 g/L 15NH 4Cl and 50 μg/ml kanamycin. Expression was induced at an OD 600 of 0.6 using 0.5 mM IPTG and incubation at 37 °C for 3 h. Purification was done as described for unlabeled sample above. Following buffer optimization using DLS and SEC-MALS, NMR experiments were performed in the optimized buffer (20 mM MES (pH 6.5), 100 mM NaCl, 5% v/v glycerol, 5 mM DTT, 10% D 2O, 100 μM NaN 3, 1 mM TCEP). Assignment of N-Myc 1-69 and N-Myc 1-100 was done at 164 µM and 98 µM, respectively, using a series of vnmrj BioPack resonance experiments (1H 15N-HSQC, CBCAcoNH, HNcaCO, HNCO, HBHAcoNH) and a HNCACB sequence retrieved from the Kay lab, recorded at 288 K on a Varian INOVA spectrometer (operating at 600 MHz) equipped with a cryogenic probe. All triple resonance experiments were non uniformly sparsely sampled to 15% and all spectra were processed using NMRPipe 83 and MddNMR 84 and visualized and manually assigned using the software NMRFAM-Sparky 85. For binding assays with AurA, unlabeled AurA was titrated to 13C 15N-labled N-Myc with a concentration of 240 μM (1-69) or 98 μM (1–100). 1H 15N-HSQC were recorded at molar ratios of 0, 7, 12, 21, and 28% AurA for N-Myc 1-69 and 0, 7, 12, 16, 23, and 30% AurA for N-Myc 1-100. I/I0 was calculated following normalization based on N-Myc concentration. CSPs were calculated as previously described 20.

The 15N longitudinal relaxation rates (R 1), rotating frame relaxation rates (R 1ρ), and {1H}-15N heteronuclear Overhauser effects (hetNOE) were measured on 250 µM N-Myc 1-69 samples in the same buffer as above, at 288 K on a Bruker Avance Neo (operating at 600 MHz) equipped with a TCI cold probe, using Bruker HSQC-detected pulse sequences: hsqct1etf3gpsitc3d (R 1), hsqcretf3gpsitc3d (R 1ρ), and hsqcnoef3gpsi3d (hetNOE), running on Topspin 4.5.0. Experiments were performed for 15N-labeled N-Myc 1-69 alone and in substoichiometric concentrations of AurA (6 µM, 15 µM and 25 µM). R 1 relaxation delays (ms) were 20, 40, 60 (triplicate), 90, 120, 200 (duplicate), 400 (duplicate), 600 and 800 ms. R 1ρ relaxation delays (ms) were 4, 24 (duplicate), 48, 72, 96 (duplicate), 140 (duplicate), 170 and 200 ms. hetNOE spectra with and without proton saturation were acquired in duplicates. Analysis of all relaxation experiments was done using PINT 86,87, including conversion of R 1ρ to transverse relaxation rates (R 2) using R 1. Error bars were calculated based on the repeats indicated above using the jackknife method embedded within PINT. The 1H 15N-HSQC of N-Myc 1-69 in the absence (1:0) and presence (1:1) of AurA (50 µM) were recorded under the same experimental conditions as the 15N relaxation data, except for the protein concentrations. Spectra were collected using a variant of hsqcetfpf3gpsi2 modified by Maxim Mayzel, obtained from the Swedish NMR center (Gothenburg, Sweden).

Hydrogen-deuterium exchange mass spectrometry (HDX-MS)
HDX-MS was performed on three separate occasions to investigate 1) N-Myc 1-100 binding to AurA, 2) N-Myc 1-69 binding to AurA, and 3) AurA binding to N-Myc 1-100 and the reverse. In run 1 and 2, 45 μM AurA was mixed 1:1 v/v with 120 μM N-Myc 1-100, 180 μM N-Myc 1-69 or TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5), yielding apo and bound AurA for comparison. For reciprocal analysis 60 μM N-Myc 1-100 and 60 μM AurA were mixed 1:1 v/v, and apo controls were prepared similarly with TBS, making it possible to qualitatively probe the protection of both proteins in the same complex and confirm consistency to previous runs. All mixtures were incubated on ice for 1 h before further handling.

HDX-MS experiments were run in triplicates using automated sample preparation on a LEAP H/D-X PAL™ platform (Trajan Scientific and medical) coupled to an Ultimate 3000 micro-LC and an Orbitrap Q Exactive Plus MS (Thermo Scientific). All reagents were from Sigma Aldrich. A 4-point calibration (pH 2, 4, 7, 10) was made prior to all measurements using a SevenCompact pH-meter with an InLab Microelectrode (Mettler-Toledo). Samples (3 µl) were diluted 10-fold with TBS (50 mM Tris-HCl, 150 mM NaCl) or a D 2O buffer of the same composition (pH 7.5 and 7.1). Exchange was carried out for t = 0 s, 30 s, 300 s, 3000 s, and 9000 s at 4 °C. The labeling reaction was quenched by a 1:1 dilution (1% TFA, 0.4 M TCEP, 4 M urea, pH 2.5) at 1 °C. Quenched samples (55 µl) were directly injected and subjected to on-line digestion at 4 °C at 50 µL/min for 4 min (0.1% formic acid (FA), pH 2.5) using an in-house immobilized 2.1 × 30 mm pepsin column (run 1-2) or a 2.1 × 20 mm Nepenthesin-2/Pepsin mixed digestion column (AffiPro, CZ) (run 3, for increased sequence overlap/resolution of the relatively small N-Myc). Peptides were subjected to on-line SPE on a PepMap 300 C18 trap column (1 mm x 15 mm) and washed with 0.1% FA for 60 s. The trap column was switched in-line with a C18 reversed-phase analytical column (Hypersil GOLD, particle size 1.9 µm, 1 × 50 mm) for separation at 1 °C (8 min 5–50% B (95% acetonitrile/0.1% FA), then 50–90% B for 5 minutes). Carry-over between injections were minimized by equilibrating the trap and column at 5% organic content, cleaning the needle port and sample loop three times after each injection (5% MeOH/0.1% FA, followed by 90% MeOH/0.1% FA and finally 5% MeOH/0.1% FA), and washing the Pepsin column by injecting 90 µl of 1% FA/4 M urea/5% MeOH. A full blank was run between each sample injection. Separated peptides were analyzed on a Q Exactive Plus MS equipped with a HESI source operated at a capillary temperature of 250 °C with sheath gas 12, Aux gas 2, and sweep gas 1 (au). MS full scan spectra were acquired at 70 K resolution, AGC 3e6, Max IT 200 ms, and scan range 300-2000.

For peptide identification, undeuterated samples were analyzed using data-dependent MS/MS with HCD fragmentation in PEAKS Studio X (Bioinformatics Solutions Inc, BSI, Waterloo, Canada) and searched against the protein sequence FASTA file (15 ppm mass error tolerance, 0.05 Da fragment mass error tolerance, allowing for fully unspecific pepsin cleavage). Peptides with a peptide score value of -log 10(P) > 25 were retained for HDX analysis in HDExaminer 3.1.1 (Sierra Analytics Inc., Modesto, US). Initial constraints disregarding modified peptides resulted in partial low coverage of the AurA sequence. Allowing for phosphorylation in the PEAKS peptide identification resulted in phosphorylated peptides across AurA residues 277-294 (data is available in PXD062561). As no significant difference in uptake was observed for phosphorylated and unmodified peptides, phosphorylated T288 (as supported by literature 28) was added to the evaluation to increased HDX-MS sequence coverage.

HDX analysis was made on charge states 1–6, allowing only for EX2, and assuming the first two residues of a peptide were unable to hold deuteration. Full deuteration was set to 75% of the maximum theoretical uptake in HDExaminer to compensate for the system wide back-exchange and best reflect the uptake dynamics without having run a fully deuturated sample. This affects the %D scaling and heatmap coloring, but does not alter the reported number of deuterons. The presented deuteration data represent averages of high/medium confidence peptides within a ±0.5 min RT window; outliers and inconsistent peptides were removed after manual inspection. In the HDX protein-protein interaction analysis, bound vs apo states were compared within each run. When comparing N-Myc 1-69 and N-Myc 1-100 binding on AurA, only AurA peptides which were consistent between runs 1 and 2 were used, resulting in a common peptide pool of 96 representative AurA peptides for a robust and direct comparison between the experiments. Heatmaps were generated using heavy smoothing and uncolored prolines. Automatically calculated significance based on volcano plot significance lines were used for generating the difference heatmaps. A summary of the HDX-MS experimental detail is reported in Supplementary Table 3. The mass spectrometry and HDExaminer analysis files have been deposited to the ProteomeXchange Consortium via the PRIDE 88 partner repository.

Biolayer interferometry (BLI)
BLI was performed using an Octet RED384 system (Forte Bio) with 150 nM biotinylated N-Myc 1-69 or N-Myc 1-100 immobilized on streptavidin (SA) biosensors (Sartorius). Binding assays were performed in the optimized buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM MgCl 2, 5% v/v glycerol, 2 mM TCEP, 0.05% Tween-20 and 0.2 mg/ml BSA) using Octet 384 tilted well microplates (Sartorius) with AurA concentrations ranging from 0-30 μM. For the competition assay, 1 μM AurA was incubated with 0-200 μM Aurkin A, and a final BSA concentration of 1 mg/ml.

Nanodifferential scanning fluorimetry (nanoDSF)
NanoDSF measurements were performed using the Prometheus NT.48 (NanoTemper Technologies) and Prometheus Standard capillaries in the optimized buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM MgCl 2, 5% v/v glycerol, 2 mM TCEP). Complex formation was done by mixing AurA at a final concentration of 20 μM, with various N-Myc variants at final concentrations of 10, 20, 40, 100 and 200 μM. Thermal unfolding was measured by monitoring the 350 nm/330 nm fluorescence ratio over a temperature gradient of 25 °C to 80 °C with an increase of 0.3 °C/minute and visualized as the first derivative of the 350 nm/330 nm curve. Measurements were done on duplicate samples, except for N-Myc alone which did not show thermal melting. The onset and thermal melting points were determined using the instrument software PR.ThermControl 2.3.1 (NanoTemper Technologies).

Aurora A kinase activity assay
Kinase activity of our recombinantly produced AurA was determined using the ADP-Glo™ Kinase Assay (#V6930) together with the Aurora A Kinase Enzyme System (#V1931) from Promega Corporation (Madison, USA). The ADP-Glo™ Kinase Assay was preformed using the substrate and reaction buffer included in the Aurora A kinase system, together with our recombinantly produced proteins, to quantify the amount of ATP consumed in the kinase reaction using luminescence detection. Briefly, the kinase reaction was performed using AurA alone or in complex with various N-Myc constructs. Complex formation was done by incubating a mixture of 3 μM AurA and 24 μM N-Myc on ice. AurA apo samples had a concentration of 3 μM. Samples were split into triplicates of 3 μl in a white 384-well plate (Corning #3824), where after the samples were mixed with 2 μl ATP/substrate mixture, giving final concentrations of 150 μM and 0.1 mg/ml for ATP and the substrate (MBP), respectively. The mixture was left for 40 min at room temperature to allow the kinase reaction to take place. Following this, ATP Depletion was done by addition of 5 μl ADP-Glo™ Reagent, after which ADP was converted to ATP using the luciferase/luciferin reaction, which generates the detectible luminescence, by addition of 10 μl Kinase Detection Reagent. To determine the ATP-to-ADP conversion in the kinase reactions, an ATP/ADP standard curve was generated by mixing different amounts of ATP and ADP and recording the luminescence for each sample. The assay was performed in a white 384-well plate (Corning #3824) on a CLARIOstar Microplate reader (BMG Labtech) using triplicates. Results are reported as % activation, calculated from the activity for AurA in complex with N-Myc compared to the activity for AurA alone.

