NMR– and MD simulation–based structural characterization of the membrane-associating FATC domain of ataxia telangiectasia mutated

Abstract

The Ser/Thr protein kinase ataxia telangiectasia mutated (ATM) plays an important role in the DNA damage response, signaling in response to redox signals, the control of metabolic processes, and mitochondrial homeostasis. ATM localizes to the nucleus and at the plasma membrane, mitochondria, peroxisomes, and other cytoplasmic vesicular structures. It has been shown that the C-terminal FATC domain of human ATM (hATMfatc) can interact with a range of membrane mimetics and may thereby act as a membrane-anchoring unit. Here, NMR structural and ¹⁵N relaxation data, NMR data using spin-labeled micelles, and MD simulations of micelle-associated hATMfatc revealed that it binds the micelle by a dynamic assembly of three helices with many residues of hATMfatc located in the headgroup region. We observed that none of the three helices penetrates the micelle deeply or makes significant tertiary contacts to the other helices. NMR-monitored interaction experiments with hATMfatc variants in which two conserved aromatic residues (Phe³⁰⁴⁹ and Trp³⁰⁵²) were either individually or both replaced by alanine disclosed that the double substitution does not abrogate the interaction with micelles and bicelles at the high concentrations at which these aggregates are typically used, but impairs interactions with small unilamellar vesicles, usually used at much lower lipid concentrations and considered a better mimetic for natural membranes. We conclude that the observed dynamic structure of micelle-associated hATMfatc may enable it to interact with differently composed membranes or membrane-associated interaction partners and thereby regulate ATM's kinase activity. Moreover, the FATC domain of ATM may function as a membrane-anchoring unit for other biomolecules.

Introduction

Ataxia telangiectasia mutated (ATM)⁴ belongs to the family of phosphatidylinositol 3-kinase–related kinases (PIKKs) that phosphorylate Ser/Thr residues of proteins regulating processes such as DNA repair, cell cycle progression, cellular senescence, apoptosis, and metabolic processes (1–4). Recently, it was found out that PIKKs also play a role in signaling in response to virus infections and during inflammation (5, 6). The function of ATM and of the related mammalian/mechanistic target of rapamycin (mTOR), a central controller of cell growth and metabolism in all eukaryotes that also has links to DNA repair signaling (7, 8), has further been related to redox signaling (9–12). Whereas the mTOR pathway negatively controls ATM (13), ATM inactivates mTORC1 in response to reactive oxygen species to induce autophagy (12), based on additional data, specifically that of peroxisomes (14). ATM also down-regulates mTORC1 under hypoxic conditions (11). Other studies indicate that ATM plays direct roles in modulating mitochondrial homeostasis (15). Activation of ATM by oxidation and other factors has been reviewed (16). Inactivation of ATM leads to ataxia-telangiectasia (A-T) disease and more generally plays a role in neuronal development and neurodegeneration (17). Because ATM controls different signaling pathways important for the DNA damage response, cell cycle checkpoints, and metabolic processes, it is considered an interesting therapeutic target with respect to cancer (18).

It has been proposed that targeted membrane localization allows spatial separation of individual signaling branches of large signaling networks, thereby potentially improving the reliability of biochemical signaling processes (19). Because PIKKs generally participate in a multitude of signaling pathways in response to ionizing radiation and other stress factors or metabolic signals (2, 5, 7, 16, 20, 21), their localization may also determine the specific signaling output (22). ATM has been found to localize and function not only in the nucleus but also in the cytoplasm (12). ATM has further been localized at microsomes (23) and cytoplasmic vesicles (24), as well as at membrane-associated vesicles (25). More recently, it has been reported that PEX5 peroxisome import receptor binds ATM to localize it to peroxisomes and that ATM can localize to mitochondria (14, 15).

Although the total length of PIKK amino acid sequences ranges from about 2500 to 4500 residues, all share a similar domain organization (5, 22). Fig. 1A shows human ATM as a representative example. The Ser/Thr kinase domain is close to the C terminus and shows homology to lipid kinases (4). Except for TRRAP, all are catalytically active (1, 4). The FRAP-ATM-TRRAP (FAT) domain resides N-terminal of the kinase (22, 26). Based on sequence analysis, the FAT and the preceding N-terminal region with only low sequence similarity between different PIKKs are mostly composed of α-helical repeat motifs that typically form platforms for protein–protein interactions (26–28). The linker region between the kinase and the FAT C-terminal (FATC) domain region varies significantly in length and sequence composition (4, 29). It has been referred to as the PIKK regulatory domain (PRD) (1, 4, 29). The highly evolutionary conserved region of the FATC domain is made up by the ∼35 C-terminal residues (PFAM domain database entry PF02660) (4, 26, 30–32). Based on mutagenesis studies and other data, this domain generally plays an important role for the regulation of PIKK function (4, 29, 31, 33–35).

Figure 1.

