TDP-43 and hnRNPA1 contain tandemly-tethered RNA-recognition-motif (RRM) domains, which not only functionally bind an array of nucleic acids, but also participate in aggregation/fibrillation, a pathological hallmark of various human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), alzheimer's disease (AD) and Multisystem proteinopathy (MSP). Here, by DSF, NMR and MD simulations we systematically characterized stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RRM domains in both tethered and isolated forms. The results reveal three key findings: (1) upon tethering TDP-43 RRM domains become dramatically coupled and destabilized with Tm reduced to only 49 °C. (2) ATP specifically binds TDP-43 and hnRNPA1 RRM domains, in which ATP occupies the similar pockets within the conserved nucleic-acid-binding surfaces, with the affinity slightly higher to the tethered than isolated forms. (3) MD simulations indicate that the tethered RRM domains of TDP-43 and hnRNPA1 have higher conformational dynamics than the isolated forms. Two RRM domains become coupled as shown by NMR characterization and analysis of inter-domain correlation motions. The study explains the long-standing puzzle that the tethered TDP-43 RRM1–RRM2 is particularly prone to aggregation/fibrillation, and underscores the general role of ATP in inhibiting aggregation/fibrillation of RRM-containing proteins. The results also rationalize the observation that the risk of aggregation-causing diseases increases with aging. TDP-43 is a well-known member of heterogeneous nuclear ribonucleoproteins (hnRNP) containing the folded RNA-recognition motif (RRM), which constitutes one of the most abundant domains in eukaryotes5. Interestingly, ~ 70 human RRM-containing proteins including FUS, TDP-43 and hnRNPA1 additionally have the intrinsically disordered prion-like domains of low-complexity sequences with amino acid compositions similar to those of the prion domains in yeast responsible for driving the formation of infectious conformers4. Remarkably, these proteins not only functionally mediate direct interactions with various nucleic acids to control both RNA processing and gene expression, but their aggregation/fibrillation is pathologically characteristic of an increasing spectrum of human diseases including ALS, frontotemporal dementia (FTD), Alzheimer’s disease (AD), chronic traumatic encephalopathy, muscle regeneration/degeneration and multisystem proteinopathy (MSP)10.Aggregation/fibrillation of TDP-43 in the cytoplasm of neurons is a pathological hallmark of ~ 97% amyotrophic lateral sclerosis (ALS) and ~ 45% frontotemporal dementia-TDP (FTLD-TDP), that lack any efficacious medicine so far11, but also extensively demonstrated to participate in disease-causing aggregation/fibrillation in additional to its C-terminal prion-like domain19. Remarkably, small molecules targeting the RRM domains of TDP-43 have been recently identified to reduce locomotor defects in drosophila model of ALS19. We also found that ATP, the universal energy currency, specifically binds the RRM domains of FUS and TDP-43 to inhibit their amyloid fibrillation22. Therefore to understand the factors and mechanisms governing their aggregation/fibrillation is not only of fundamental interest, but represents an essential step for further development of therapeutic strategies/molecules to treat these diseases.414-Residue TDP-43 contains two folded RRM domains  of 55 °C, lower than that of the human γS-crystallin with Tm of 71 °C24, but also resulting from its high conformational dynamics, that allow the dynamic opening of the structure to expose its central hydrophobic regions for aggregation/fibrillation23. Consequently, although ATP induces no enhancement of the thermal stability of the FUS RRM domain, it is sufficient to kinetically inhibit the amyloid fibrillation through weak but specific binding with a Kd of 3.8 ± 0.5 mM to a pocket within the conserved nucleic-acid-binding surfaces, which thus leads to blocking the dynamic opening of the structure19.Structurally, two TDP-43 RRM domains adopt the same overall fold shared by all RRM domains, which consists of a four-stranded β-sheet and two perpendicular α-helices Fig. B. Recent11 while the isolated RRM1 and RRM2 domains appeared to be stable and soluble as evidenced by their NMR structures determination at high protein concentrations by RIKEN Structural Genomics/Proteomics Initiative (PDB ID of 2CQG and 1WF0). We