Molecular modeling using AFsample
To explore the conformational space comprehensively and increase the likelihood of identifying accurate structural models, we generated 10,000 structural models using the AFsample algorithm 56 without incorporating any templates. This large number of models allows for extensive sampling of possible conformations and enhances the probability of capturing the native-like structure of the proteins. To assess the fit of the generated models to the Small-Angle X-ray Scattering (SAXS) data, we employed Pepsi-SAXS 57. This facilitated the evaluation of how well each model conformed to the experimental SAXS data (χ2) and computed Radius of gyration (Rg) for each model. The buried surface area formed by the interaction between N-Myc and AurA was calculated using Naccess 58. This calculation involved the following steps: the solvent-accessible surface area (SASA) of each individual subunit (N-Myc and AurA) was computed separately, the SASA of the N-Myc-AurA complex was determined, and the difference in SASA between the individual subunits and the complex was calculated for each residue to ascertain the buried surface area. The total absolute buried surface areas were reported in square angstroms (Å2). To investigate specific contacts between N-Myc and AurA, a residue-residue contact map was constructed, reflecting the frequency of contact between specific residues in N-Myc and AurA within a subset of the ensemble which had a χ2 < 1.1 to the experimental SAXS scattering profile for the respective complex. The ranking confidence was calculated as 0.8 ipTM + 0.2 pTM, where ipTM is the pTM for the interface, and pTM is the individual chains. To visualize the generated ensembles, the 31 top ranking models for N-Myc 1-69 and N-Myc 1-100, respectively, were aligned in PyMol based on the AurA structure (Supplementary Data 6-7).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Hultman et al perform an integrative study of the N-Myc – Aur interaction, providing insights into the\narchitecture of this complex and the molecular determinants for complexation. The manuscript is generally well constructed,\nand the set of experiments performed is logical. However, in places there is a lack of detail which may hamper a non-expert\nfrom being able to understand the data presented. My area of expertise lies in the application of HDX-MS and other MS tools\nfor structural applications, and as a result my comments are primarily aligned with this portion of the work.\n- The authors perform extensive SEC-MALS and SEC-SAXS analysis. They present the resultant data in various ways – e.g.\nGuinier analysis(line 154) and p(r) analysis (line 156). It would be helpful for the authors to provide further detail as to how\nthe data presented support their conclusions. For example, this could be included in the appropriate figure legends. As\nwritten, for a non expert in these techniques it is hard to understand how the authors have drawn the conclusions presented.\n- Why are the authors showing two runs of AurA data in Figure 4C? This is confusing to the reader and is not discussed in\nthe text/Methods. They are pretty well identical and im not sure both are needed to be shown here.\n- More labels are needed in Figure 4. For example it is not clear to me the colouring shown in the top part of panel A. The\nauthors state that the colouring here represents the innate HDX pattern, but that is a vague statement and further details are\nneeded. I assume this has to do with percentage uptake from maximal deuteration but the ‘key’ on the right of the figure\nshould be labelled to indicate this. The authors also need to state why they have chosen a ’75 %’ uptake (from Methods) to\nmean 100 % uptake for such an analysis. This is non-standard in the field from my experience.\n- Label scale bars shown in lower part of 4A and Figure 4B. Might also be useful to add labels to the structures shown in\nFigure 4D given that they refer to key regions in the structure in the text.\n- The authors state different residues are involved in interactions – e.g. 162-184 (line 239). But the data where protection is\nreferred to doesn’t directly correlate with these exact residue numbers (at least in terms of the labels shown). How were\nthese numbers selected?\n- How do the authors define ‘strong’ and ‘weak’ HDX protection (e.g. line 240 and Figure 4D)? This needs clarification and a\nmetric applied. Was any statistical analysis used to identify protected regions?\n- Given the difference in affinities, which the authors measure here, what is the difference in percentage bound during the\nHDX experiments for the two Myc constructs studied? This could be calculated and be helpful in interpreting the data\nshowed. This should be stated in the text and a discussion of this included.\n- The authors display an output from PEAKS in Supplementary Figure 4 where putative phosphorylation sites are indicated.\nIn my experience, PEAKS can annotate very low confidence PTM sites in these types of reports, where the MS/MS data do\nnot provide the requisite support for the presence of PTMs at a given residue. The authors should investigate their data\nfurther, and include MS/MS spectra for all phosphosites that are confidently identified. I would\nrecommend changing this figure to one that only shows the localisation of confident phosphosites after this manual data curation has been performed.\n- In the methods indicate the protein concentrations used – this is critical for understanding the percentage bound in these\nexperiments.\n- What do the authors mean by the FASTA file only containing the RDB sequence (line 703)?\n- The authors must indicate the PRIDE dataset identifier in their manuscript – at the moment this is missing (line 699)\n- The authors must include experimental details for their phosphosite analysis.\nMinor comments:\nLine 121 – add ‘mass’ before spectrometry.\nIn several places references are missing. E.g. line 478 and line 715. Please thoroughly proof-read the manuscript.", "reviewer_2": "The N-Myc MB0-MBI region forms a specific, functional and fuzzy complex with the N-lobe of Aurora kinase A, Hultman et al.\nThe manuscript describes the interaction between N-Myc and Aurora A. Disordered protein regions, like those found in Myc,\nare increasingly being viewed as potential drug targets. Given its near ubiquitous role in many cancers, Myc is frequently\ncited as a potential target. Finding interfaces that are formed when an IDR binds to another protein are an attractive\nalternative to simply trying to identify small molecules that can bind to IDRs in isolation. The work presented here not only\ndoes a great job in characterizing the specific interaction between Myc and Aurora A, but also lays out a straight-forward\n(albeit using a number of biophysical techniques) approach for characterizing such interfaces in general. The manuscript is\nwritten in a clear and succinct fashion and is easy to read and follow. The figures are all well organized and clear (I\nparticularly appreciated the unified color things throughout the entire paper).\nThe authors use a combination of multiple, complementary, biophysical techniques that both report on structural features\nand affinities. Given what is required to obtain good quality NMR spectra on a protein like Aurora, I appreciated the use of\nthe HDX as a more cost-effective and simpler (albeit lower resolution) alternative. With some revisions, this manuscript is\ncertainly worthy of publication.\n1. The use of “fuzzy” and “dynamic” to describe the complex, while quite possibly true, is not heavily supported in the data\npresented. These terms explicitly describe a dynamic process, and the vast majority of data in the paper is ensemble\naveraged and does not directly report on dynamics. The one exception is the HDX. I think the interpretation of the time\nscales of protection do hint at some dynamics in the complex, but the long time point (9000 s) is really quite long and I’m not\nsure how these exchange dynamics would compare to any complex, dynamic or no, with micromolar affinity. The authors\nalso cite other NMR studies of IDPs (such as Milles et al) to suggest that their NMR data is in agreement with other studies of\ndynamic complexes. What is missing in this comparison is the careful NMR relaxation dynamics experiments that are\npresent in those papers. If the authors were to include R1 R2 hetNOE and relaxation dispersion analysis, the point would be\nsignificantly stronger. This data would also significantly enhance the paper's conclusions.\n2. The authors conclude that the significant line broadening on addition of Aurora is the result of intermediate exchange (and\nthus also indicative of a dynamic complex). While it is possible that this is true, a more likely explanation is simply that\nresidues in complex with Aurora are tumbling with a much different tau_c and are broadened relative to the other disordered\nresidues. This is quite common in complexes of IDRs with larger, folded, binding partners regardless of the exchange\nregime. In order to make this point, again, a much more detailed accounting of the NMR relaxation should be done.\n3. The conclusion that the complex is 1:1 is based on SEC-MAL measurements where the starting stoichiometry was only\n2:1. It isn’t clear to me that either 1. There isn’t evidence of a higher MW complex at the front edge of the complex elution\npeak or 2. That there couldn’t be a higher molecular weight complex where a second MYC (or second kinase) is added with\na much lower affinity. For example, this paper from Ben Schuler’s group describes and archetypal dynamic complex:\nhttps://www.nature.com/articles/nature25762 . The original work concluded it was a 1:1 complex. However, later\ninvestigation at different concentrations revealed the existence of trimers and even phase separated droplets:\nhttps://www.nature.com/articles/s41586-023-06329-5 . I think that it is likely fair to conclude that a 1:1 complex largely\ndominates the measurements shown here, but I don’t know if its reasonable to conclude that a 1:1 complex is the only\ncomplex that can form. I agree, with the authors that the SAXS data as shown also suggests a 1:1 complex. However, we\ndo not see what the elution profiles look like for these data and the same concentration dependence would apply here as does\nfor the SEC-MALs.\n4. The nano DSF data is very compelling. It is very clear that it correlates with the HDX and show specific residues that are\ncritical to the interactions. It is also great to see that this correlates with activity. One minor comment is that this should be\nreferred to as “DSF” data and not “Prometheus” data in the text.\n5. I liked the structural modeling section. Perhaps I have missed this in the literature, but, to my knowledge, this is the first\nuse of AF structures in this way. That said, I think the more appropriate way to have approached this (as opposed to sorting\nby how well individual structures fit the experimental data) would be to use one of the methods that is established in the IDP\nfield for fitting saxs data to ensembles. Either the EOM method included in the ATSAS package or any type of ensemble\nreweighting (https://pubs.acs.org/doi/10.1021/acs.jctc.8b01231 for example). Given that the structural details seemed quite\nsimilar for the full ensembles versus the filtered, I don’t suspect there would be a huge difference in the conclusions for this\npaper, but the method would be more appropriate and the final results could be a bit cleaner.\n6. Finally, I think that it is worth noting that the introduction contained a great level of detail that keys the reader in to why this\nparticular interaction could be important in a broader biological setting. This is not always the case in biophysics papers.\nA few small details\n“most human cancers” in the introduction – I would prefer most specifics\nLine 121 “several additional techniques” – there aren’t many more to list, perhaps just list them\n145 – MYC is an IDR based on DLS? Also, based on the long retention time on the SEC column, presumably the SAXS\nprofile and the NMR HSQC peak dispersion\n163 – SAXS Kratky analysis is referenced, but is not explained\n167 – It is worth noting that the two MYC constructs overlay nearly perfectly, also supporting the fact that they are disordered\nand there are no significant long-range interactions", "reviewer_3": "The manuscript by Hultman and coworkers show an integrative structural biology approach to describe the complex\nbetween the first 100 residues in the transcription factor N-Myc and the Aurora A kinase. They conclude that the complex is\n“fuzzy” at 1:1 stoichiometry and formed with a strength of 1-4 micromolar. They show that binding to Aurora A leads to its\nactivation and that binding can be inhibited by a competitive binding partner. The data have been carefully collected, and the\nmanuscript is well written and provide new insight into the system under investigation. However, I have some reservations\nabout the conclusion they reach - that the data supports a complex of multiple exchanging bound states; a conclusion – if\ntrue, that will need much more data to be confirmed, as detailed below. Also, there are already some studies on dynamic IDP\n– kinase interactions that have not been mentioned and referred to, but which are highly relevant for this study.\nMy main reservation is related to the statement of this complex being dynamic and fuzzy. This conclusion in reached from\ndisappearance of the peaks from the NMR spectrum and lack of protection against exchange monitored by HDX-MS. This\ndoes not necessarily mean that the complex is disordered. The problem is that we cannot observe the bound state (see\ncomments to HDX-MS below). First, there are no experimental data on the dynamics (with is provided by rates) of this\ncomplex, so how can the authors interpreted it as dynamic? Not forming secondary structure in binding can be a case where\nno protection will emerge in HDX MS. The disappearance of the NMR peaks may simply be due to exchange broadening\nbetween two states – a free and a (single) bound state – caused by the exchange rate being in the intermediate exchange\nregime for NMR and not due to the presence of multiple states in the bound state (fuzzy). The conclusion that these regions\n“shows extensive dynamics” are not supported by the data.\nIt will be necessary to record data on the bound state to provide insight into the structure and the dynamics of it, and not infer\nits properties from lack of signals, which can have many origins depending on the exchange rate, size of complex etc. Insight\ninto the dynamics can be done either by titration of the complex, so that R1rho and/or R2s can be obtained under sub-\nsaturation (5% bound, 10% bound, 20% bound) as done e.g. by the Blackledge group, or recording of CMPG.