NMR structure of the micelle-associated FATC domain of human ATM and comparison with that earlier determined for yeast TOR1. A, human ATM (hATM) consists of a long N-terminal region and a FAT domain, which are both made up of helical repeats, the catalytic Ser/Thr kinase domain, and the FATC domain (26). To structurally characterize the C-terminal FATC domain (residues 3024–3056, Uniprot accession number Q13315), it was fused to the B1 domain of streptococcal protein G (GB1, 56 residues, = hATMfatc). B, superposition of the amino acid sequences of the FATC domains of hATM and yeast TOR1 and schematic representation of the secondary structure content in DPC micelles (schematic representation to the right). Aromatic residues are shown in light (Phe, His, and Tyr) and dark (Trp) green, hydrophilic ones in light blue, except Lys and Arg in blue and Asp and Glu in red, and those containing methyl groups in dark blue. Prolines are colored purple and glycines in orange. C, the NMR structure of hATMfatc in the presence of micelles (150 mm DPC) consists of three helices with a central dynamic linker and a less dynamic C-terminal one (Table 1, PDB code 6HKA, BMRB accession number 27167 (67)). The structure is overall different from that of the micelle-immersed oxidized yeast TOR FATC domain (y1fatc, PDB code 2KIO) (44). For the ribbon representations, the 20 lowest-energy structures have been superimposed for the indicated regions. D, each helical stretch of micelle-associated hATMfatc is shown in line mode. The backbone of the helical stretches is depicted in red. The color coding of the side chains is the same as in B except that charged side chains are also colored light blue. All structure pictures were made with the program MolMol (86). E, information about the backbone dynamics from a plot of the {¹H}-¹⁵N NOE values of hATMfatc with 150 or 200 mm DPC as a function of the residue sequence position. No data can be obtained for prolines or overlapping peaks (Fig. S1). Values around 0.6–0.8 are typical for structured proteins. Lower NOE values indicate increased backbone flexibility. Fig. S1 shows the 2D {¹H}-¹⁵N NOE and reference spectra, and Fig. S2 additionally shows the ¹⁵N-T₁ and -T₂ relaxation times at 200 mm DPC as well as a plot of the ¹⁵N relaxation data for the full hATMfatc-gb1ent fusion protein.

Obtaining high-resolution structural data for PIKKs is challenging due to their large size. For ATM, EM structural data at lower resolution provided insights into the interaction with DNA (36), the inhibitory effect of dimerization of human ATM (37), and the dimerization properties of the yeast ATM homolog Tel1 (38, 39). In 2017, higher-resolution EM structures of the closed ATM dimer (PDB code 5NP0, 5.7 Å resolution) and the presumably more active open ATM dimer (PDB code 5NP1, 5.7 Å resolution for combined data) were published (40).

The FATC domains of all PIKKs are rather hydrophobic and rich in aromatic residues (ATM and TOR; see Fig. 1B). In line with this, all interact with membrane mimetics, although with somewhat different preferences for membrane features such as surface charge, curvature, or packing density, and thus may all act as membrane-anchoring units (32). Like TOR, ATM binds to all tested membrane mimetics, including dodecylphosphocholine (DPC) micelles, bicelles composed of dimyristoyl and diheptanoylphosphocholine (DMPC, DihepPC), and small unilamellar vesicles (SUVs) made of DMPC (32). An estimate of the secondary structure content based on secondary chemical shifts suggested that the micelle-immersed human ATM FATC domain contains three helical stretches (32). Here, we present the determined NMR structure and an analysis of the relative orientation on the micelle and its membrane immersion properties and dynamics based on NMR and molecular dynamics (MD) data. We further prepared mutants by replacing conserved aromatic residues that are expected to be important for the membrane affinity and tested their ability to interact with DPC and DihepPC micelles, DMPC/DihepPC bicelles, and DMPC SUVs. Overall, the data provide a better understanding of the role of the AMT FATC domain in regulating the kinase function as well as the observed localization to specific membrane compartments.

Results

The NMR structure of micelle-immersed hATMfatc consists of three helices with a central flexible linker and a less flexible C-terminal linker

The three-dimensional structure of the human ATM FATC domain (hATMfatc) associated with DPC micelles (PDB code 6HKA) was determined by multidimensional heteronuclear NMR spectroscopy in solution. Because cleavage of the GB1 tag is not efficient and results in very low yields, all structural restraints were derived from NMR data recorded for the GB1-tagged form (hATMfatc-gb1ent) (32). Previous studies have shown that the GB1 tag does not affect the association with membrane mimetics and results only in little overlap of NMR signals of the GB1 tag and the FATC domain (41). The structural statistics are given in Table 1.

Table 1.

Statistics for the 20 lowest-energy NMR structures of the micelle-associated human ATM FATC domain (PDB code 6HKA)

Experimental restraints
Distance restraints	All (assigned + ambiguous)
Total	748 (707 + 41)
15N NOESY	332 (313 + 19)
13C NOESY aliphatic/aromatic	366 (345 + 21)/26 (25 + 1)
Assigned short/medium/long rangea	630/61/2
Ambiguous short/medium/long rangea	≤62/≤17/≤3
Hydrogen bond	24
φ + ψ angle restraints	39
Structural statisticsb
RMSDs from experimental restraints
Distance (Å)	0.0240 ± 0.0030
Dihedral angle (degrees)	0.1937 ± 0.1399
RMSDs from idealized geometry
Bonds (Å)	0.0032 ± 0.0002
Angles (degrees)	0.3709 ± 0.0177
Improper (degrees)	0.2409 ± 0.0145
Lennard–Jones energy (kcal mol−1)c	−141 ± 31
Procheck (85) statisticsd
Residues in most favored regions (%)	85.5
Residues in additional allowed regions (%)	14.5
Residues in generously allowed regions (%)	0.0
Residues in disallowed regions (%)	0.0
Average RMSD to mean structure
Residues 70/3026–100/3056 (backbone/heavy) (Å)	4.47/5.26
Residues 73/3029–83/3039 (backbone/heavy) (Å)	0.37/0.89
Residues 87/3043–99/3055 (backbone/heavy) (Å)	1.32/2.33
Residues 87/3043–91/3047 (backbone/heavy) (Å)	0.22/1.35
Residues 95/3051–99/3055 (backbone/heavy) (Å)	0.27/1.72

^a Short: i, i or i ± 1; medium: i, i ± 2 or 3 or 4; long range, here only i, i ± 5. Note that for ambiguous restraints, two options were taken into account, thus the less than/equal to sign before the given numbers.

^b None of the structures had distance restraint violations >0.5 Å or dihedral angle violations >5°; PDB code 6HKA.

^c Calculated for the full 100 residue protein hATMfatc-gb1ent, but residues 1–67 had not been restrained and thus should not contribute significantly to the calculation.

^d For the well-structured regions 72–83, 86–92, and 94–100.