also observed that in the buffer with pH and salt concentration close to in vivo conditions (10 mM sodium phosphate at pH 6.8 with 150 mM NaCl and 10 mM DTT), the tethered TDP-43 RRM1–RRM2 could form amyloid fibrils within several days while the isolated RRM1 and RRM2 failed even after a month22. In particular, we found that under exactly the same conditions, the tethered RRM1–RRM2 of TDP-43 has only one transition with Tm of only 49 °C, lower than Tm of the isolated RRM1 (57 °C). On the other hand, the binding affinity of ATP has been characterized to be higher to the tethered RRM1 than the isolated RRM122.It has been a long-standing puzzle that the tethered RRM domains of TDP-43 were particularly prone to aggregationSo far, it remains completely unexplored for the relationship of thermodynamic stability, conformational dynamics and ATP-binding of RRM domains between the tethered and isolated forms. In the present study, with experimental methods including DSF and NMR as well as molecular dynamics (MD) simulations, we aimed to address this problem by characterizing the thermal stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RRM domains in both tethered and isolated forms. The most unexpected finding is that upon tethering, TDP-43 RRM1 and RRM2 become highly coupled to behaving as one denaturing unit with the stability significantly reduced. By contrast, no significant destabilization was observed upon tethering of two RRM domains of hnRNPA1. Our study further showed that the tethering-induced effects mainly result from the inter-domain interactions as detected by NMR characterization and analysis of inter-domain correlation motions calculated from MD simulations. Moreover, we found that ATP can also specifically bind the hnRNPA1 RRM2 but not RRM1 domain with the affinity and complex structure highly similar to those for FUS and TDP-43 RRM domains. Intriguingly, the previous and present results together revealed that ATP bind the RRM domains of both TDP-43 and hnRNPA1 with the affinity slightly higher for the tethered than for the isolated ones. Our study provides the first mechanistic insight into the tethering-induced effects on the tandem RRM domains, and highlights the general role of ATP in inhibiting aggregation/fibrillation of RRM-containing proteins, which extensively causes various human diseases by “loss of functions” or/and “gain of function”.25. As such, here we used the NMR structure (PDB ID of 4BS2) in complex with RNA11, whose Cα and Cβ chemical shifts indicative of secondary structures were shown to be very similar to those in the free state25. As shown in Fig. 11.Due to the presence of dynamics and aggregation at high concentrations, the structure of the tethered TDP-43 RRM1–RRM2 could not be determined in the free state by NMR or X-ray crystallographyHere we cloned and expressed the tethered RRM1–RRM2 of TDP-43 (102–269), as well as its isolated RRM1 (102–191) and RRM2 (191–269). Indeed as extensively observed, the tethered RRM1–RRM2 protein was prone to aggregation even at concentrations of ~ 100 μM but the isolated RRM1 and RRM2 proteins showed no significant aggregation at concentrations of ~ 1 mM. Nevertheless, the tethered RRM1–RRM2 protein has a well-dispersed HSQC spectrum typical of a well-folded protein at 50 μM in 10 mM sodium phosphate buffer containing 10 mM DTT and 150 mM NaCl (pH 6.8) Fig. : (1) HSQInterestingly, we have further acquired HSQC spectrum of the mixture of the isolated RRM1 and RRM2 at an equal molar ratio (1:1), which is very different from that the tethered RRM1–RRM2 Fig.  but almo14.Due to the critical role of TDP-43 RRM domains in amyloid fibrillation associated with various neurodegenerative diseases, previously their thermodynamic stability has been extensively characterized by a variety of biophysical methods which monitor distinctive probes associated with the secondary and tertiary structures. Nevertheless, despite exhaustive studies, it remains challenging for understanding the relationship between structure, stability and fibrillation. For example, previous far-UV CD studies in the low salt buffer indicated that TDP-43 RRM domains showed no complete denaturation of secondary structures even at 90 °C, implying that TDP-43 RRM domains might directly assemble into soluble β-rich oligomers from partially-unfolded intermediates23, the thermal denaturation is irreversible because the spectra of the samples cooled down to 25 °C