\nMore appropriately, it should be attempted to access the bound state structure by e.g. CEST to get the 13C chemical shifts\nunder similar sub stoichiometric conditions. Running CD would also be a possibility to access if there are folding-upon\nbinding of part of the N-Myc as suggested by the ITC. Is it not likely that the two transient helices of N-Myc would fold upon\nbinding as they are involved in the contacts? A mutation to break the helices would add some information.\nWhat are the protein concentrations in the HDX-MS experiments? It is stated that the complex is in a 1:3 molar ratio, but\nthere is no information on concentration only volume. The ratio of 1:3 means that there are lots of free N-Myc present.\nDepending on the concentration, with a Kd of 1 micromolar, working e.g. at 1 micromolar concentrations will only produce\nabout 40% bound state. Thus, the contribution to protection will be dominated by the free N-Myc.\nIf this is a disordered complex and if the complex is dynamic, effects on mutation of the different sites should be relatively\nindependent. Thus, recording NMR relaxation on the variants that abolished some of the main sites (FYPD, FYFG and\nWKKF), would support this. It is interesting that in the context of the long N-Myc variants, there is less loss of intensity,\nsuggesting a shift in the exchange regime by moving to higher affinity, again questioning the extreme dynamics of the\ncomplex.\nDynamic complexes with kinases have been shown before and is likely not as novel as suggested by the authors, and the\ndynamics in these kinase complexes serve different functional roles, e.g., facilitating multisite phosphorylations (e.g., Orand\n& Jensen 2025; Kragelj Biomolecules 2021; Hendus-Altenburger et al., BMC biology 2018) – is this the case here as\nwell?\nIs N-Myc a substrate of active Aurora? Or does the first 100 residues of N-Myc serve as anchoring for substrate sites in the\nremaining N-Myc?\nThe presence of FXF sites in N-Myc resembles sites for ERK2 and JNK interactions as shown in Orand & Jensen 2025;\nKragelj Biomolecules 2021; Hendus-Altenburger 2018 and more. Does N-Myc phosphorylation contribute to the activity\nassay conducted? Not clear how this assay works from the description in the manuscript. How does these sites in N-Myc\ncompare to the FXFP sites for MAPK interactors generally observed, should be discussed (described by the Kornfeld and\nthe Ghoose groups in the 90'ies). A discussion on motifs is missing.\nTargeting kinases have shown quite dramatic side effects as these molecules have multiple partners. The suggestion of\ntargeting the aromatic pockets to inhibit binding of N-Myc will also affect all other partners binding to this site. Thus, there are\nexpectedly several side effects associated with such a drug scheme. Should be considered in the discussion (see e.g. work\non JAK inhibitors)\nThe authors used the term “touchpoint” to describe the interaction. It is not clear what defines a touchpoint - is this an\naccepted term? They go on to mention “dynamic touchpoints”, how are they different from touchpoints? Why not use\ncommonly used descriptions of interactions?\nThe abstract states this study goes against the “current dogma” – but the current dogma is not clear? Several disordered\ncomplexes involving IDPs have been described, and the current view is that retained disordered in complexes covers a\ncontinuum from fully folded upon binding to fully disordered. It requires some explanation and definitions to follow what the\nauthors mean regarding “current dogma”.\nMinors:\nIt is concluded that the stability of the complex correlate with affinity, but no correlation plots or statistics have been done to\nsupport this. Can this be included to support this conclusion? Is unfolding reversible?\nThe sequence in Fig. S3 should be included in the main figures, avoid spaces in the sequence, this is confusing.\nFigure 5C, move data to SI and show the bar graphs with uncertainties instead.\nNumber of repetitions of the ITC should be at least n=3. Consider the numbers and the relevance (e.g. 3.98 +/- 0.45 should\nbe 4 +/- 0.5). Uncertainties in the ITC should be on the figures\nWhy are both DDT and TCEP added?\nMore description of the kinase assay is needed to understand the experiment\nPost transcriptional versus post translational – why use these two terms?\nLine 173, some underline under Che should be removed\nLine 183, what is residue motifs?\nLine 477 a-> alpha\nLine 478 (REF Diana)"}, "author_response": "Point-by-point response to Reviewers 1-3\nWe thank the Reviewers for their helpful, generous and critical advice, which we feel has\nsignificantly strengthened this manuscript. Importantly, the Reviewer comments have\nprompted the inclusion of new data that has further strengthened the conclusions of our work.\nIn summary, the new data include:\n• A full set of fast relaxation experiments by NMR on N-Myc at several substoichiometric\nconcentrations of Aurora A, where the very rapidly increasing R supports a dynamic\n2\ninteraction. (New Fig. 4)\n• NMR data of a 1:1 complex of N-Myc – Aurora A saturated to 93% still shows no recovery of\nsignals line broadened by intermediate or fast exchange or increased molecular weight,\nwhich together with the rapidly increasing R indicates that the interaction is dynamic on a\n2\ntime scale higher than the T2 timescale (0.1-0.2 s). (New Supplementary Fig. 5)\n• A new set of HDX-MX experiments to analyse the complex at 1:1 stochiometry and high\nsaturation in order to evaluate the protection of bound N-Myc. We find that the protection\nobserved is well below what would be expected for a stably bound complex, suggesting\nretained dynamics (New Fig. 5E,F, Supplementary Fig. 5B). This and previous data is jointly\nuploaded to PRIDE as described below and in the manuscript.\n• As requested by Reviewer #3, we have increased the number of replicates for the ITC\nexperiments and revised figures as advised; overall results were unchanged (Fig. 6D and\nSupplementary Fig. 9).\nFurther to this, we have included:\n• a correlation plot, requested buy reviewer #3, between the K ’s and the DT s as presented in\nD m\nFig. 5E showing a R value of 0.95 supporting the correlation between N-Myc interaction\na\\inity and thermal stabilization of Aurora A (New Fig. 5E)\n• Full SEC-MALS elution profiles as requested by reviewer #2 are now provided in\nSupplementary Fig 1 and 3 supporting monodispersity of our investigated complexes.\nThe entire manuscript has been edited for clarity with special attention to items raised by the\nReviewers. In response to Reviewers 2 and 3, we have carefully revised wordings referring to\ndynamic properties and decided to entirely avoid the term “fuzzy complex” even in the title due\nto some unsettlement still in the field regarding its definition in molecular terms. We are still\nconvinced, and even more so with the new experimental data, that the complex is highly\ndynamic, especially as several orthogonal approaches strongly point in this direction. However,\nthe collected argumentation and discussion on this is now entirely in the discussion.\nOur point-by-point response to all Reviewer’s comments is appended below.\nWith best regards, and for all co-authors,\nJohanna Hultman Alexandra Ahlner Maria Sunnerhagen\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Hultman et al perform an integrative study of the N-Myc – Aur interaction,\nproviding insights into the architecture of this complex and the molecular determinants for\ncomplexation. The manuscript is generally well constructed, and the set of experiments\nperformed is logical. However, in places there is a lack of detail which may hamper a non-\nexpert from being able to understand the data presented. My area of expertise lies in the\napplication of HDX-MS and other MS tools for structural applications, and as a result my\ncomments are primarily aligned with this portion of the work.\nReviewer #1, Comment 1:\n- The authors perform extensive SEC-MALS and SEC-SAXS analysis. They present the resultant\ndata in various ways – e.g. Guinier analysis (line 154) and p(r) analysis (line 156). It would be\nhelpful for the authors to provide further detail as to how the data presented support their\nconclusions. For example, this could be included in the appropriate figure legends. As written,\nfor a non expert in these techniques it is hard to understand how the authors have drawn the\nconclusions presented.\nResponse:\nWe agree that the results from the SEC-MALS and SAXS data needed further explaining in order\nto guide the reader and make the conclusions more readily available.\nWe have now clarified the data interpretation in the main text, added an appropriate reference\nfrom the Svergun lab, and revised the figure legend of Fig. 2.\nReviewer #1, Comment 2:\n- Why are the authors showing two runs of AurA data in Fig. 4C? This is confusing to the reader\nand is not discussed in the text/Methods. They are pretty well identical and im not sure both are\nneeded to be shown here.\nResponse:\nDue to the heavy use of the national infrastructure, the two HDX-MS experiments included in\nthe first submission of this manuscript were independently scheduled and performed\nseparately. To enable comparison between the experiments, corresponding free AurA states\nwere run within each set of experiments. We appreciate that Reviewer #1 agrees with us that\nthe free AurA runs are indeed highly similar, or even “pretty well identical”. Still, to ensure data\nintegrity and correct interpretation, we would prefer to show the free AurA HDX-MS results for\nboth experiments in Fig 5C (former Fig 4C) to emphasize the reproducibility, increase data\ntransparency and allow the reader to make their own judgement based on the data provided.\nTo increase clarity for the reader, we have revised the main text, figure legend and annotations\nof Fig 5C (previously 4C). In the methods section, we have further clarified the experimental\nsetup of the two experimentally equal but timely distinct HDX-MS runs.\nReviewer #1, Comment 3:\n- More labels are needed in Figure 4. For example it is not clear to me the colouring shown in the\ntop part of panel A. The authors state that the colouring here represents the innate HDX pattern,\nbut that is a vague statement and further details are needed.\nI assume this has to do with percentage uptake from maximal deuteration but the ‘key’ on the\nright of the figure should be labelled to indicate this.\nResponse:\nAgreed. We have now clarified the figure legend to state that “The bars show the HDX-MS\nsequence coverage and are colored by the average deuterium uptake over all investigated\ntimepoints”. The label “ % Deuterium uptake” has been added to the color key. To ensure clarity,\nwe have followed this up with corresponding clarifications in Methods.\nReviewer #1, Comment 4:\nThe authors also need to state why they have chosen a ’75 %’ uptake (from Methods) to\nmean 100 % uptake for such an analysis. This is non-standard in the field from my experience.\nResponse:\nIn our setup, as in others, achieving 100% deuteration is impossible both due to the dilution of a\n1H O-based sample with D₂O (1:10) and as deuteration levels are commonly further reduced by\n2\nback-exchange occurring within the system. To account for this, we have set the maximum\ndeuteration level to 75% in HDExaminer and we ensure that no peptide exceeds 100%\ntheoretical uptake. The set value of 25% for system wide back-exchange is based upon the\nexperience of having used this particular HDX-MS approach for >200 HDX projects within the\nSciLifeLab Structural Proteomics Swedish national infrastructure over the last 5 years (for list of\ncitations see https://www.scilifelab.se/units/structural-proteomics/). While this approach may\nresult in a slight over- or underestimation of the actual uptake, it is important to note that this\ncorrection in HDExaminer only a\\ects the color scale of the % deuteration heatmap. The\ncomparative analysis is always based on relative comparisons of uncorrected deuteron uptake\nbetween bound and unbound states as determined from residual butterfly plots. Our approach\nallows us to obtain an accurate and reliable visualization of the overall protein without the need\nfor doing a fully deuterated control. For the experts to scrutinize all details of our analysis, we\nhave added all the visual exports from HDExaminer to the table in PRIDE, and in the manuscript\nwe have significantly detailed the description of the Methods section for the HDX-MS\nexperiments.\nReviewer #1, Comment 5:\nLabel scale bars shown in lower part of 4A and Figure 4B. Might also be useful to add labels to\nthe structures shown in Figure 4D given that they refer to key regions in the structure in the text.\nResponse:\nWell noted; we have revised Fig. 5 (previously Fig. 4) as proposed.\nReviewer #1, Comment 6:\n- The authors state different residues are involved in interactions – e.g. 162-184 (line 239). But\nthe data where protection is referred to doesn’t directly correlate with these exact residue\nnumbers (at least in terms of the labels shown). How were these numbers selected?\nResponse:\nWe thank the reviewer for pointing this out, and we agree that how the different residue\nstretches highlighted in our main text were selected was not made clear in the first manuscript\nversion.\nThe protected regions were identified based on statistics as described in our response to\nComment 7 below. Protection data is visually summarized in heatmaps, such as chiclet plots\nas shown in Fig. 5B, but we also show examples of uptake plots from key regions (Fig. 5C), and\nthe total deuterium uptake over all investigated timepoints (Supplementary Fig. 6). Detailed\nexamination of the HDX results, including the identity of each peptide in the analysis, can be\ngained by the reader from the uptake plots for the individual peptides attached as\nsupplementary files with the manuscript; these are also available on the PRIDE repository.\nTo help the reader traverse the difference between sequence labelling in the fragment map (Fig.\n5A) and peptide # labelling in the figures where protection is visualised (Fig. 5B, Supplementary\nFig. 6), we have revised the sequence labels in Fig. 5A to preferentially highlight the peptide\nsegments in Fig. 5B that correspond to structural elements in AurA (Fig. 5A). In Fig. 5B, and\nrelated to Supplementary Fig. 6, we found a typo in the Fig. 5B label, where 208-225 should\nhave been 208-236. This has now been corrected, and more thorough labelling has been added\nto the figures. In addition, we have revised the main text to better guide the reader and kept\nfocus on our key findings.