The structure of micelle-associated hATMfatc, residues Thr⁶⁸–Val¹⁰⁰ in hATMgatc-gb1ent corresponding to Thr³⁰²⁴–Val³⁰⁵⁶ in full-length human ATM, consists of three helical regions (Fig. 1, B–D). The structure is overall well-defined with 85.5% of the residues in the well-structured region (Val⁷²–Ala⁸³, Pro⁸⁶–Leu⁹², Pro⁹⁴–Val¹⁰⁰) occupying the most favored region of the Ramachandran plot (Table 1). Based on the 20 lowest energy structures, the N-terminal longer α-helix (α1) is on average formed by residues Gly⁷³–Ala⁸³ of the 100-residue fusion protein, which corresponds to residues Gly³⁰²⁹–Ala³⁰³⁹ of full-length human ATM. Following a three-residue linker (Ile^84/3040-Asp^85/3041-Pro^86/3042), a second α-helix (α2) is on average formed by residues Lys^87/3043-Arg^91/3047. Following another proline-containing short linker (Leu^92/3048-Phe^93/3041-Pro^94/3049), the C-terminal helix follows. Although tertiary contacts between the helices could not be observed, their orientation with respect to each other is not completely random. This may be explained by the presence of a proline in each linker. Based on the analysis of the conformations of short peptides based on residual dipolar couplings, prolines increase local order (42).

The central flexible linkage between α1 and α2 results in a high RMSD value of 4.47 Å for residues 70/3026–100/3056 compared with very low ones for the single-helical regions (α1, 0.37 Å; α2, 0.22 Å; α3, 0.27 Å; Table 1) or the region including α2 and α3 (residues 87–99, 1.32 Å). This can be explained by the fact that no interhelical NOE contacts could be observed (Table 1). In line with the dynamic linkage between α1 and α2 around Asp^85/3041, it shows a {¹H}-¹⁵N NOE value of only 0.32 ± 0.10 at 150 mm DPC and 298 K and 0.38 ± 0.11 at 200 mm DPC and 298 K (Fig. 1E). The linkage between α2 and α3 is also not rigid, but the {¹H}-¹⁵N NOE values in the linker region are not as low (Leu^92/3048, 0.42; Phe^93/3049, 0.48 at 150 mm DPC and 298 K) and of similar size as the ones for Lys^97/3053 (0.43) and Trp^99/3055 (0.48) in the short C-terminal helix. A more detailed description of the ¹⁵N relaxation data is provided in the supporting information (text and Figs. S1 and S2).

Consistent with the differences in the amino acid sequence to the FATC domain of yeast TOR1 (y1fatc; Fig. 1B, bottom), the overall structural appearance is quite different. Y1fatc contains two conserved cysteines that can form a disulfide bond, and oxidized y1fatc in the absence of membrane mimetics forms already an α-helix that is followed by a C-terminal disulfide bonded loop (43). In contrast, hATMfatc in buffer is largely unstructured, and most backbone amide signals are not visible in the ¹H-¹⁵N HSQC spectra (32), which hampers its resonance assignment.

Compared with micelle-associated hATMfatc, micelle-associated oxidized y1fatc adopts only a single α-helix that is followed by a disulfide bonded loop or bulb (Fig. 1C, right) (44). Even in the reduced micelle-associated state of y1fatc, the C terminus folds back, and the whole structure is less dynamic than that of hATMfatc (44). The {¹H}-¹⁵N NOE values of oxidized and reduced micelle-associated y1fatc at 298 K are more uniform in the well-structured region and do not show residues with significantly lower values in between (44), as observed for micelle associated hATMfatc (Fig. 1E).

The surface of micelle-associated hATMfatc (Fig. 2, top) is largely hydrophobic except for a positively charged surface region formed by Lys^87/3043, Arg^91/3047, and Lys^97/3053 that is in the neighborhood of a small negatively charged surface region formed by Asp^85/3041. In contrast, that of micelle-immersed y1fatc (Fig. 2, bottom) shows a large acidic region toward the N terminus and only two small positively charged regions around Lys²⁴⁴⁸ and Arg²⁴⁵⁸. The C-terminal loop of y1fatc is largely hydrophobic (44).

Figure 2.

The surface charge distribution of micelle-associated hATMfatc differs from that earlier determined for oxidized y1fatc (see also Fig. 1, B and C) (44). Positively charged surface areas and side chains in stick mode in the ribbon representation are colored blue, and negatively charged ones are shown in red. All structure pictures were made with the program MolMol (86).

The helical regions of micelle-associated hATMfatc can move with respect to each other

Additional insights into the dynamics as well as immersion properties (see below) of micelle-associated hATMfatc were obtained from molecular dynamics simulations. In total, three runs of MD simulations were performed using as starting structure for each one of the three lowest-energy NMR structures (Fig. 3A). The simulation time for each run was 1.3 μs. The first 300 ns were discarded as equilibration time. As illustrated by the pictures of the simulated micelle-associated hATMfatc of each run (Fig. 3B), the relative orientation of the three helices with respect to each other varies in each due to the dynamic linkages between the helices, especially the one between α1 and α2. Additional information about the conformational freedom is provided based on RMSD and RMSF (global and local) values in the supporting information (text and Fig. S4 (A–C)). Consistent with the fact that no interhelical NOEs could be observed, distances below 0.5 nm between residues on different helices were only rarely and for very short simulation time periods observed (Fig. S4D). Plots of the angle between α1 and α2 as well as between α2 and α3 (Fig. 3C and Fig. S5A) as a function of time show that these angles can vary significantly as a function of the simulation time. Even with a simulation time of 1.3 ms, such simulations usually do not converge. In summary, the ¹⁵N relaxation data and the MD simulations suggest that the reorientation of the helices with respect to each other due to the dynamic central linkage between α1 and α2 and less dynamic linkage between α2 and α3 is the major contribution to their dynamic properties, rather than more local conformational changes.