after treatment at 95 °C are different from those initial spectra at 25 °C. The melting temperature (Tm) was estimated to be 55 °C for TDP-43 RRM1. Noticeably, the tethered RRM1–RRM2 sample has a Tm of 45 °C while the mixed RRM1 and RRM2 sample shows a Tm of 55 °C. As RRM2 has no Trp intrinsic fluorescence, this observation suggests that only upon covalent connection to RRM2, RRM1 is significantly destabilized with a Tm decrease of 10 °C.As the high-salt buffer with 150 mM NaCl we used triggered unacceptable noise for far-UV CD spectroscopy, we therefore conducted the thermal denaturation monitored by the intrinsic Trp UV fluorescence for the tethered TDP-43 RRM1–RRM2, isolated RRM1 and RRM2 domain, as well as their mixture at 1:1 Fig. . Due to 21 and isolated RRM122 of TDP-43 by differential scanning fluorimetry (DSF) which reports the increase of binding of the fluorescent dye due to the exposure of hydrophobic patches in the partially-unfolded intermediates which might directly assemble into β-rich oligomers. Interestingly the tethered RRM1–RRM2 has only one thermal unfolding transition with Tm of 49 °C, which increased to 54 °C with addition of ATP. The isolated RRM1 also has only one thermal unfolding transition but with Tm of 57 °C, which increased to 60 °C with addition of ATP. To understand this unexpected observation, here we measured the thermal stability of the isolated RRM2 under the same conditions. Interestingly, the isolated RRM2 has also only one thermal unfolding transition with Tm of 59 °C, which is not affected by the addition of ATP even up to 15 mM , as well as its isolated RRM1 (5–95) and RRM2 (94–184). Different from what was observed on the TDP-43 RRM domains, the tethered RRM1–RRM2 is highly soluble and has a well-dispersed HSQC spectrum typical of a well-folded protein at 50 μM Fig. : (1) CSDWe also carried out the thermal denaturation monitored by the intrinsic Trp UV fluorescence for the tethered hnRNPA1 RRM1–RRM2, isolated RRM1 and RRM2 domain, as well as their mixture at 1:1 Fig. . Due to 28. One the other hand, the isolated RRM2 has one thermal unfolding transition with Tm of 60 °C, which increased to 63 °C with the addition of ATP. The results together suggest that the tethering of hnRNPA1 RRM1 and RRM2 domain led to no significant destabilization.We further characterized the thermal stability and ATP binding of the tethered and isolated RRM domains of hnRNPA1. The tethered RRM1–RRM2 has only one thermal unfolding transition with Tm of 55 °C, which increased to 58 °C with addition of ATP Fig. A. IntrigWe further characterized the binding of ATP to the tethered and isolated RRM domains. As shown in Fig. 29, we determined Kd value of the ATP binding to hnRNPA1 RRM2 to be 4.9 and 7.7 mM respectively for the tethered and isolated forms . Interestingly, as observed on TDP-43 RRM domains, the ATP binding affinity to the RRM domains of hnRNPA1 is also higher for the tethered form than for the isolated form.With the same method we previously used to characterize the ATP-binding to the FUS and TDP-43 RRM domainsrms Fig. A, B. The31, as we extensively conducted before on the non-classic ATP-protein complexes30.Because of the extremely low binding affinity with Kd of ~ mM, it is impossible to determine the three-dimensional structure of the ATP-RRM2 complexes by the classic methods of NMR spectroscopy or X-ray crystallography. So here to visualize the complex structure, we utilized the NMR-binding derived constraints to guide the molecular docking with the well-established HADDOCK programFigure So far, we have studied the binding complexes of ATP to FUS and TDP-43 RRM domains, as well as non-classic AcD domains which are all capable of binding nucleic acids. ATP appears to always occupy the pockets within their interfaces utilized for binding nucleic acids. Although ATP has no complete binding pocket on the RRM1 domain of hnRNPA1, the three residues with large shifts of HSQC peaks induced by adding ATP are also located within the conserved surfaces for RRM domains to bind nucleic acids Fig. G.34 as we previously showed on other proteins38.To understand the dynamic basis underlying the coupling of the tethered RRM1–RRM2 of TDP-43, we conducted molecular dynamics (MD) simulations for the tethered as well as isolated RRM1 and RRM2 of TDP-43 with three parallel 50-ns simulations for each constructs. Molecular dynamics simulation is a powerful tool which can not only provide