\nReviewer #1, Comment 7:\n- How do the authors define ‘strong’ and ‘weak’ HDX protection (e.g. line 240 and Figure 4D)?\nThis needs clarification and a metric applied. Was any statistical analysis used to identify\nprotected regions?\nResponse:\nTo identify significant HDX protection, we used the built-in statistics in HDExaminer with the\nmost stringent significant criterion. In the revision, this is now more carefully described in\nMethods, and the corresponding significance results for our study are explicitly described in\ndetail in Table S1 available in the PRIDE repository.\nTo visualise the protected areas on the AurA structure in Fig. 5D (previously Fig. 4), we classified\nsignificant protection at early time points as ‘strong” if it persisted over several of the time\npoints measured, and ‘weak’ if it persisted over only one time point. Protected areas were only\nmapped onto the AurA structure if they were persistent in several peptide fragments in the same\nregion, as shown in Fig. 5B. To distinguish between early and late protection, we choose a\nblue/pink color scheme. In the revision, we decided to not subclassify late protection as\n“strong” or “weak”, as their patterns were quite similar. All this has now been clarified in the\nMain text and in the corresponding figure legend.\nReviewer #1, Comment 8:\n- Given the difference in affinities, which the authors measure here, what is the difference in\npercentage bound during the HDX experiments for the two Myc constructs studied? This could\nbe calculated and be helpful in interpreting the data showed. This should be stated in the text\nand a discussion of this included.\nResponse:\nBased on the equilibrium K s obtained by ITC, the percentage bound in the two HDX-MS\nD\nexperiments after the 1:10 D O dilution was 87% (AurA Run1), 69% (AurA Run2). A higher\n2\nsaturation is obtained for the slightly stronger N-Myc binder. To be noted is that in the mix-and-\ndilute HDX experiment, a ten-fold dilution step into D O is an essential component of the HDX\n2\nexperiment, but as AurA alone is unstable at concentrations above 60 µM, together with the\nmagnitude of the K s this limits the final concentration of the complex obtainable during\nD\nexchange conditions.\nIn this revision, we included a third HDX-MS experiment with the aim to better characterise any\nprotection on N-Myc in the complex. We here reached 77% bound in a 1:1 N-Myc : AurA\n1-100\ncomplex. All the information above is included and briefly discussed in text and methods.\nReviewer #1, Comment 9:\n- The authors display an output from PEAKS in Supplementary Fig. 4 where putative\nphosphorylation sites are indicated. In my experience, PEAKS can annotate very low confidence\nPTM sites in these types of reports, where the MS/MS data do not provide the requisite support\nfor the presence of PTMs at a given residue. The authors should investigate their data\nfurther, and include MS/MS spectra for all phosphosites that are confidently identified. I would\nrecommend changing this figure to one that only shows the localisation of confident\nphosphosites after this manual data curation has been performed.\nResponse:\nAs the reviewer correctly states PEAKS can be a bit optimistic in assigning PTM’s, however, this\nwas not the main purpose of this analysis. Rather, it was discovered that there was a lack of\nHDX peptide coverage in the AurA active loop region, which is known to hold phosphorylation\nsites at T287 and T288 (Bayliss et al., Mol Cell 2003). The phosphorylation analysis was done on\nthe undeuterated MS/MS samples used for generation of a HDX peptide pool and helped\nidentify peptides in this region. For the purposes of increasing HDX-MS sequence coverage, we\ntherefore show the “uncorrected” coverage as it is not needed to have 100% secure site\nassignment. Furthermore, as stated in the text there was no significant di\\erence between the\nprotection of phospho- and non-phospho peptides. To address any possible e\\ects on N-Myc\nbinding from AurA active loop phosphorylation, we designed AurA mutants and found that\nphosphorylation at these sites has very little or no e\\ect on N-Myc binding (Supplementary Fig.\n7B). A dedicated phosphosite analysis would need to have been done using ETD on a di\\erent\nMS instrument, but given our results at this level of analysis and the already well-established\nAurA phosphorylation pattern for both eucaryotic and E. coli-produced protein, we found such\nanalysis to be of little relevance to the conclusions in this work and therefore outside the scope\nof this study.\nWe have revisited the data, curated it manually and based on this analysis revised\nSupplementary Fig 7. In this revision, we have revisited the data, curated it manually and based\non this analysis revised Supplementary Fig. 7A to include only the phosphorylation sites in the\nregion spanning the AurA active loop. In the text, we have clarified that this analysis was done\nmerely for the purpose of increasing peptide coverage and not to determine the exact\nphosphorylated residues. The data is included in deposited files in PRIDE.\nReviewer #1, Comment 10:\n- In the methods indicate the protein concentrations used – this is critical for understanding the\npercentage bound in these experiments.\nResponse:\nWe agree with the reviewer; this was an unfortunate omission on our side. We have now added\nthe protein concentrations to the text including an estimated stoichiometry based on the\naffinities measured by independent techniques (ITC and BLI). See also our response to\nComment 8 above.\nReviewer #1, Comment 11:\n- What do the authors mean by the FASTA file only containing the RDB sequence (line 703)?\nResponse:\nThis is an error on our side; the text should read “the targeted protein sequence” instead of “the\nRGB sequence”. This has now been corrected.\nReviewer #1, Comment 12:\n- The authors must indicate the PRIDE dataset identifier in their manuscript – at the moment\nthis is missing (line 699)\nResponse:\nThe PRIDE dataset was deposited on the Editor’s request after our recommended manuscript\ntransfer from Nature SMB to Nature Comm was completed. At that stage we could not make\nany alteration to the already submitted manuscript text, but we submitted the update with\nPRIDE identifiers within the Data Code Availability pdf as required, and assumed this would\nalso reach the Reviewers. The data set has been in the PRIDE database since March 4th.\nIn the current revision, we have now been able to properly include this information in the Data\nAvailability Section. We have also deposited the new HDX-MS data recorded during the revision\nprocess under the same identifier.\nReviewer #1, Comment 13:\nThe authors must include experimental details for their phosphosite analysis.\nResponse:\nThis has now been described in detailed in Methods under the HDX-MS section and is\nmentioned in Results, please see our response to Comment 9 above.\nReviewer #1, Minor comments:\nLine 121 – add ‘mass’ before spectrometry.\nIn several places references are missing. E.g. line 478 and line 715. Please thoroughly proof-\nread the manuscript.\n• We apologize for these oversights and thank the reviewer for pointing this out. We have\nnow corrected these errors and thoroughly proof-read the revised manuscript.\nReviewer #2 (Remarks to the Author):\nThe N-Myc MB0-MBI region forms a specific, functional and fuzzy complex with the N-lobe of\nAurora kinase A, Hultman et al.\nThe manuscript describes the interaction between N-Myc and Aurora A. Disordered protein\nregions, like those found in Myc, are increasingly being viewed as potential drug targets. Given\nits near ubiquitous role in many cancers, Myc is frequently cited as a potential target. Finding\ninterfaces that are formed when an IDR binds to another protein are an attractive alternative to\nsimply trying to identify small molecules that can bind to IDRs in isolation. The work presented\nhere not only does a great job in characterizing the specific interaction between Myc and Aurora\nA, but also lays out a straight-forward (albeit using a number of biophysical techniques)\napproach for characterizing such interfaces in general. The manuscript is written in a clear and\nsuccinct fashion and is easy to read and follow. The figures are all well organized and clear (I\nparticularly appreciated the unified color things throughout the entire paper).\nThe authors use a combination of multiple, complementary, biophysical techniques that both\nreport on structural features and affinities. Given what is required to obtain good quality NMR\nspectra on a protein like Aurora, I appreciated the use of the HDX as a more cost-effective and\nsimpler (albeit lower resolution) alternative. With some revisions, this manuscript is certainly\nworthy of publication.\nReviewer #2, Comment 1:\nThe use of “fuzzy” and “dynamic” to describe the complex, while quite possibly true, is not\nheavily supported in the data presented. These terms explicitly describe a dynamic process,\nand the vast majority of data in the paper is ensemble averaged and does not directly report on\ndynamics. The one exception is the HDX. I think the interpretation of the time scales of\nprotection do hint at some dynamics in the complex, but the long time point (9000 s) is really\nquite long and I’m not sure how these exchange dynamics would compare to any complex,\ndynamic or no, with micromolar affinity.\nResponse:\nWe thank Reviewer #2 for appreciating our work, including our choice of HDX as an option to\nanalyse binding effects on AurA. As explained, we used HDX to identify any protection patterns\non/in AurA on binding MYC, and, as the reviewer mentioned, we also recognize that HDX will not\nresolve local flexibilities on ps-ms timescales. However, HDX has been well validated and\nappreciated as a technique able to capture changes in dynamics in a time window of seconds\nthat pertains to both folding and collective motions within, or between, larger protein domains\n(Henzler-Wildman & Kern, Nature 2007, Masson et al., Nature Methods 2019 and more). Having\nsaid this, we do agree that the longest time point (9000 s) is “really quite long” and in our\ninterpretation of data we therefore do not consider any protection/deprotection that only relies\non this, or any single, time point. In response to Reviewer #2, we have clarified in this\nmanuscript re-submission that our definition of strong and weak effects in HDX requires\nsignificant differences in deuterium uptake over at least two time points.\nReviewer #2, Comment 2:\nThe authors also cite other NMR studies of IDPs (such as Milles et al) to suggest that their NMR\ndata is in agreement with other studies of dynamic complexes. What is missing in this\ncomparison is the careful NMR relaxation dynamics experiments that are present in those\npapers. If the authors were to include R1 R2 hetNOE and relaxation dispersion analysis, the\npoint would be significantly stronger. This data would also significantly enhance the paper's\nconclusions.\nThe authors conclude that the significant line broadening on addition of Aurora is the result of\nintermediate exchange (and thus also indicative of a dynamic complex). While it is possible that\nthis is true, a more likely explanation is simply that residues in complex with Aurora are\ntumbling with a much different tau_c and are broadened relative to the other disordered\nresidues. This is quite common in complexes of IDRs with larger, folded, binding partners\nregardless of the exchange regime. In order to make this point, again, a much more detailed\naccounting of the NMR relaxation should be done.\nResponse:\nWe agree entirely with the Reviewer that a comprehensive NMR dynamic evaluation would\nincrease our understanding of the highly clinically relevant N-Myc-AurA complex. However, with\nN-Myc–AurA, our accessible experimental conditions are quite limited. Due to the very\nsignificant line broadening on AurA binding, N-Myc resonance signals in binding regions as\ndefined by CSPs are lost already at 10% AurA and do not recover at saturation, which precludes\ndirect NMR analysis of N-Myc dynamics in the bound state. In the relaxation analysis that is\ndone in for example Milles et al (2018) significant signal remains at 20% saturation, enabling\ntheir NMR studies. To explicitly demonstrate the effects of N-Myc line broadening to the reader,\nthe NMR data is now complemented by a 15N-1H HSQC for the 1:1 complex of 15N N-Myc and\n1-69\nunlabeled AurA, showing that N-Myc peaks in the binding regions are not recovered at near-\nsaturation (93%, 50uM of each component) well above the K (4uM) of the complex, performed\nD\nat the highest concentration obtainable for AurA in buffers and temperature where N-Myc is\nalso happy.\nWithin this experimentally accessible window, we were still able to determine NMR fast\nrelaxation rates for all N-Myc residues with substoichiometric amounts of AurA, as suggested\nby both Reviewers 2 and 3, but only up to a degree of saturation of 6% AurA, after which some\nsignals were too small to evaluate reliably. These data, recorded at 600 MHz, are now included\nin the Revision. We find, and show, that while N-Myc R s are not much affected by the addition\n1\nof AurA in agreement with R rates in general being small for 15N in biomolecular systems, R s\n1 2\nare very much increased as expected for a system in intermediate to fast exchange on the T\n2\ntimescale. (Baldwin and Kay, Journal of Biomolecular NMR, 2013)\nAs the reduction of the signal is much higher than proportional to the amount of ligand added,\nwe can exclude that the exchange is slow. If the system is in intermediate or fast exchange with\nrespect to T relaxation, resonances will be broadened beyond detection because of exchange\n2\nbroadening owing to the much smaller and dominant T of the larger component, the theoretical\n2\nbackground of which has been carefully described elsewhere by the Perham group (Howard et\nal, JMB 2000). With respect to the R s, we therefore find ourselves caught in a dynamic region\n2\nwhere we, at this stage, will not be able to evaluate T s to the level required for a complete\n2\ndynamic analysis, and we are also not able to measure R s or hetNOEs for the 1:1 complex due\n1\nto the extreme line broadening.