Figure 3.

Characterization of the dynamic properties of the micelle-associated FATC domain of human ATM (hATMfatc) from MD simulations. A, ribbon representations of the three lowest-energy structures that were used as starting structures for three independent MD simulation runs. B, representative structure pictures of the MD simulations of hATMfatc in the presence of DPC micelles. The structures shown are the conformations of hATMfatc, which are the closest to the middle of the most populated cluster of structures for a given run. The DPC micelle is shown as a transparent averaged density iso-surface, and the peptide is shown in a ribbon representation. In both A and B, negatively charged side chains or residues are shown in red, positively charged ones in blue, polar ones in green, and aliphatic ones in gray. For simplicity, only the residue numbering as used in the PDB entry (PDB code 6HKA) is given. The N and C termini are indicated by the letters N and C. C, information about the movement of the three helices with respect to each other from an analysis of the variation of the angle between helices 1 and 2 as well as the one between helices 2 and 3 for run 1 (for runs 2 and 3, see Fig. S5A). Plots of the secondary structure content as a function of the simulation time are shown in Fig. S3. Plots of the RMSF (global and local) and the backbone and side chain RMSD values as a function of the residue sequence position, a superposition of the top 10 cluster structures, and an analysis of interhelical distances as a function of the simulation time are shown in Fig. S4.

The hATM FATC domain resides largely in the headgroup region of DPC micelles

Based on the MD simulations of the three lowest-energy NMR structures of micelle-associated hATMfatc in the presence of DPC micelles (Fig. 3), the three helices immerse mostly into the headgroup region. To probe this experimentally, we recorded ¹H-¹⁵N HSQC spectra of hATMfatc-gb1ent in the presence of DPC micelles (50 mm) and increasing amounts of paramagnetic 5- or 16-doxyl stearic acid (5-/16-SASL) (Fig. 4A and Fig. S5B). Earlier MD simulations of DPC micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed (45). The MD data further confirmed that the doxyl group of 5-SASL, as expected (46), resides also near the DPC headgroups (45). Residues of hATMfatc that localize close to the headgroups should thus show the strongest spectral changes. The doxyl label of 5- or 16-SASL results in paramagnetic relaxation enhancement (PRE), which reduces the signal intensity of nearby amide groups with an r⁻⁶ distance dependence (47). To better correlate the PRE effects with the average chemical shift changes (Figs. S6 and S7) as well as with the distance from the micelle center of mass (COM) (Fig. 4C and Fig. S8) and the surface area that is covered by DPC (Fig. 4D and Fig. S9) from the MD simulation, we plotted 1 − I (x mm SASL)/I (0 mm SASL), which we refer to as 1-PRE. Thus, the stronger the PRE effect, the closer to 1 is the 1-PRE value (Fig. 4B and Figs. S6 and S7). As for the FATC domain of yeast TOR1, the PRE effects with 16-SASL are stronger than with 5-SASL (45). 1 mm 16-SASL results overall in somewhat larger 1-PRE values than 2 mm 5-SASL. Because the nitroxide spin label usually does not induce significant pseudocontact shifts, the observed chemical shift changes arise from a change in the chemical environment. Because the GB1 tag does not directly interact with the micelle, it shows only small 1-PRE values and average ¹H-¹⁵N chemical shift changes (Figs. S6 and S7). For the micelle-associating hATMfatc part, the changes with 5- and 16-SASL show overall a similar trend as a function of the residue sequence position (Fig. 4, A and B). Strong PRE effects (1-PRE ≥ 0.4) at 2 mm 5-SASL or 1 mm 16-SASL are seen for Val⁷², Ile⁸⁴, Asp⁸⁵, Phe⁹³, and Gly⁹⁵ and, in the case of 1 mm 16-SASL, additionally for Gly⁷³, Leu⁷⁸, Leu⁷⁹, Gln⁸¹, Gln⁸², Ala⁸³, Leu⁹², Lys⁹⁷, Ala⁹⁸, and Trp⁹⁹. Not surprisingly, many of them show also significant chemical shift changes (Fig. 4A and Figs. S6 and S7). Because the listed residues based on the NMR data should localize near the headgroups, they should have a distance from the micelle COM that is in a similar range as the phosphorus atom of the DPC headgroup. Fig. 4C shows the average distance from the COM of each residue of hATMfatc to the micelle COM for run 1. Those for runs 2 and 3 are displayed in Fig. S8. The profiles as a function of the sequence look very similar for all runs. In line with the 5- and 16-SASL NMR data, residues that show strong PRE effects (Fig. 4B) have mostly distances to the micelle COM that are similar to that of the DPC phosphorus atom (dotted line at 1.914 nm). In contrast, residues that show a small PRE effect, such as Thr^68/3024 at the N terminus of hATMfatc, show a larger distance to the micelle if they are more solvent-accessible or a closer one such as for example Leu^92/3046 if they reside deeper in the micelle. From a plot of the solvent-accessible surface area covered by DPC that was derived from the three MD runs (Fig. 4D and Fig. S9), it can further be estimated which residues immerse deeper and which should still be solvent-accessible. As expected, those residues showing a short distance to the micelle COM (≤1.6 nm in Fig. 3C and Fig. S8) are 80% or more covered by DPC (e.g. for run 1 Val⁷², Val⁷⁶, Leu⁷⁹, Leu⁸⁹, and Phe⁹³). As expected, these residues are mostly hydrophobic.

Figure 4.