insights into the conformational dynamics that underlies protein functions, but also detect long-range inter-domain correlation motionsI of Fig. Figure 33. Briefly, this approach utilizes second-order terms from the configurational entropy expansion, called the mutual information, to identify pairs of residues with correlated conformations, or correlated motions. Figure MutInf represents an entropy-based approach to analyze ensembles of protein con-formers, such as those from molecular dynamics simulations by using internal coordinates and focusing on dihedral angles. In particular, this approach is particularly applicable for those in which conformational changes are subtleWe also conducted 50-ns molecular dynamics (MD) simulations for the tethered as well as isolated RRM1 and RRM2 of hnRNPA1 with three parallel simulations for each constructs. I of Fig. Figure Figure E. coli50. Out of various factors that modulate aggregation/fibrillation of the folded proteins, two key determinants are thermodynamic stability and conformational dynamics. Nevertheless, despite exhaustive studies, the relationship between thermodynamic stability and conformational dynamics still remains largely elusive. In human genome, many RRM-containing proteins such as TDP-43 and hnRNPA1 have two tethered RRM domains, which have been demonstrated to play a key role in the disease-causing aggregation/fibrillation. Although the previous observations imply that the tethered TDP-43 RRM domains are particularly prone to aggregation/fibrillation, so far there has been no systematic study to understand the underlying mechanisms, which, however, are of both fundamental and therapeutic interest.Protein aggregation/fibrillation has been now established to be the universal hallmark of an increasing spectrum of human diseases beyond neurodegenerative diseases, which also include cardiac dysfunction, eye cataract, degeneration of muscle and bone, as well as aging down to 54, as exemplified by the ubiquitination-induced destabilization of the modified proteins, which was proposed to function to facilitate their degradation54. Interestingly, the tethering-induced destabilization has been proposed to evolve from the trade-offs for functions53. In this regard, it is of great interest in the future to define the functional role of the unique destabilization for tandem TDP-43 RRM domains. Nevertheless, this unexpected destabilization certainly contributes to the unusually high tendency of TDP-43 in aggregation/fibrillation, which has been well established to lead to a variety of human diseases by “loss of function” or/and “gain of function”.In the present study, by both experimental and computational approaches we conducted a systematic study to characterize the thermal stability and ATP-binding by DSF and NMR, followed by MD simulations to assess the conformational dynamics of two RRM domains of TDP-43 and hnRNPA1 in both tethered and isolated forms. Very unexpectedly, the results showed that the isolated TDP-43 RRM1 and RRM2 domains have Tm of 57 and 59 °C respectively, while the tethered form has only one denaturation transition with Tm significantly reduced to only 49 °C. This set of results indicates that the tethering induced the significant coupling of two RRM domains of TDP-43, as well as dramatic destabilization. Intriguingly, no significant destabilization was observed for the tethering of two RRM domains of hnRNPA1. The results thus underscore the extreme complexity of the tethering-induced effects even for the tandem RRM domains of the different members within the same hnRNP protein family. In a general context, the dramatic destabilization for the tethered TDP-43 RRM domains is also very unusual because previously the tethering was demonstrated to have either no significant effect or even to stabilize the tethered domains. Only recently it was found that the tethering may also destabilize the protein domains55, the conformational dynamics appear to operate through a global network and the perturbation by a mutation or binding without altering the average structure will trigger extensive reorganization of the whole network. Therefore, it still remains an extreme challenge in the future to integrate the results of thermodynamic stability and conformational dynamics to understand why the inter-domain connection significantly destabilize the two RRM domains of TDP-43 but lead to even a slight stabilizing effect on the RRM1 