\nWhat we can conclude is that N-Myc is in intermediate or fast exchange with respect to T\n2\nrelaxation, but we cannot define whether such exchange is only between the free and one\nbound state or if this also includes exchange between several bound states (avidity). However,\ndeletion of the third C-terminal N-Myc interaction with AurA (as in N-Myc ) has very similar\n1-69\neffects on AurA as judged by HDX, and furthermore, this three-touchpoint rapidly exchanging\nsystem migrates as a 1:1 complex on a gel filtration column. This suggests that the three N-Myc\ninteracting regions bind AurA and can at least partly compensate for each other with regards to\nAurA binding, suggesting allovalency and/or avidity. This is also independently concluded from\nthe SAXS-based modelling of the complex, although at low resolution. While we are committed\nto resolving the dynamic features of this complex in detail, this is outside the scope of the\ncurrent manuscript as it will require comprehensive NMR experimental optimization\n(temperatures, magnetic fields and pulse programs) and theoretical analysis, together with\nsignificantly extended mutational analysis and careful biophysics.\nWith all this in mind, and while taking care not to overinterpret data, in this revision we have\nincluded, and commented on, the fast relaxation results in the Main. In the discussion, we\ncomment on the plausible allovalency within the N-Myc - AurA complex and its agreement with\nour other biophysical data, as a basis for further investigation.\nReviewer #2, Comment 3:\nThe conclusion that the complex is 1:1 is based on SEC-MAL measurements where the starting\nstoichiometry was only 2:1. It isn’t clear to me that either 1. There isn’t evidence of a higher MW\ncomplex at the front edge of the complex elution peak or 2. That there couldn’t be a higher\nmolecular weight complex where a second MYC (or second kinase) is added with a much lower\naffinity. For example, this paper from Ben Schuler’s group describes an archetypal dynamic\ncomplex:\nhttps://www.nature.com/articles/nature25762 .\nThe original work concluded it was a 1:1 complex. However, later investigation at different\nconcentrations revealed the existence of trimers and even phase separated\ndroplets: https://www.nature.com/articles/s41586-023-06329-5 . I think that it is likely fair to\nconclude that a 1:1 complex largely dominates the measurements shown here, but I don’t know\nif its reasonable to conclude that a 1:1 complex is the only complex that can form. I agree,\nwith the authors that the SAXS data as shown also suggests a 1:1 complex. However, we do\nnot see what the elution profiles look like for these data and the same concentration dependence\nwould apply here as does for the SEC-MALs.\nResponse:\nSEC-MALS elution profiles are now included in Supplementary Fig. 1 and 3, and SEC-SAXS\nelution profiles in Supplementary Fig. 2 and 3 with the respective SAXS-scattering data\n(alsodeposited in SASBDB). None of these profiles show any indication of a heavier complex\nwith similar or higher affinity. The SAXS experiment is very sensitive to formation of higher-\nmolecular weight assemblies but shows no such indications in the scattering curve (linear at\nlow q) or Guinier analysis (molecular weight corresponds to 1:1), or in the P(r), which goes\nnicely down to 0 at longer D .\nmax\nAs in every biophysical investigation, we cannot exclude that much weaker additional binding\nevents occur. However, any biological effects of such binding would require unphysiologically\nhigh concentrations of both N-Myc and AurA. As described above, detailing significantly lower\naffinities than the low-micromolar K s identified there would be experimentally challenging due\nD\nto aggregation at higher concentrations as described above. For completeness, being in the IDP\ncommunity, we are of course aware of the possibility that phase separated droplets could\noccur when working with MYC, but we have not seen any indications of such in the N-Myc –\nAurA system.\nThe limitation of AurA concentration has been included in Methods and is referred to in the\nmain text.\nReviewer #2, Comment 4:\nThe nano DSF data is very compelling. It is very clear that it correlates with the HDX and show\nspecific residues that are critical to the interactions. It is also great to see that this correlates\nwith activity. One minor comment is that this should be referred to as “DSF” data and not\n“Prometheus” data in the text.\nResponse:\nWe acknowledge the Reviewer’s appreciation of the nanoDSF data, and we agree with the\nreviewer that we should not refer to the technique by the brand name. We have changed the\ntext to nanoDSF and refer to a methods review of the technique to assist the reader.\nReviewer #2, Comment 5:\nI liked the structural modeling section. Perhaps I have missed this in the literature, but, to my\nknowledge, this is the first use of AF structures in this way. That said, I think the more\nappropriate way to have approached this (as opposed to sorting by how well individual\nstructures fit the experimental data) would be to use one of the methods that is established in\nthe IDP field for fitting saxs data to ensembles. Either the EOM method included in the ATSAS\npackage or any type of ensemble reweighting\n(https://pubs.acs.org/doi/10.1021/acs.jctc.8b01231 for example). Given that the structural\ndetails seemed quite similar for the full ensembles versus the filtered, I don’t suspect there\nwould be a huge difference in the conclusions for this paper, but the method would be more\nappropriate and the final results could be a bit cleaner.\nResponse:\nWe thank the reviewer for raising this important point. We are, of course, aware of established\nensemble approaches such as EOM and ensemble reweighting (see our work in Caporaletti et\nal., Biophys J 2023; Salomonsson et al., Protein Science 2024), and we also tested such\nmethods in our analysis. In fact, when applying reweighting across the full pool of ~10,000\nAlphaFold-generated models, we could reach χ² values essentially at 1.0. However, in this\ncase, we judged this approach to be heavily overdetermined, since the SAXS data provides only\nlimited constraints and a sufficiently large pool of conformers allows almost any profile to be\nmatched extremely well. For this reason, we deliberately chose a stricter criterion where only\nthose individual conformations with χ² < 1.1 were retained. This procedure effectively defines\nan ensemble that is consistent with the SAXS data, while avoiding potential artifacts of\nexcessive reweighting. We view this strategy as more conservative and transparent, since it\nprovides a direct link between physically plausible models and experimental agreement,\ninstead of relying on an optimized weighted mixture over thousands of possibilities. In our\nrevision, we have pointed this item out in Methods, and, briefly, in Main.\nReviewer #2, Comment 6:\nFinally, I think that it is worth noting that the introduction contained a great level of detail that\nkeys the reader in to why this particular interaction could be important in a broader biological\nsetting. This is not always the case in biophysics papers.\nResponse:\nWe thank the reviewer for recognizing our effort on putting our work in a broader biological\ncontext. In the revision, due to lack of space, we have however had to slightly reduce some\ndetail in the introduction, but we hope this is still sufficient to key the reader in the right\ndirection as the Reviewer suggests.\nReviewer #2, A few small details\n“most human cancers” in the introduction – I would prefer most specifics\n• MYC is indeed overwhelmingly critical for nearly all human cancers, as recently\nreviewed in Nature Rev Clin Oncol, and at least 27 out of the 33 human cancer types in\nthe Cancer Genome Atlas are associated with MYC (new references added in revision).\nAs this is a molecular study, there is limited space to expand on this with more clinical\nperspectives. To meet the Reviewer, we have polished this statement and added two\ncomprehensive review articles to support it, but for specifics we will have to refer the\nreader to selected reviews.\nLine 121 “several additional techniques” – there aren’t many more to list, perhaps just list them\n• Completed\n145 – MYC is an IDR based on DLS? Also, based on the long retention time on the SEC column,\npresumably the SAXS profile and the NMR HSQC peak dispersion\n• Indeed; yes we have included these as well in the same context.\n163 – SAXS Kratky analysis is referenced, but is not explained\n• A short explanation has been included in the Supplementary/Methods\n167 – It is worth noting that the two MYC constructs overlay nearly perfectly, also supporting the\nfact that they are disordered and there are no significant long-range interactions\n• Indeed, we have included this in the revision.\n• Corrected.\nReviewer #3 (Remarks to the Author):\nReviewer #3; Comment 1:\nThe manuscript by Hultman and coworkers show an integrative structural biology approach to\ndescribe the complex between the first 100 residues in the transcription factor N-Myc and the\nAurora A kinase. They conclude that the complex is “fuzzy” at 1:1 stoichiometry and formed\nwith a strength of 1-4 micromolar. They show that binding to Aurora A leads to its activation and\nthat binding can be inhibited by a competitive binding partner. The data have been carefully\ncollected, and the manuscript is well written and provide new insight into the system under\ninvestigation. However, I have some reservations about the conclusion they reach - that the\ndata supports a complex of multiple exchanging bound states; a conclusion – if true, that will\nneed much more data to be confirmed, as detailed below. Also, there are already some studies\non dynamic IDP – kinase interactions that have not been mentioned and referred to, but which\nare highly relevant for this study.\nResponse:\nWe thank the Reviewer for appreciating our integrated structural biology approach. N-Myc\nbinding to AurA is a critical system for cancer research and holds high therapeutic potential but\nhas hitherto resisted biophysical investigation in solution. We are committed to explore and\nunderstand its molecular mechanisms.\nReviewer #3; Comment 2:\nMy main reservation is related to the statement of this complex being dynamic and fuzzy. This\nconclusion in reached from disappearance of the peaks from the NMR spectrum and lack of\nprotection against exchange monitored by HDX-MS. This does not necessarily mean that the\ncomplex is disordered. The problem is that we cannot observe the bound state (see\ncomments to HDX-MS below). First, there are no experimental data on the dynamics (with is provided by\nrates) of this complex, so how can the authors interpreted it as dynamic? Not forming\nsecondary structure in binding can be a case where no protection will emerge in HDX MS.\nResponse:\nWe appreciate the Reviewer’s request for clarity in this respect. The reviewer is correct in that\nwe had weak HDX-MS information about the dynamics of the bound state due to the presence\nof free N-Myc in the HDX-MS runs in the submitted manuscript (1:3 ratio AurA:N-Myc). In the\npresented experiment it is true that the excess of free N-Myc would make it appear as largely\nunprotected in HDX-MS. However, given the large and clear perturbation on AurA it was still\nvery surprising that N-Myc appeared to be so dynamic while 1/3 is bound. On the contrary, if N-\nMyc would have been a strong binder with a low koff it should be possible to observe a bimodal\n(1:3) isotope distributions for the interacting peptides, but this was not seen for N-Myc. With\nexperience from hundreds of projects over 8 years, our HDX-MS expert Dr Simon Ekström at the\nSwedish National infrastructure (https://www.scilifelab.se/units/structural-proteomics/) has\ndone quite a few IDP interaction studies and bimodality is something he has seen in many HDX-\nMS IDP interaction projects before, and where there is a stronger interaction, it has even been\npossible to deduce binding stoichiometry. The HDX data presented here are actually the first\ntime ever that Dr Ekström has seen an IDP that retains this high uptake while bound; indeed,\neven peptide binders usually have a clearer protection than observed for here for N-Myc.\nIn response to the reviewer, we have now complemented the HDX-MS data with a run at a 1:1\nratio N-Myc:AurA at 77 % saturation. While the same pattern of large HDX-MS perturbations\nwere observed on AurA, only very weak perturbations were observed on N-Myc and only at the\nshortest time point. Essentially, we see the same thing for N-Myc as in our first set of\nexperiments: regardless of free or bound form, the uptake is very high over the entire sequence.\nThis is a clear sign that from an HDX perspective, N-Myc has a very high o\\ rate while\nmaintaining a high “occupancy” presumably due to high association rate on AurA, which agrees\nwith the large observed perturbation of DHDX for AurA (1-3 Da) while DHDX for N-Myc is\nsignificantly lower (0.2-0.6 Da).\nOf note, the effects on AurA as judged from HDX are very similar for N-Myc1-69 and N-Myc .\n1-100\nThis implies that including the third binding site in N-Myc as evident by NMR I/I0 and CSPs does\nnot seem to add another binding site on AurA, suggesting possible allovalent properties in this\ninteraction. In agreement, the SAXS-based ensemble model suggest that all N-Myc binding\nregions contact the same regions on AurA irrespective of # binding sites, and the SAXS data is\nalso not consistent with any single conformation where all binding regions on N-Myc are\nsimultaneously bound to AurA. Together with the observation that the N-Myc-AurA complexes\nstudied here elute as 1:1 on a gel filtration column despite the low-micromolar affinities\nobserved, this suggests allovalency which is a heterogeneous binding model common to\ndynamic IDP interactions. Together with the orthogonal evidence from other methods clearly\npointing in the same direction, this is a clear hallmark of a dynamic interaction.\nResponding to Reviewer #2, in this revision we have included a new HDX-MS data set further\nvalidating the statement of a dynamic complex as described above and present the\nconclusions in Results. To help the reader, in the Discussion we now review all the data in our\nstudy that support allovalent binding properties of this interaction, encouraging the Reader to\nmake their own judgement.\nReviewer #3 Comment 3:\nThe disappearance of the NMR peaks may simply be due to exchange broadening between two\nstates – a free and a (single) bound state – caused by the exchange rate being in the\nintermediate exchange regime for NMR and not due to the presence of multiple states in the\nbound state (fuzzy). The conclusion that these regions “shows extensive dynamics” are not\nsupported by the data.