Analysis of the membrane immersion properties of hATMfatc by NMR and MD simulations. A, superpositions of the ¹H-¹⁵N HSQC spectra of hATMfatc-gb1ent in the presence of DPC micelles and 1 or 2 mm paramagnetically tagged 5-SASL (left) or 16-SASL (right) with 3 and 4 mm (see Fig. S5B). The hATMfatc signals are labeled by the residue sequence position in hATMfatc-gb1ent/human ATM and the one-letter amino acid code. The color coding is indicated at the top. B, diagrams of the PRE effects due to the presence of 5- or 16-SASL as a function of the residue sequence position. To better compare the PRE effects with the average chemical shift changes (Figs. S6 and S7), 1-PRE (= 1 − I (x mm SASL)/I (0 mm SASL)) was plotted. Accordingly, the larger the PRE effect, the higher the 1-PRE value. The sequence is given at the bottom, and a schematic representation of the location of the helices is shown at the top. C, diagram of the average distance of the COM of each residue of hATMfatc to that of the DPC micelle for run 1 (for runs 2 and 3, see Fig. S8). The averaging was performed over the last microsecond of the run. The dotted line corresponds to the average distance of the phosphor atom of the DPC headgroup to the micelle COM (1.914 nm). D, diagram of the percentage of the solvent-accessible surface area (SAS) of each residue of hATMfatc covered by DPC from run 1 (for runs 2 and 3, see Fig. S9). Residues with shorter distances to the micelle COM in C usually show a higher percentage in D.

In summary, the NMR data using spin-labeled micelles and the MD simulations of micelle-associated hATMfatc show that it binds the micelle by a dynamic assembly of three helices with many residues localizing in the headgroup region. None of the three helices penetrates the micelle very deeply or makes significant tertiary contacts to the other helices.

Mutation of Phe^93/3049 and Trp^96/3052 to alanine abrogates the association of hATMfatc with neutral SUVs but not with micelles and bicelles

Tryptophans but also other aromatic residues are known to play an important role for the affinity between membranes and proteins in general (48, 49). For the FATC domain of TOR, we have shown previously that replacement of up to 6–7 residues did not result in a significant abrogation of the association with micelles or bicelles. However, replacement of only one tyrosine (Tyr²⁴⁶³) or one tryptophan (Trp²⁴⁶⁶) resulted in an impairment of the interaction with SUVs (50). To find out whether aromatic residues of hATMfatc at equivalent positions (Phe^93/3049 and Trp^96/3052) have a similar effect, we mutated them individually and at the same time to alanine residues. The free states of the mutant proteins show even fewer peaks and thus appear to be more dynamic than WT hATMfatc (Fig. S10, left). This may be explained with the smaller size of alanine compared with tyrosine or tryptophan. In the presence of 50 mm DPC, they adopt as the WT a more ordered structure, which reduces the backbone dynamics such that the backbone and side chain amide signals become clearly visible (Fig. S10, right). The interaction with neutral membrane mimetic micelles composed of DPC or the short chain diacyl lipid DihepPC, neutral bicelles composed of DMPC for the planar region and DihepPC for the rim, and small unilamellar vesicles composed of DMPC was further monitored by recording ¹H-¹⁵N HSQC spectra in the absence and presence of these membrane mimetics (Fig. 5 and Figs. S11 and S12). As indicated by the observed spectral changes, neither of these mutations abolished the interaction with micelles or bicelles (Table 2). Whereas the single mutants F93A/3049A and W96/3052A could still interact with neutral DMPC SUVs (Figs. S11 and S12, green plus signs), a lack of spectral changes for the double mutant F93/3049A-W96/3052A (Fig. 5, bottom right, red minus sign) indicates that it could not (Table 2).

Figure 5.

Characterization of the role of conserved aromatic residues in the membrane affinity of hATMfatc. The pictures show superpositions of the ¹H-¹⁵N HSQC spectra of the double mutant hATMfatc-gb1ent-F93/3049A-W96/3052A in the absence (black) and presence (red) of DPC micelles (upper left), DihepPC micelles (upper right), bicelles (q = 0.2, [DMPC] = 0.04 m, and [DihepPC] = 0.20 m, cL 15%, bottom left) or DMPC liposomes (<50 mm DMPC, bottom right). Analogous superpositions for each single mutant (F93/3049A and W96/3052A) are shown in Figs. S11C and S12B, respectively. A green plus sign below a schematic representation of the used membrane mimetic indicates strong spectral changes and thus interactions, and a red plus sign indicates no significant changes and thus no significant interactions. To better identify the signals of the ATM FATC part, the spectrum of the GB1 tag followed by a thrombin and factor Xa site (= GB1xa) is additionally shown in green at the top. Accordingly, peaks with a green peak at the top belong to the GB1 tag, which does not interact with membrane mimetics (41). In the spectrum in the presence of DPC, hATMfatc signals (red) are labeled by the one-letter amino acid code and the sequence position based on similarity to the respective WT spectrum. Small letters indicate the side chain signals. A question mark behind the label indicates tentative assignments. Note that some red peaks that appear at new positions could not be assigned in this way and thus have no label. Superpositions of the ¹H-¹⁵N HSQC spectrum of the WT protein with that of each mutant are given in Fig. S10, and superpositions in the presence of stepwise increasing concentrations of DPC for F93/3049A-W96/3052A are given in Fig. S11A, for F93/3049A in Fig. S11B, and for W96/3052A in Fig. S12A.

Table 2.

Summary of the NMR-monitored interaction analysis of mutant hATMfatc-gb1ent proteins with different neutral membrane mimetics

Mutation	DPC micelles (50 mm)	DihepPC micelles (50 mm)	DMPC/DihepPC bicelles (q = 0.2, [DMPC] = 0.04 m, and [DihepPC] = 0.20 m, cL 15%)	DMPC SUVs (<50 mm DMPC)
F93/3049A	+	+	+	+
W96/3052A	+	+	+	+
F93/3049A–W96/3052A	+	+	+	−

+, strong spectral changes indicating an interaction with the respective membrane mimetic; −, no significant spectral changes/interaction.