domain of hnRNPA1.The tethering-induced effects for TDP-43 and hnRNPA1 RRM domains appear to result mainly from their inter-domain connection. Residue-specific NMR data showed that the dissection-induced perturbation for TDP-43 RRM domains is much more profound than that for hnRNPA1 RRM domains, while MD simulations further revealed that the inter-domain correlation motions of TDP-43 RRM domains are more extensive than those for hnRNPA1 RRM domains. Therefore, experimental and simulation results together rationalize the observation that the tethered TDP-43 RRM domains could become highly coupled with only one denaturation transition with Tm much lower than those of their isolated ones. On the other hand, even only based on our previous studies58, although the majority of ATP needs to be produced by very complex supramolecular machineries embedded in membranes56. Only recently, it was decoded that ATP with concentrations > 5 mM acts to hydrotropically dissolve liquid–liquid phase separation (LLPS), aggregation/fibrillation57, which appears to operate at a proteome-wide scale58. We further found that by weak but specific binding with Kd of ~ mM, ATP is also able to biphasically modulate LLPS of intrinsically disordered domains60. Remarkably, ATP can inhibit amyloid fibrillation not only for the tethered TDP-43 RRM1–RRM2 by enhancing its thermal stability21, but also for the single FUS RRM domain by blocking the dynamic opening of the RRM fold without alternation of its thermal stability20. Here, again we found that ATP can specifically bind the hnRNPA1 RRM2 domain in both tethered and isolated contexts with the affinity and complex structure very similar to those of the FUS and TDP-43 RRM domains. Moreover ATP can even enhance the thermal stability of the hnRNPA1 RRM1–RRM2 domains. Interestingly, our previous and current results together suggest that the affinities of the ATP binding to the tethered forms of both TDP-43 and hnRNPA1 RRM domains are slightly higher than those to the isolated forms. This may be mainly due to the higher conformational dynamics of the tethered RRM domains than those of the isolated forms as uncovered by MD simulations, which thus provides ATP the higher dynamic accessibility to the binding pockets in the tethered RRM domains.Mysteriously, all cells maintain very high ATP concentrations of 2–12 mM, much higher than those required for its previously-known functions14. We also showed that the relatively high conformational dynamics of FUS RRM domain appear to be sufficient to allow the opening of the RRM fold, thus leading to aggregation/fibrillation. Furthermore, the ATP binding to FUS RRM even without enhancing its stability is sufficient to kinetically block the conformational opening. Therefore, the results here that ATP can bind the conserved pockets of TDP-43 and hnRNPA1 RRM domains again highlight that ATP may play a general role in preventing the pathological aggregation/fibrillation of the RRM-containing proteins containing more than one RRM domain.Previous studies implied that TDP-43 RRM domains might start to assemble into amyloid structures without needing the complete denaturation54.In summary, in the present study we showed that upon tethering, two TDP-43 RRM domains become highly coupled but dramatically destabilized with the Tm reduction of ~ 8 °C. On the other hand, no significant destabilization occurs for the tethering of two hnRNPA1 RRM domains. Mechanistically, the tethering-induced effects appear to mainly result from the inter-domain connection between two RRM domains as reflected by NMR and MD simulation results. Moreover, we showed that ATP can specifically bind TDP-43 and hnRNPA1 RRM domains with the affinity to the tethered forms slightly higher than to the isolated forms. Results together thus suggest that ATP, the universal energy currency, may also play a general role in preventing aggregation/fibrillation of RRM-containing proteins, which has been extensively identified to cause an increasing spectrum of human diseases beyond neurodegenerative diseases. Therefore, our results imply a potential mechanism to rationalize the observation that upon being aged, the risk of protein aggregation-causing diseases increases most likely also because ATP concentrations gradually reduce in all cells during aging61. On the other hand, the expression vector of the tethered RRM1–RRM2 (5–184) of hnRNPA1 were purchased from a local company (Bio Basic Asia