\nIt will be necessary to record data on the bound state to provide insight into the structure and\nthe dynamics of it, and not infer its properties from lack of signals, which can have many origins\ndepending on the exchange rate, size of complex etc. Insight into the dynamics can be done\neither by titration of the complex, so that R1rho and/or R2s can be obtained under sub-\nsaturation (5% bound, 10% bound, 20% bound) as done e.g. by the Blackledge group, or\nrecording of CMPG.\nMore appropriately, it should be attempted to access the bound state structure by e.g. CEST to\nget the 13C chemical shifts under similar sub stoichiometric conditions.\nResponse:\nWe agree entirely with the Reviewer that a comprehensive NMR dynamic evaluation would\nincrease our understanding of the highly clinically relevant N-Myc-AurA complex. However, with\nN-Myc–AurA, our accessible experimental conditions are quite limited.\nDue to the very significant line broadening on AurA binding, N-Myc resonance signals in binding\nregions as defined by CSPs are lost already at 10% AurA and do not recover at saturation, which\nprecludes direct NMR analysis of N-Myc dynamics in the bound state. In the relaxation analysis\nthat is done in for example Milles et al (2018) or Delaforge et al (2018) significant signal remains\nat 20% and 10% saturation, enabling their NMR studies. To explicitly demonstrate the effects of\nN-Myc line broadening to the reader, the NMR data is now complemented by a 15N-1H HSQC for\nthe 1:1 complex of 15N N-Myc and unlabeled AurA, showing that N-Myc peaks in the binding\n1-69\nregions are not recovered at near-saturation (93%, 50µM of each component) well above the KD\n(4uM) of the complex. These experiments were performed at the highest concentration\nobtainable for AurA in buffers and temperature where N-Myc is also happy.\nWithin our experimentally accessible window, we were still able to determine NMR fast\nrelaxation rates for all N-Myc residues with substoichiometric amounts of AurA, as suggested\nby both Reviewers 2 and 3, but only up to a degree of saturation of 6-10% AurA, after which\nsome signals were too small to evaluate reliably. These data, recorded at 600 MHz, are now\nincluded in the Revision. We find, and show, that while N-Myc R s are not much affected by the\n1\naddition of AurA in agreement with R rates in general being small for 15N in biomolecular\n1\nsystems, R s are very much increased as expected for a system in intermediate to fast exchange\n2\non the T timescale. (Baldwin and Kay, Journal of Biomolecular NMR, 2013)\nwe can exclude that the exchange is slow. If the system is in intermediate or fast exchange with\nrespect to T relaxation, resonances will be broadened beyond detection because of exchange\n2\nbroadening owing to the much smaller and dominant T of the larger component, the theoretical\n2\nbackground of which has been carefully described elsewhere by the Perham group (Howard et\nal, JMB 2000). With respect to the R s, we therefore find ourselves caught in a dynamic region\n2\nwhere we, at this stage, will not be able to evaluate T s to the level required for a complete\n2\ndynamic analysis, and we are also not able to measure R s or hetNOEs for the 1:1 complex due\n1\nto the extreme line broadening.\nWhat we can conclude is that N-Myc is in intermediate or fast exchange with respect to T\n2\nrelaxation, but we cannot define whether such exchange is only between the free and one\nbound state or if this also includes exchange to several bound states (avidity). However, as\ndescribed in “Response to R3;2” above, the presence of two (N-Myc ) or three (N-Myc )\n1-69 1-100\ninteracting regions has very similar effects on AurA as judged by HDX-MS, and furthermore,\nboth these constructs similarly migrate as 1:1 complexes on a gel filtration column as detected\nby SEC-SAXS. This suggests that the N-Myc interacting regions can at least partly compensate\nfor each other with regards to AurA binding, suggesting allovalency and/or avidity. While we are\ncommitted to resolving the dynamic features of this complex in detail as proposed by Reviewer\n#2, this is outside the scope of the current manuscript as it will require comprehensive NMR\nexperimental optimization (temperatures, magnetic fields and pulse programs) and theoretical\nanalysis, together with significantly extended mutational analysis and careful biophysics.\nAs advised by Reviewers #2 and #3, in the revision we have now added new NMR data showing\ndramatically increased N-Myc R s under sub-stoichiometric AurA conditions (2, 6 and 10%),\n2\nsuggesting intermediate to fast exchange relative to the T relaxation timescale.\nand its agreement with NMR our other biophysical data, as a basis for further investigation.\nTaking care not to overinterpret data, and in the absence of a full CPMG/CEST NMR analysis, we\nhave toned down the conclusions regarding the dynamic state of the interaction throughout the\nmanuscript.\nReviewer #3 Comment 4:\nRunning CD would also be a possibility to access if there are folding-upon binding of part of the\nN-Myc as suggested by the ITC. Is it not likely that the two transient helices of N-Myc would fold\nupon binding as they are involved in the contacts?\nResponse:\nIndeed, as judged by CheSPI there is a very small propensity for helicity in segments comprising\nresidues 48-53 in N-Myc , as well as in residues 75-86 in N-Myc . In the longer N-Myc\n1-69 1-100\nfragment, the helicity in residues 48-53 in N-Myc is slightly decreased and no longer\n1-69\nvalidated by the CheSPI DSSP prediction (Supplementary Fig. 4). In the complex, as judged by\nNMR (very small CSPs) and HDX-MS (no reduced accessibility in N-Myc), the two possibly\ntransient helical segments do not seem prone to folding-on-binding. By CD, the already very\nlarge amount of helix in AurA would make it very difficult to measure a small helical increase on\nbinding, and even if such changes would be measurable, they would not be possible to directly\nattribute to N-Myc as CD cannot distinguish between helicity increase in the two binding\npartners. Furthermore, as the MBI region is extremely conserved across MYC homologues, any\ndesigned mutation here, and in particular to proline, would likely affect N-Myc interactions in\nunforeseen ways, rendering such an experiment inconclusive to address the Reviewer’s\nconcerns.\nTo respond to the Reviewer’s concerns regarding whether there is folding-upon-binding of part\nof the N-Myc, we have for this revision performed HDX-MS experiments optimized for detecting\nN-Myc in the bound state. This method has been validated to recognize the formation of helical\nstructure in INCENP binding to Aurora B, where significant changes in the HDX-MS pattern of\nthe INCENP “IN-box” peptide revealed folding-on-binding onto the N-lobe (Segura-Peña et al.,\neLife 2023). In contrast, our N-Myc optimized HDX-MS measurements on N-Myc – AurA\nperformed at 77% saturation in a 1:1 molar ratio shows very similar rapid H/D exchange in the\nfree and bound states of N-Myc, which indicates that N-Myc folding-on-binding into secondary\nstructure is not a primary mechanism here (see also “Response to comment 2” above). While\nthis may seem contradictive to our ITC measurements, we need to keep in mind that ITC cannot\nresolve between these effects on a molecular level, as calorimetry considers the system as a\nwhole.\nIn the revised text, the comparison with INCENP mentioned above has been introduced into the\nmanuscript, and the text regarding transient helical segments has been revised to be more\nprecise. To avoid overinterpretation, the thermodynamic profiles are now only pointed out as\nbeing similar, leaving out any molecular interpretations as these can not be distinguished from\nthe thermodynamics alone\nReviewer #3 Comment 5:\nWhat are the protein concentrations in the HDX-MS experiments? It is stated that the complex\nis in a 1:3 molar ratio, but there is no information on concentration only volume. The ratio of 1:3\nmeans that there are lots of free N-Myc present. Depending on the concentration, with a KD of 1\nmicromolar, working e.g. at 1 micromolar concentrations will only produce about 40% bound\nstate. Thus, the contribution to protection will be dominated by the free N-Myc.\nResponse:\nWe have now provided details on the saturation levels in the HDX-MS experiments, including\nconcentrations and level of saturation, both in Methods and in Results. The HDX-MS\nexperiments optimized to detect AurA protection, as described in the first submission, were\nperformed at three-fold excess of N-Myc at concentrations ensuring saturation levels on AurA\nat 69 and 87% for N-Myc and N-Myc , respectively, in the D O-diluted sample intrinsic to\n1-69 1-100 2\nthe HDX experiment. Thus, the contribution to AurA protection will be dominated by bound N-\nMyc, despite the experimental limitations imposed and the solubility limit for AurA (50 µM).\nFurthermore, in this revision, and to resolve any protection of N-Myc contributed by AurA\nbinding, we have included an additional HDX-MS experiment at 1:1 ratio of N-Myc to AurA, at a\nconcentration ensuring 77% saturation. The protection of AurA is very similar, and the\nexperiment allowed us to evaluate the possible protection of N-Myc in the complex with very\nlittle contribution from free N-Myc.\nReviewer #3 Comment 6:\nIf this is a disordered complex and if the complex is dynamic, effects on mutation of the\ndifferent sites should be relatively independent. Thus, recording NMR relaxation on the variants\nthat abolished some of the main sites (FYPD, FYFG and WKKF), would support this.\nResponse:\nWe are not sure what the Reviewer means by that the effects should be “relatively\nindependent”. We also do not state or believe that the complex is entirely disordered; we\napologize if our writing has been unclear here. However, we do agree that IF the complex would\nhave been entirely disordered, it would – according to current knowledge in the field – most\nlikely be driven by electrostatics and allovalency, and if so mutating single interaction sites\nwould not affect binding. But in our case here, such mutations reduce affinity significantly and\nimpairs AurA activity. The reduced affinity would make conclusive NMR relaxation on the\nvariants studied here even more difficult. While further mutational and dynamic analysis of this\ninteraction is a worthy objective, given the hardships of studying this particular system by NMR,\nwe believe that such studies need to be very carefully designed to answer specific questions in\na separate study.\nReviewer #3 Comment 7:\nIt is interesting that in the context of the long N-Myc variants, there is less loss of intensity,\nsuggesting a shift in the exchange regime by moving to higher affinity, again questioning the\nextreme dynamics of the complex.\nResponse:\nHere, we respectfully disagree with the Reviewer as it is not clear to us which residues\nReviewer#3 is looking at. Within MB0 and MBI, the reduction pattern in I/I is very similar in the\n0\ntwo constructs, with final intensities differing by <5% in the few cases where there is still any\nintensity remaining at 0.3 mol equivalents of AurA. Outside MB0 and MBI, in contrast to\nReviewer statement there is a notable (10-25%) higher loss of intensity in the longer construct.\nWe propose that this increased loss of intensity in N-Myc could results from higher affinity\n1-100\n(K 0.9 µM vs 4.0 µM) together with increased tumbling time due to increased M and\nD W\nsignificantly increased apparent volume in the N-Myc complex as shown by SAXS (R and\n1-100 g\nPorod volume; Suppl Table 1) and SEC-MALS (increased elution volume; Fig. 3A).\nReviewer #3 Comment 8:\nDynamic complexes with kinases have been shown before and is likely not as novel as\nsuggested by the authors, and the dynamics in these kinase complexes serve different\nfunctional roles, e.g., facilitating multisite phosphorylations (e.g., Orand & Jensen 2025; Kragelj\nBiomolecules 2021; Hendus-Altenburger et al., BMC biology 2018) – is this the case here as\nwell?\nResponse:\nWe apologize for our unclear writing in the Discussion. It was certainly not our intention to\noverlook previous critical dynamics work in the kinase field; indeed, we bring up several\nexamples of such dynamic IDP investigations later in the paragraph, including the suggested\nOrand & Jensen (2025) as well as Csizmok (2017) and, earlier, Xie et al (2020). In this revision,\nwe have also included the comprehensive review by Camacho-Zarco et al (Chemical Reviews,\n2021) which focuses on NMR evaluations with several examples of kinases.\nHowever, our aim in this paragraph of the discussion was to review cases where IDP\ninteractions have been shown to affect enzyme activity, as to date a majority of the IDP\ninteractions characterized at molecular and structural level seem to effect biology by serving as\na scaffold to bring proteins together to form a complex, rather than by directly activating\nenzymatic function. Indeed, thanks to works in the groups of Blackwell and Jensen pointed out\nby Reviewer#3, the molecular interaction landscape of mitogen-activated protein kinases\n(MAPK) kinases has been resolved in structural and dynamic detail. However, Aurora kinases\nbelong to the distinct protein kinase A (PKA) family, as structurally resolved in groundbreaking\nworks by the Taylor group and others. Within this subgroup, the Aurora kinases have quite\nspecific structural and dynamic features, including a distinct regulatory spine as described by\nthe Levinson group. Furthermore, in contrast to MAKPs, Aurora kinases have been shown to be\npredominantly activated through interactions with its N-terminal lobe as well as through ROS-\nenabled cysteine-governed dimerization. Direct comparisons with the MAPKs and their\ninteraction motifs are therefore difficult to do and would not add to the understanding of the N-\nMyc-AurA system in the current context, although we agree that this would be well considered\nin a future review on kinase-IDP interactions.\nDue to space constraints given the added data required by reviewers in this revised version of\nthe manuscript, we have been forced to limit our referencing to include only the most relevant\nkinase complexes both in the introduction and discussion, and primarily need to focus on\nreferencing reviews. However, related to this we agree with Reviewer #3 that it is relevant to\ndiscuss the function of dynamics in complexes with Aurora A including possible binding motifs;\nfor this please see our response to Reviewer #3 Comment 10. In the discussion, we also\ndedicate a MYC-centred paragraph to the discussion of tunable interactions, such as by\nphosphorylation, and how this could play significant functional roles in shifting the populations\nof alternative binding modalities.