Discussion

ATM is not only involved in DNA repair; it has also been suggested that ATM plays a role in the oxidative stress response and as a link between genome stability and carbon metabolism (9, 10). In line with this, it was recognized that ATM localizes not only in the nucleus but also at cytoplasmic vesicles (24) and more specifically peroxisomes (14) and microsomes (23), the plasma membrane (25), and mitochondria (15). NMR- and CD-monitored interaction studies showed that the ATM FATC domain can interact with all tested membrane mimetics and thus may, besides protein–protein interactions, also mediate direct membrane interactions of ATM (32). The presented NMR structure of the micelle-associated state of the FATC domain of human ATM (Figs. 1, B–D and 2) and the characterization of its dynamic and immersion properties from MD and further NMR data (Figs. 3 and 4) show that membrane association is mediated by three rather flexibly linked helices that immerse largely in the headgroup region and slightly below and that do not significantly interact with each other. This dynamic assembly may enable specific interactions with other membrane-embedded proteins. Recruitment to the plasma membrane involves interactions of the protein CKIP-1 (casein kinase-2 interaction protein-1) that is involved in muscle differentiation and the regulation of the actin cytoskeleton with the ATM C-terminal region, including the catalytic and the FATC domain (51). In addition, interactions with the PEX5 peroxisome import receptor localize ATM to peroxisomes (14).

The presence of a C-terminal positively charged surface patch next to a smaller negatively charged one in the micelle-associated ATM FATC domain (Fig. 2) may not only mediate interactions with the positively and negatively charged headgroup components of neutral phosphatidylcholines or phosphatidylethanolamines but additionally enable interactions with negatively charged phosphatidylserine in the PM or phosphorylated phosphoinositol lipids at mitochondria (52). The folding-upon-binding to a membrane mimetic, which has, for example, also been detected for the 30-residue-long glucagon-like peptide 1 (GLP-1) (53), and the resulting dynamic helix assembly of membrane-associated hATMfatc may further be helpful to interact with specific receptors or membranes with different packing densities, which depends, for example, on the amount of cholesterol or the ratio of saturated versus unsaturated fatty acids in the lipids (52). Based on the membrane catalysis hypothesis, initial interactions of signaling peptides with the bilayer increase their local concentration and allow them to adopt a structure that is recognized by the target membrane–resident receptors (53). The same may be true for specific protein domains or segments.

Based on mutagenesis studies, the FATC domains of all PIKKs play an important role for their regulation (26, 30, 31, 33–35, 54) and share the ability to interact with different membrane mimetics, albeit with varying preferences for specific membrane properties (32, 44). The observed evolutionarily conserved sequence differences (e.g. Fig. 1B, ATM and TOR) may result in different membrane-associated conformations that determine the specific membrane-targeting behavior (32). In line with this, micelle-associated hATMfatc adopts a rather dynamic assembly of three helices with a hydrophobic N-terminal region and a more polar C-terminal one, whereas the structure of the yeast TOR1 FATC domain consists of a single helix followed by a loop that in the oxidized state contains a disulfide bond and where the N-terminal region is acidic and the C-terminal one largely hydrophobic (Figs. 1C and 2). Consistent with this, the FATC domain of ATM cannot replace the one of ATR (ataxia- and Rad3-related) (29) or TOR (33).

The FATC domains of all PIKKs share conserved sequence features (e.g. Fig. 1B, ATM and TOR) that may be important for the regulatory function and especially for directly mediating membrane interactions (32). The conserved glycine near the C terminus of TOR (Gly²⁴⁶⁵ of y1fatc (Fig. 1B), Gly²⁵⁴⁴ in human TOR) appears to facilitate the back bending of the C-terminal region to the α-helix (Fig. 1C, right). Except for TRRAP, this glycine is also conserved in all other PIKKs (32) and, in the case of ATM, participates in the flexible linkage between α2 and α3 (Fig. 1C, center). The evolutionarily also completely conserved proline 3042 (= Pro⁸⁶), aspartate 3041 (= Asp⁸⁵), and alanine 3039 (= Ala⁸³) of hATM form together with Ile³⁰⁴⁰ (= Ile⁸⁴) in humans but a methionine in other organisms, the flexible linker between α1 and α2. The proline and the aspartate are also found in the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) amino acid sequences from most organisms (human Asp⁴¹¹³ and Pro⁴¹¹⁴) and, based on ¹³C^α secondary shifts, also disrupt two helical stretches (32). Interestingly, alanine 3039 of hATMfatc is also in most other PIKKs conserved, including TOR, in which the helix of the micelle-associated oxidized and reduced states is distorted around this residue (44).