Pacific Pte Ltd), which was subsequently dissected into the isolated RRM1 (5–95) and RRM2 (94–184) by PCR with designed primers.Previously the expression vectors of TDP-43 RRM1–RRM2 (102–269), RRM1 (102–191) and RRM2 (191–269) have been constructed and their expression and purification were establishedE. coli BL21 cells with IPTG induction at 20 °C overnight. They were found all in the supernatant, and therefore were purified by a Ni2+-affinity column (Novagen) under native conditions. Subsequently the on-gel cleavage by thrombin was conducted and the eluted fractions containing the RRM proteins were further purified by a heparin column to remove nucleic acids followed by FPLC purification with either a Superdex-75 or a Superdex-200 column.The six recombinant proteins were expressed in 61. Briefly, the bacteria were grown in M9 medium with addition of (15NH4)2SO4 for 15 N-labeling. The protein concentrations were determined by the UV spectroscopic method in the presence of 8 M urea, under which the molar extinct coefficient at 280 nm of a protein can be calculated by adding up the contribution of Trp, Tyr, and Cys residues62.Here we followed our previous protocol to generate isotope-labeled proteins for NMR studies2 was added into ATP for stabilization by forming the ATP-Mg complex57. The fluorescent dye SYPRO Orange (S5692-50UL) was purchased from Sigma-Aldrich. The protein samples, as well as ATP, were all prepared in 10 mM sodium phosphate buffer containing 10 mM DTT and 150 mM NaCl with a final pH adjusted to 6.8.ATP was purchased from Sigma-Aldrich with the same catalog numbers as previously reported. MgCl23.To monitor the thermal denaturation by intrinsic Trp fluorescence for the tethered RRM1–RRM2, isolated RRM1 and RRM2 domains as well as their mixture at 1:1 of both TDP-43 and hnRNPA1 at 10 μM in 10 mM sodium phosphate buffer containing 10 mM DTT and 150 mM NaCl (pH 6.8), their emission spectra of UV fluorescence were acquired on a Jasco J-1500 spectropolarimeter from 25 to 95 °C at a 5-degree interval with the excitation wavelength at 280 as previously described and the fluorescence intensity was reported in arbitrary unit24, ATP triggered very high non-specific noise in CD spectroscopy and quenched the intrinsic fluorescence of exposed Trp residues, here again we used differential scanning fluorimetry (DSF) as we previously reported to determine the thermodynamic stability of RRM1–RRM2, RRM1 and RRM2 domains of TDP-43 and hnRNPA1 at 10 μM in 10 mM sodium phosphate buffer containing 10 mM DTT and 150 mM NaCl (pH 6.8) with addition of ATP at different concentrations.Furthermore, as we previously showed24. Briefly, in a single well of a 384-well PCR plate, a 10 µl reaction solution was placed, which contains the RRM12, or RRM1 or RRM2 domain at 10 µM, ATP at different concentrations, and 10× SYPRO Orange in 10 mM sodium phosphate buffer containing 150 mM NaCl (pH 6.8). The program in Real-Time PCR instrument was set to be SYBR green and run temperature scan from 30 to 90 °C with the increment of 1 °C per minute. Upon completion, the obtained thermal unfolding curves were displayed as the first derivatives (dF/dT) by the RT-PCR software Bio-Rad CFX Manager 3.0.DSF experiments were performed using the CFX384 Touch Real-Time PCR Detection System from BIO-RAD, following the SYBR green melting protocol to obtain Tm value24. For NMR HSQC titration studies of the interactions of RRM1–RRM2, RRM1 and RRM2 with ATP, two dimensional 1H-15 N NMR HSQC spectra were collected on the 15 N-labelled samples at a protein concentration of 50 µM in 10 mM sodium phosphate buffer containing 10 mM DTT and 150 mM NaCl (pH 6.8) at 25 °C in the absence and in the presence of ATP at concentrations of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, and 20.0 mM.All NMR experiments were acquired at 25 °C on an 800 MHz Bruker Avance spectrometer equipped with pulse field gradient units and a shielded cryoprobe as we described previouslySequential assignments of the tethered and isolated RRM1 and RRM2 domains of TDP-43 and hnRNPA1 were achieved based on the previously deposited chemical shifts: the tethered TDP-43 RRM1–RRM2 domain (BMRB ID of 19290 and 27613), the isolated RRM1 (BMRB ID of 18765) and RRM2 (BMRB ID of 19922), as well as the tethered hnRNPA1 RRM1–RRM2 domain (BMRB ID of 18728).Due to the fast exchange with bulk water, or/and μs-ms dynamics, or/and overlap for some, particularly loop residues, the 