\nReviewer #3 Comment 9:\nIs N-Myc a substrate of active Aurora? Or does the first 100 residues of N-Myc serve as\nanchoring for substrate sites in the remaining N-Myc?\nDoes N-Myc phosphorylation contribute to the activity assay conducted? Not clear how this\nassay works from the description in the manuscript.\nResponse:\nN-Myc is not a known substrate for AurA, within the first 100 residues or elsewhere. The\nconsensus phosphorylation motifs for AurA are [R/K/N]-R-X-[S/T]-B, where X stands for any\namino acid and B stands for any hydrophobic residue [AFILMV] except Pro, reported by Ferrari\net al (Biochem J 2005)1, and R-X-S/T-[ILV] reported by Ohashi et al (Oncogene 2006), neither of\nwhich is present in the N-Myc sequences used here. However, c-Myc has recently been\nidentified as a substrate for Aurora B, with phosphorylation at c-Myc-Ser67 (Jiang et al., Cancer\nCell 2020). This c-Myc serine, boldfaced below and directly C-terminal to MBI, is not conserved\nin N-Myc:\nc-Myc: 58-TPPLSPSRRSGLC\nN-Myc: 58-TPPLSPSRGFAEH\nThe AurA kinase assay was performed with the ADP-GloTM kinase Assay from Promega\nCorporation (Madison, USA) to determine the activity of our recombinantly produced AurA.\nDuring the first step of the activation assay, the kinase is incubated with substrate, ATP in a\nbu\\er including MgCl , in the presence or absence of N-Myc, and the remaining ATP after\n2\ncompletion of this reaction is depleted. The generated ADP from this step is then converted to\nATP and quantified using the luciferase/luciferin reaction. This has all been detailed in\nMethods.\nOf note, none of our biophysics experiments are conducted in the presence of ATP, thus,\nphosphorylation will not be possible during our NMR, SAXS, HDX-MS or biophysics experiments.\nReviewer #3 Comment 10:\nThe presence of FXF sites in N-Myc resembles sites for ERK2 and JNK interactions as shown in\nOrand & Jensen 2025; Kragelj Biomolecules 2021; Hendus-Altenburger 2018 and more.\nHow does these sites in N-Myc compare to the FXFP sites for MAPK interactors generally\nobserved, should be discussed (described by the Kornfeld and the Ghoose groups in the 90'ies).\nA discussion on motifs is missing.\nResponse:\nAs AurA is not a MAP kinase, N-Myc is not a substrate of AurA, and MAPKs to our knowledge are\nnot active on N-Myc, a detailed comparison with described features of MAPK interactors, we\nfind it difficult to see how such a comparison would increase the understanding of the N-Myc-\nAurA system in the context of this study. However, we have shown earlier that around 50% of\nmore than 350 interactors depend on one or several conserved so-called MYC boxes (Kalkat et\nal., Mol Cell 2018; Figure 1). Beyond these conserved MYC boxes, comprising 20-50 residues\neach, interaction patterns/motifs in MYC are not yet resolved, and it is still very open whether\nthe MYC boxes comprise embedded SLIM-type motifs or if interactions rely on entire or multiple\nMYC boxes. A comparative discussion on such specific motifs is therefore difficult to make.\nHowever, we still agree with Reviewer #3 that it is relevant to discuss and compare binding\nmotifs. To this end we compared the N-Myc binding regions identified in this study with Aurora\nbinding regions identified in TPX2 (AurA), INCENP (AurB) and CEP192 (AurA) co-crystal\nstructures. Like in our study, their interacting regions all feature aromatic/aliphatic residues\nflanked mainly by charged residues, most often negatively charged which makes sense as the\nN-lobe of the Auroras is highly positively charged. However, we could not identify any unifying,\ndistinct SLIM-like motifs common to these interactors and N-Myc. Obviously this needs more\nstructural and dynamic data to be completely evaluated. We still briefly mention this\ncomparison in the discussion in the context of contextual modulation and competitive binding,\ntogether with the need for further mapping of the structural and dynamic diversity of these\ncomplexes.\nReviewer #3 Comment 11:\nTargeting kinases have shown quite dramatic side effects as these molecules have multiple\npartners. The suggestion of targeting the aromatic pockets to inhibit binding of N-Myc will also\naffect all other partners binding to this site. Thus, there are expectedly several side effects\nassociated with such a drug scheme. Should be considered in the discussion (see e.g. work\non JAK inhibitors)\nResponse:\nWe appreciate that Reviewer#3 is already considering implementations of drug design based on\nour current model study. Current drug targeting efforts of AurA has so far been limited to direct,\nATP-like active-site inhibitors disrupting the enzymatic function of AurA. As described and\nreferenced in the manuscript, although such inhibitors have shown certain effects in preclinical\nstudies, their efficacy in clinical trials is poor, and it appears that simply blocking Aurora kinase\nactivity is usually not enough to achieve maximum therapeutic efficacy. Alternative strategies\nto target AurA therefore need to be explored, including targeting of other regulatory interaction\nsites.\nAlthough our study bears promise towards this direction, we believe that it is way too early to\ndiscuss pros/cons of potential future drug schemes in more detail here. Evaluating such an\ninhibitor for cross-reactivity to other kinases would be a classic step in kinase inhibitor drug\ndevelopment. Only when such an inhibitor is developed, can this specificity issue be\naddressed.\nReviewer #3 Comment 12:\nThe authors used the term “touchpoint” to describe the interaction. It is not clear what defines\na touchpoint - is this an accepted term? They go on to mention “dynamic touchpoints”, how are\nthey different from touchpoints? Why not use commonly used descriptions of interactions?\nResponse:\nWe appreciate the reviewer’s concern for clarity and have revised to commonly used\ndescriptions of interaction depending on context.\nReviewer #3 Comment 13:\nThe abstract states this study goes against the “current dogma” – but the current dogma is not\nclear? Several disordered complexes involving IDPs have been described, and the current view\nis that retained disordered in complexes covers a continuum from fully folded upon binding to\nfully disordered. It requires some explanation and definitions to follow what the authors mean\nregarding “current dogma”.\nResponse:\nAgain, we apologize for lack of clarity. We used “current dogma” in the previous version related\nto the previous model of N-Myc-AurA interaction, which confines this to the N-Myc segment.\n61-89\nThis model has become somewhat of a “dogma” as it led to a series of publications focused on\ninteraction to this region, and our results go against this model in several ways. We have now\nrevised our wording regarding this to exclude the wording “current dogma” and instead highlight\nit in the discussion as an alternative model that may be relevant in specific situations as part of\na contextual modulation by multiple binding sites on N-Myc and AurA.\nReviewer #3 Minors:\nIt is concluded that the stability of the complex correlate with affinity, but no correlation plots or\nstatistics have been done to support this. Can this be included to support this conclusion? Is\nunfolding reversible?\n• In the first version, we did not include a correlation plot as the data points (# mutations,\nconstructs) are too few to support any statistical evaluation. However, with the data we\nhave, the correlation is close to linear and is now included in Main for completeness. In\nthe nanoDSF setup, unfolding of AurA is not reversible, which is commonly observed in\nthis setup, in contrast to a DSC experiment optimised to obtain equilibrium at each step\nof the denaturation and with optimized cell surfaces to resist aggregation.\nThe sequence in Fig. S3 should be included in the main figures, avoid spaces in the sequence,\nthis is confusing.\n• In the reviewed manuscript, the sequence in Supplementary Fig. 3 is that of N-Myc\nwhereas the HDX-MS experiment in Main Figure - which we assume the Reviewer is\nreferring to - instead shows the AurA protection pattern, where, due to space\nconstraints, only amino acid numbers and secondary structure elements of AurA is\nincluded. With the new HDX-MS experiment performed to target N-Myc protection (see\nabove), both Fig. 5 and Supplementary Fig. 6 in the revised manuscript have been\nremade with special attention to clarity.\nFigure 5C, move data to SI and show the bar graphs with uncertainties instead.\nNumber of repetitions of the ITC should be at least n=3. Consider the numbers and the\nrelevance (e.g. 3.98 +/- 0.45 should be 4 +/- 0.5). Uncertainties in the ITC should be on the\nfigures\n• We have now contributed triplicates of the ITC measurements in Fig. 5C, moved the data\nto Supplementary Fig. 9 and report the requested bar graph. Regarding accuracy, we\nhave revised to two significant digits for all reported ITC parameters.\nWhy are both DDT and TCEP added?\n• Both TCEP and DDT are strong reducing agents. We use both to take advantage of the\nstability of TCEP, making it suitable for long-term measurements and storage, while the\nless bulky structure of DDT can sometimes allow for more e\\iciency. DDT is also more\ncost e\\icient.\nMore description of the kinase assay is needed to understand the experiment\n• The kinase assay, which is a commercial assay extensively used in the field, is now\ndescribed in detail in Methods; see also our response above.\nPost transcriptional versus post translational – why use these two terms?\n• We agree and have revised to post-translational throughout.\nLine 173, some underline under Che should be removed\nLine 183, what is residue motifs?\nLine 477 a-> alpha\nLine 478 (REF Diana)\n• These unclarities have now been revised.\nREVIEWER COMMENTS\nPoint-2-point response on review after first revision\nReviewer #1 (Remarks to the Author):\nThe authors have thoroughly addressed all of my comments in this revision. The manuscript\noverall significantly advances our understanding of the interaction mode between AurA and\nN-Myc, and is suitable for publication in its current form.\nAuthors response:\nWe thank Reviewer #1 for the kind appreciation of our work, and for insightful and helpful\nsuggestions during the review process.\nReviewer #3 (Remarks to the Author):\nHultman and coworkers have presented a revised manuscript where they have gone far to\nprovide the additional experimental data requested which aide to support their conclusions.\nI appreciate the responsiveness of the authors to the suggestions made and in clarifying the\ntext, not overinterpreting their findings. The manuscript is clearly improved with the new\ndata, and with the attention to the textual formulation. However, I find that there are still a\nfew issues and comments remaining that must be addressed:\nReviewer #3 Comment 1:\nThere is some inconsistency in the SEC-SAXS and the ITC, that is not reflected upon. While\nSEC SAXS suggest a 1:1 complex, the ITC is probing a 2:1 complex (molar ratio 0.5). Weak\nbinding as 5 micromolar are often difficult to capture in co-elution on the SEC, so what is the\norigin of this discrepancy? Other disordered complexes form higher order complexes, and\nthis may be very relevant to address here as well. Some explanation is warranted.\nAuthors response:\nWe acknowledge that applying the 1:1 ITC model is a first approximative description of the\nlandscape of bound states, and that the ITC N-value ranges from 0.45 to 0.7 even with our\nextensive experimental optimisation. The N-value in ITC only properly reflects stoichiometry\nif the number of distinct binding events is identical to those reflected in the model. If we have\nmultiple binding sites of varying affinities and thermodynamic profiles (low/high DH) in the\ntitrating molecule, the N value will be lower than 1 when fitting a 1:1 model to the data, as\nadditional sites will then contribute to early saturation (see for example Watson et al., JMB\n2022). This agrees with the presence of several binding regions as suggested from our NMR\nand HDX data. More complex multisite models in the ITC fitting slightly increase the total N-\nvalue (N1+N2 etc), but many of the parameters are then poorly determined. As not to\noverinterpret our data, and to allow direct comparisons between constructs that may\ncontain different numbers of binding regions, we have applied the 1:1 model to all our ITC\ndata.\nOur combined data suggests an interaction involving multiple binding sites on both\ninteraction partners, however regarding stoichiometry, this can still be 1:1, as any or all sites\ncan be occupied by the same receptor at any given time, even if the affinity of each site is\nweak. This would be consistent with co-elution on a SEC column, which could be facilitated\nby the multiple interaction sites maintaining complex formation despite the relatively low\naffinities. As reported by us in detail in Revision#1, all our stochiometric data (SAXS, SEC-\nMALS) are consistent with a 1:1 stoichiometry, with no evidence of higher stoichiometries\nsuch as 2:1. Therefore, our data gives us no scientific support to suggest higher AurA\noccupancy than 1:1.\nAction:\nIn Results, we note the lower-than-one N-value and provide a short explanation based on\nmultiple binding regions, with reference to Watson et al JMB 2022. We also explain how this\nis consistent with co-elution on SEC. In Methods, we include and describe our choice of\nbinding model with motivation as above.\nReviewer #3 comment 2:\nCan NMR data help here, in the sense that the peak movements – even at the border of the\ndisappearing regions – may respond differently at the different stoichiometric ratios. This\nwill add to the complexity of the binding mode, but given the disordered nature of it, perhaps\nnot too surprising.\nAuthors response:\nWhile assessing the N-Myc- AurA interaction by NMR we lose peak intensity at very low\nstoichiometries in a non-recoverable manner for residues both in and adjacent to the\nbinding region, with intensity remaining only at the N- and C-termini at 1:1 (see Revision #1).\nAs this limits the range of stoichiometries that we can access by NMR, we cannot confidently\nrefine our binding model to higher degrees of complexity using peak movements at this stage.