All PIKK FATC domains contain at least one or more tryptophans and/or one or more tyrosine or phenylalanine and several aliphatic hydrophobic residues (32). Tryptophans are often found at the interface between the apolar interior and the polar aqueous environment (48, 49), and many hydrophobic residues show a positive free energy contribution for the transfer of a model peptide from a lipid bilayer to water (55). Replacement of either Tyr²⁴⁶³ or Trp²⁴⁶⁶ (Tyr²⁵⁴² and Trp²⁵⁴⁵ in human TOR) is already sufficient to abrogate the interaction of y1fatc with DMPC SUVs (50). For hATMfatc, Phe³⁰⁴⁹ (= Phe⁹³) and Trp³⁰⁵² (= Trp⁹⁶) at equivalent positions have to be replaced simultaneously to achieve the same effect (Fig. 5 and Figs. S11 and S12). However, the respective mutations did not abrogate the interaction with micelles and bicelles (Fig. 5 and Figs. S10–S12). The different association behavior of FATC mutants with micelles and bicelles compared with SUVs can be explained 2-fold. First, in the case of micelles or bicelles, the total concentration of the used detergents or lipids and thus the available membrane mimetic surface area is much higher than for SUVs. The used concentration of DPC and DihepPC was 50 mm, and the total lipid concentration in the DMPC/DihepPC bicelle samples was ∼240 mm. However, due to the preparation method, the total lipid concentration in the SUV samples is significantly below 50 mm because pure DMPC cannot easily be resuspended at high concentrations, and in addition, some of the lipid is lost when separating the small from larger uni- and multilamellar vesicles by centrifugation. Whereas one-dimensional ¹H NMR spectra can be used to compare the lipid content in samples with micelles and bicelles, this is due to the large size of SUVs and corresponding line broadening not possible. However, besides established methods such as dynamic light scattering, the CD signal can be used to compare different liposome preparations (50, 56). A second reason for the different binding behavior of the FATC domain to micelles and bicelles versus SUVs may further be a higher affinity for curved membrane surfaces. DPC micelles are rather small, spherical particles that contain ∼50–60 DPC molecules, resulting in a molecular mass of ∼19 kDa and a radius of gyration of about 17–18 Å (57, 58). Bicelles have a rather planar bilayer region that is formed by a long-chain phospholipid, such as DMPC. However, the rim that is formed by a short-chain lipid such as DihepPC shows as micelles also a high curvature. The molecular mass of bicelles is usually >∼250 kDa, and the diameter of the planar region ranges from 21 to 77 Å for q values between 0.2 and 1 (59, 60). Even liposomes of the SUV type are much larger than bicelles. Although they are as micelles approximately spherical, they are less curved because the radius is much larger (around 20–100 nm) (61). We already suggested earlier that for proteins with a rather high and broad affinity for different membrane mimetics, SUVs at the usually used lower concentrations are better suited to detect mutants that may also abrogate membrane association in localization studies in cells (50).

The characterization of the membrane association of ATM by NMR and MD simulations was done looking only at the FATC domain (Figs. 1–4). This raises the question of how accessible it is for membrane interactions in the context of the full-length protein. Based on recent cryo-EM data, ATM was suggested to be in an equilibrium between a closed, inactive dimer (resolution ∼4–6 Å) with restricted access to the substrate-binding site and an open, active dimer (resolution ∼11.5 Å; if combined data used, 5.7 Å) with good access to the substrate-binding side (40). Whereas the FATC domain appears not well-accessible in the closed, inactive ATM dimer, it appears well-accessible for interactions with membranes and regulatory proteins in the open, active state. The conformation of the FATC domain in the provided cryo-EM structures is similar to the micelle-associated conformation presented here and also consists to two α-helices and a C-terminal helix or turnlike structure (40). Early studies suggested that DNA damage induces Ser¹⁹⁸¹ autophosphorylation and that this results in active, monomeric ATM (62), in which the FATC domain is expected to be even more accessible than in the open dimer. Moreover, studies of oxidative activation of ATM indicated that oxidation of Cys²⁹⁹¹, which is about 60–80 residues N-terminal of the FATC domain, results in dimerization (63). Thus, future membrane interaction studies may analyze the effect of reactive oxygen species or oxidized lipids on Cys²⁹⁹¹. Future localization studies of ATM in cells have to clarify how it varies in response to specific signals and which interactions with specific, especially membrane-localized, proteins such as the PEX5 peroxisome import receptor (14), as well as membrane regions and specific signaling lipids it involves. Moreover, the oligomerization state under different signaling conditions and the resulting cellular localizations have to be determined. Finally, acetylation of lysine 3016 (54) N-terminal of the highly conserved FATC region of ATM may affect the described localization at the plasma membrane, mitochondria, peroxisomes, microsomes, and other cytosolic vesicles (12, 14, 15, 23–25). As suggested for the FATC domain of TOR, the one of ATM may also be fused to other proteins or peptides or other substances to tether them to membrane mimetics (64).

Experimental procedures

Plasmid cloning, protein expression, and purification

The coding sequence for the FATC domain of human ATM (residues 3024–3056, UniProt accession number 13315) was cloned into GEV2 (65). The resulting fusion protein consisting of GB1 (residues 1–56), a linker region composed of a thrombin (residues 57–62) and an enterokinase (residues 63–67) protease recognition site, and the C-terminal 33 residues of human ATM (residues 68–100) (= hATMfatc-gb1ent) was overexpressed in Escherichia coli BL21 at 37 °C. Because proteolytic removal of the GB1 tag is inefficient and the GB1 tag does not disturb the interaction with membrane mimetics and its monitoring, the fusion protein was used for the described NMR studies (32, 41). Uniformly ¹⁵N– or ¹⁵N-¹³C–labeled proteins were prepared in M9 minimal medium containing ¹⁵NH₄Cl and/or [¹³C]glucose as the sole nitrogen and carbon sources. The expression and the purification by a heating step (66) and IgG affinity chromatography were done as described previously (32, 67).

Site-directed mutagenesis

Replacement of one or more aromatic residue in hATMfatc-gb1ent by alanine (Fig. 1B, F93A = F3049A, W96A = W3052A, F93A/W96A = F3049A/W3052A) was achieved by following the QuikChange site-directed mutagenesis protocol (Stratagene). The success of the mutagenesis was verified by DNA sequencing. Cells expressing mutant hATMfatc-gb1ent were lysed by sonication in 50 mm Tris, 150 mm NaCl, 2 mm benzamidine, and 0.3 mm EDTA, pH 7.6. Mutant proteins were purified by IgG affinity chromatography as described for the WT and in the manufacturer's manual (32, 67). Fractions containing the target protein were pooled, washed with NMR buffer (50 mm Tris, 100 mm NaCl, pH 7.4, for the single mutants F93A = F3049A, W96A = W3052A or pH 6.5 for the double mutant F93A/W96A = F3049A/W3052A), and concentrated.

Preparation of membrane mimetics

DPC, DihepPC, DMPC, 5-SASL, and 16-SASL were purchased from Avanti Polar Lipids and/or Sigma-Aldrich. Deuterated DPC (DPC-d₃₈) was obtained from Cambridge Isotopes.