168-residue TDP-43 RRM1–RRM2 containing 6 Pro residues had 142 peaks detected and assigned. The 90-residue TDP-43 RRM1 containing 3 Pro residues had 82 peaks detected and assigned, while the 79-residue TDP-43 RRM2 containing 3 Pro residues had 72 peaks detected and assigned. The 184-residue hnRNPA1 RRM1–RRM2 containing 6 Pro residues had 155 peaks detected and assigned. The 96-residue hnRNPA1 RRM1 containing 5 Pro residues had 86 peaks detected and assigned, while the 90-residue hnRNPA1 RRM2 containing 1 Pro residues had 76 peaks detected and assigned.15 N-labeled RRM1–RRM2, RRM1 and RRM2 domains collected in the absence and in the presence of ATP at different concentrations. Subsequently, the shifted HSQC peaks could be identified and further assigned to the corresponding RRM residues based on the sequential assignments. The chemical shift difference (CSD) was calculated by an integrated index calculated by the following formula:To calculate chemical shift difference (CSD), the HSQC spectra were superimposed for the 29 with the following formula:In order to obtain residue-specific dissociation constant (Kd), we fitted the shift traces of the residues with significant shifts of HSQC peaks (CSD > average + STD), by using the one binding site modelHere, [P] and [L] are molar concentrations of RRM domains and ligands (ATP) respectively.31, which makes use of CSD data to derive the docking with various degrees of flexibility. Briefly, five residues of hnRNPA1 RRM2 with significant CSD values were set to be active residues and HADDOCK docking procedure for the complexes was performed at three stages: (1) randomization and rigid body docking; (2) semi-flexible simulated annealing; (3) flexible explicit solvent refinement. The ATP-RRM2 structure with the lowest energy score were selected for the detailed analysis and displayed by Pymol .The structure model of the ATP-RRM2 complex of hnRNPA1 was constructed by use of HADDOCK softwareFor MD simulations, the NMR structures of RRM1–RRM2 of TDP-43 (PDB ID of 4BS2) and hnRNPA1 (PDB ID of 2LYV) were selected as the tethered models while their isolated RRM1 and RRM2 models were obtained by dissecting the two structures into the isolated RRM domains.38. Briefly, the simulation cell is a periodic cubic box with a minimum distance of 10 Å between the protein and the box walls to ensure the protein would not directly interact with its own periodic image given the cutoff. The water molecules, described using the TIP3P model, were filled in the periodic cubic box for the all atom simulation. To neutralize the system, some Na+ and Cl− ions were randomly placed far away from the surface of the protein.The simulation setting was previously reported63 with the AMBER-03 all-atom force field64. The long-range electrostatic interactions were treated using the fast particle-mesh Ewald summation method, with the real space cutoff of 9 Å and a cutoff of 14 Å was used for the calculation of van der Waals interactions. The temperature during simulation was kept constant at 300 K by Berendsen's coupling. The pressure was held at 1 bar. The isothermal compressibility was 4.5 × 10−5 bar−1. The time step was set as 2 fs. Prior to MD simulations, all the initial structures were relaxed by 1000 steps of energy minimization using steepest descent algorithm, followed by 100 ps equilibration with a harmonic restraint potential applied to all the heavy atoms of the proteins.Three independent 50-ns MD simulations were performed for each of six constructs: namely the tethered RRM1–RRM2, isolated RRM1 and RRM2 of TDP-43 as well as of hnRNPA1 by the program GROMACS34. MutInf represents an entropy-based method to analyze ensembles of protein conformers, including those from MD simulations by using internal coordinates and focusing on dihedral angles. This method is particularly applicable for subtle conformational changes by utilizing second-order terms of the configurational entropy expansion called mutual information to pinpoint the pairs of residues with correlated conformations, or correlated motions, in an equilibrium ensemble. Here, the normalized matrix values were used and the threshold value was set up to 0.3 for determining the pairs of highly correlated residues.To identify the coupling of RRM1 and RRM2 domains of both TDP-43 and hnRNPA1, we calculated their correlation matrices of three independent simulations of the tethered RRM1–RRM2 by MutInfSupplementary Figures.