\nReviewer #3 Comment 3:\nIf you have multiple binding sites in N-Myc that are non-identical, binding to multiple non-\nidentical sites on Aurora A (discussion p. 17, line 508), is it then allovalency? (see e.g.\nLocasale, Allovalency revisited: an analysis of multisite phosphorylation and substrate\nrebinding. J Chem Phys 128:115106. doi:10.1063/1.2841124). In my opinion, and according\nto the definitions, it is not. It appears instead to be a case of multivalency. Please reconsider\nand relate to the definitions of different binding mechanism described in the literature.\nAuthors response:\nIn Revision #1, as requested by Reviewers #2 and #3, we paid special attention to revise\nwording referring to dynamic properties of the complex. We agree that definition of\nallovalency - with multiple identical binding sites - is not the case here although all binding\nsites include aromatic residues that affect binding when mutated. We have therefore now\nalso removed the word “allovalent” from the mentioned sentence in the discussion, which\nis the only time this word is used in the manuscript.\nHow to describe and understand IDP interactions is vividly discussed in the field and the\nliterature classifying different binding models is vast and diverse. Within the scope and\nspace of this article, we do not have space to include specific descriptions of the definitions,\nbut refer to recent, comprehensive work to clarify these concepts for IDPs (Holehouse and\nKragelund, Nature Rev Mol Cell Biol 2024). “Multivalency” within the IDP field is currently\nmainly used in the context of phase separation. The “fuzzy” definition broadly encompasses\nmuch of the IDP binding events but has little mechanistic detail. Within the limits of our\nexperimental data, the N-Myc-AurA complex can be described as multivalent with high\ndynamics, but it could just as well be described as fuzzy. As we do not yet have any direct\nkinetic data to inform us about the specific binding mechanism, we prefer not to introduce\na mechanistic discussion in this work as this would risk overinterpretation of our current\ndata.\nAction:\nIn the discussion we summarise our experimental data and conclude that “all these results\nhint at a mode of interaction where several regions in N-Myc contact multiple binding sites\non AurA in a dynamic manner, resulting in a complex with multivalent and/or fuzzy\nproperties.”\nReviewer #3 Comment 3:\nIt seems that the region of MB0 needs adjusting based on the R2s and on the I/I0 – it appears\nto extend on the C-terminal side, please rethink this region.\nResponse and action:\nAs for MYC regions MBI-MBIV, the MB0 region is defined based on the conservation between\nthe different myc family proteins and their participation in protein interactions (Lourenco et\nal., Nat Rev Cancer, 2021; Kalkat et al., Mol Cell 2018). MB0 is thus not defined by the Aurora\nA interaction site. To support the reader, we have included a sequence alignment in Suppl\nFig 1 where MB0 and MBI regions are annotated in detail.\nReviewer #3 Comment 4:\nAre the R2 changes “dramatic” – or just what is expected from binding to a larger kinase?\nPlease rethink.\nAuthors response:\nThe word “dramatic” was not used in the manuscript, only in the Reviewer response in\nRevision#1 to describe the fast increase in R2 relative to the amount of added AurA. We\nagree that this wording was unscientific, and we apologize.\nIn response to the Reviewer’s concerns, it is indeed reasonable to expect that the R₂ values\nin the bound state are substantially higher than in free N-Myc, due to the anticipated\nincrease in correlation time associated with the molecular weight (M ) of the kinase.\nW\nComparing with for example Delaforge et al (JACS 2018), they determined R rates of\n2, bound\n60–200 s⁻¹ in a system size wise similar to ours.\nIf we assume that the observed increase in relaxation rate is solely attributable to the higher\nM of the IDP–kinase complex and not to conformational exchange between free and bound\nW\nstates or to internal dynamics within the bound state, then the loss of signal intensity should\nscale proportionally with the amount of added kinase. However, this is not what we observe:\nfor some residues, 100% of the signal is lost at only 10% kinase added, which is a more\nunproportional response than what was reported by Delaforge et al. This suggests a\nsignificant, additional contribution to R by fast exchange and/or multiple binding modes,\n2\nalthough our current data cannot distinguish between these possibilities. A direct\ncomparison between our data and the data in Delaforge et al is further challenged by\ndifferent experimental conditions, such as different magnetic field strengths and\ntemperatures; moreover, exchange rates are likely to differ between these two systems.\nStill, we can convincingly conclude from our NMR analysis that three distinct regions in N-\nMyc are similarly perturbed by AurA binding, as shown by CSPs, line broadening, and\nincreased R values.\nWe have clarified language in the Results section “Three distinct N-Myc regions interact with\nAurA” to ensure that the Reader notices that several factors contribute to line broadening\nand increased R2: correlation time, conformational exchange between free/bound and\nexchange processes in the bound state."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoroughly addressed all of my comments in this revision. The manuscript overall significantly advances our understanding of the interaction mode between AurA and N-Myc, and is suitable for publication in its current form.", "reviewer_3": "Hultman and coworkers have presented a revised manuscript where they have gone far to provide the additional experimental data requested which aide to support their conclusions. I appreciate the responsiveness of the authors to the suggestions made and in clarifying the text, not overinterpreting their findings. The manuscript is clearly improved with the new data, and with the attention to the textual formulation. However, I find that there are still a few issues and comments remaining that must be addressed:\nThere is some inconsistency in the SEC-SAXS and the ITC, that is not reflected upon. While SEC SAXS suggest a 1:1 complex, the ITC is probing a 2:1 complex (molar ratio 0.5). Weak binding as 5 micromolar are often difficult to capture in co-elution on the SEC, so what is the origin of this discrepancy? Other disordered complexes form higher order complexes, and this may be very relevant to address here as well. Some explanation is warranted. Can NMR data help here, in the sense that the peak movements – even at the border of the disappearing regions – may respond differently at the different stoichiometric ratios. This will add to the complexity of the binding mode, but given the disordered nature of it, perhaps not too surprising.\nIf you have multiple binding sites in N-Myc that are non-identical, binding to multiple non-identical sites on Aurora A (discussion p. 17, line 508), is it then allovalency? (see e.g. Locasale, Allovalency revisited: an analysis of multisite phosphorylation and substrate rebinding. J Chem Phys 128:115106. doi:10.1063/1.2841124). In my opinion, and according to the definitions, it is not. It appears instead to be a case of multivalency. Please reconsider and relate to the definitions of different binding mechanism described in the literature.\nIt seems that the region of MB0 needs adjusting based on the R2s and on the I/I0 – it appears to extend on the C-terminal side, please rethink this region. Are the R2 changes “dramatic” – or just what is expected from binding to a larger kinase? Please rethink."}, "author_response": "We acknowledge that applying the 1:1 ITC model is a first approximative description of the landscape of bound states, and that the ITC N-value ranges from 0.45 to 0.7 even with our extensive experimental optimisation. The N-value in ITC only properly reflects stoichiometry if the number of distinct binding events is identical to those reflected in the model. If we have multiple binding sites of varying affinities and thermodynamic profiles (low/high DH) in the titrating molecule, the N value will be lower than 1 when fitting a 1:1 model to the data, as additional sites will then contribute to early saturation (see for example Watson et al., JMB 2022). This agrees with the presence of several binding regions as suggested from our NMR and HDX data. More complex multisite models in the ITC fitting slightly increase the total N-value (N1+N2 etc), but many of the parameters are then poorly determined. As not to overinterpret our data, and to allow direct comparisons between constructs that may contain different numbers of binding regions, we have applied the 1:1 model to all our ITC data.\nOur combined data suggests an interaction involving multiple binding sites on both interaction partners, however regarding stoichiometry, this can still be 1:1, as any or all sites can be occupied by the same receptor at any given time, even if the affinity of each site is weak. This would be consistent with co-elution on a SEC column, which could be facilitated by the multiple interaction sites maintaining complex formation despite the relatively low affinities. As reported by us in detail in Revision#1, all our stochiometric data (SAXS, SEC-MALS) are consistent with a 1:1 stoichiometry, with no evidence of higher stoichiometries such as 2:1. Therefore, our data gives us no scientific support to suggest higher AurA occupancy than 1:1.\nAction:\nIn Results, we note the lower-than-one N-value and provide a short explanation based on multiple binding regions, with reference to Watson et al JMB 2022. We also explain how this is consistent with co-elution on SEC. In Methods, we include and describe our choice of binding model with motivation as above.\n\nWhile assessing the N-Myc- AurA interaction by NMR we lose peak intensity at very low stoichiometries in a non-recoverable manner for residues both in and adjacent to the binding region, with intensity remaining only at the N- and C-termini at 1:1 (see Revision #1). As this limits the range of stoichiometries that we can access by NMR, we cannot confidently refine our binding model to higher degrees of complexity using peak movements at this stage.\n\nIn Revision #1, as requested by Reviewers #2 and #3, we paid special attention to revise wording referring to dynamic properties of the complex. We agree that definition of allovalency - with multiple identical binding sites - is not the case here although all binding sites include aromatic residues that affect binding when mutated. We have therefore now also removed the word “allovalent” from the mentioned sentence in the discussion, which is the only time this word is used in the manuscript.\nHow to describe and understand IDP interactions is vividly discussed in the field and the literature classifying different binding models is vast and diverse. Within the scope and space of this article, we do not have space to include specific descriptions of the definitions, but refer to recent, comprehensive work to clarify these concepts for IDPs (Holehouse and Kragelund, Nature Rev Mol Cell Biol 2024). “Multivalency” within the IDP field is currently mainly used in the context of phase separation. The “fuzzy” definition broadly encompasses much of the IDP binding events but has little mechanistic detail. Within the limits of our experimental data, the N-Myc-AurA complex can be described as multivalent with high dynamics, but it could just as well be described as fuzzy. As we do not yet have any direct kinetic data to inform us about the specific binding mechanism, we prefer not to introduce a mechanistic discussion in this work as this would risk overinterpretation of our current data.\nAction:\nIn the discussion we summarise our experimental data and conclude that “all these results hint at a mode of interaction where several regions in N-Myc contact multiple binding sites on AurA in a dynamic manner, resulting in a complex with multivalent and/or fuzzy properties.”\n\nAs for MYC regions MBI-MBIV, the MB0 region is defined based on the conservation between the different myc family proteins and their participation in protein interactions (Lourenco et al., Nat Rev Cancer, 2021; Kalkat et al., Mol Cell 2018). MB0 is thus not defined by the Aurora A interaction site. To support the reader, we have included a sequence alignment in Suppl Fig 1 where MB0 and MBI regions are annotated in detail.\n\nThe word “dramatic” was not used in the manuscript, only in the Reviewer response in Revision#1 to describe the fast increase in R2 relative to the amount of added AurA. We agree that this wording was unscientific, and we apologize.\nIn response to the Reviewer’s concerns, it is indeed reasonable to expect that the R₂ values in the bound state are substantially higher than in free N-Myc, due to the anticipated increase in correlation time associated with the molecular weight (M ) of the kinase. Comparing with for example Delaforge et al (JACS 2018), they determined R rates of 2, bound 60–200 s⁻¹ in a system size wise similar to ours.\nIf we assume that the observed increase in relaxation rate is solely attributable to the higher M of the IDP–kinase complex and not to conformational exchange between free and bound states or to internal dynamics within the bound state, then the loss of signal intensity should scale proportionally with the amount of added kinase. However, this is not what we observe: for some residues, 100% of the signal is lost at only 10% kinase added, which is a more unproportional response than what was reported by Delaforge et al. This suggests a significant, additional contribution to R by fast exchange and/or multiple binding modes, although our current data cannot distinguish between these possibilities. A direct comparison between our data and the data in Delaforge et al is further challenged by different experimental conditions, such as different magnetic field strengths and temperatures; moreover, exchange rates are likely to differ between these two systems.\nStill, we can convincingly conclude from our NMR analysis that three distinct regions in N-Myc are similarly perturbed by AurA binding, as shown by CSPs, line broadening, and increased R values. 2\nAction:\nWe have clarified language in the Results section “Three distinct N-Myc regions interact with AurA” to ensure that the Reader notices that several factors contribute to line broadening and increased R2: correlation time, conformational exchange between free/bound and exchange processes in the bound state."}]

Decision letter