Membrane mimetic DPC micelles were prepared by placing a defined amount of DPC from a concentrated stock solution in chloroform (0.5 m) in a glass vial and drying under a stream of nitrogen gas. The resulting DPC film was dissolved in NMR buffer containing the protein of interest. For NMR titration experiments, samples containing >30 mm DPC were prepared in the same way, whereas those with DPC concentrations <30 mm were prepared by diluting a 100 mm DPC stock in buffer with a protein stock and buffer. Note that a separate sample was prepared for each DPC concentration to avoid dilution effects and thus to keep the protein concentration constant. DihepPC micelles were prepared by diluting a 0.25 m stock solution in NMR buffer to 50 mm with the respective protein solution and buffer. Micelles form above the critical micelle concentration, which is 1.1 mm for DPC (68) and 1.4–1.8 mm for DihepPC (69, 70).

DMPC/DihepPC bicelles (q = 0.2, [DMPC] = 0.04 m, and [DihepPC] = 0.20 m, cL 15%) were prepared as follows. An appropriate amount of a 0.25 m stock of the long-chain phospholipid (DMPC) in chloroform was placed in a glass vial, and the chloroform was blown away under a stream of nitrogen gas. To form bicelles, the right amount of the short-chain lipid (DihepPC) in NMR buffer was added, and the vial was vortexed until a clear solution was obtained. Last, the protein solution was added to the concentrated bicelles solution.

Liposomes were prepared by drying an appropriate amount of DMPC in chloroform (0.25 m) under a stream of nitrogen gas. The resulting pellet was dissolved in NMR buffer to obtain a 100 mm solution. To dissolve the pellet, it was exposed to seven cycles of freezing in liquid nitrogen, thawing by incubation in a water bath at 40 °C, and thorough vortexing. To induce the formation of SUVs from large uni- and multilamellar vesicles, the DMPC suspension was incubated in an ultrasonication bath for 30 min. A clear supernatant containing only SUVs was obtained after centrifuging the lipid mixture at about 15,000 rpm in a table top centrifuge for 5 min. The resulting white precipitate at the bottom corresponds to large uni- and multilamellar vesicles. For the preparation of a protein sample in the presence of liposomes, only the clear supernatant containing small unilamellar vesicles was diluted 1:1 with protein solution. Thus, using the 100 mm stock solution, the final sample contained <50 mm DMPC.

NMR sample preparation

For the NMR resonance assignment and structural characterization, samples containing ∼0.5 mm of ¹⁵N– or ¹⁵N-¹³C–labeled hATMfatc-gb1ent in 50 mm Tris, 100 mm NaCl, 10 mm tris(2-carboxyethyl)phosphine, 0.02% NaN₃ (95% H₂O, 5% D₂O), pH 6.5, 150 mm DPC-d₃₈ were used. We also prepared samples with 200 mm DPC (180 mm DPC-d₃₈ and 20 mm DPC), but the spectral quality was overall lower (broader signals, more noise signals) than for samples with 150 mm DPC (e.g. see Fig. S1). The 10% ¹³C-labeled hATMfatc-gb1ent sample used for the stereospecific assignment of valine and leucine methyl groups contained 170 mm DPC-d₃₈ micelles.

The samples of hATMfatc-gb1ent mutants contained about 0.15 mm ¹⁵N-labeled protein in NMR buffer. For the double mutant (F93A/W96A = F3049A/W3052A), the pH was 6.5, as for the WT. The single mutants (F93A = F3049A, W96A = W3052A) were measured at pH 7.4 to improve their solubility and stability.

For NMR experiments with spin-labeled DPC micelles, samples containing about 0.1 mm protein and 50 mm undeuterated DPC were prepared. The concentration of 5-/16-SASL was increased by the stepwise addition of a small amount (1–4 mm) from a 0.25 m stock solution in chloroform.

NMR spectroscopy

NMR spectra were recorded on Bruker Avance 500-, 600-, and 900-MHz spectrometers at a temperature of 298 K. The 500- and 900-MHz spectrometer were equipped with cryogenic probes. Data were processed with NMRPipe (71) and analyzed using NMRView (72). Assignments for the ¹³C, ¹⁵N, and ¹H nuclei of micelle-immersed hATMfatc fusion protein were based on 2D ¹H-¹⁵N and various ¹H-¹³C HSQC spectra, partially without decoupling, 3D HNCA, HNCACB, CCONH-TOCSY, HCCH-TOCSY, HNHA, HNHB, and ¹⁵N- and ¹³C-edited NOESY spectra as described (67). The NOESY mixing times were 90 ms for the ¹⁵N-edited NOESY and 100 ms for the aromatic and aliphatic ¹³C-edited NOESY experiments. Information about backbone dynamics was derived from the analysis of ¹⁵N relaxation data, including T₁ (spin-lattice longitudinal relaxation rate), T₂ (spin-spin or transverse relaxation rate), and {¹H}-¹⁵N NOE.

The immersion depth properties of hATMfatc in DPC micelles were derived by recording ¹H-¹⁵N HSQC and 1D ¹H NMR spectra of samples containing increasing amounts of paramagnetic 5- or 16-SASL. The resulting PRE effects were derived based on the observed changes in signal intensity. Because the spectral changes with 5-SASL were overall smaller compared with 16-SASL, 1-PRE values for 2 mm were analyzed for 5-SASL, and values for 1 mm were analyzed for 16-SASL. In addition, the average chemical shift changes of the backbone amide nitrogen and proton (Δδ(N,H)_av) were calculated based on the formula, ((Δδ_HN)² + (Δδ_N/5)²)^½. The interaction of mutant proteins with membrane mimetics was monitored by recording 1D ¹H and 2D ¹H-¹⁵N HSQC spectra.

Structure calculation

All structure calculations were performed with XPLOR-NIH (73) using molecular dynamics in torsion angle and Cartesian coordinate space and the standard force field with the parameter files topallhdg_new.pro and parallhdg_new.pro. Distance restraints were generated in NMRView (72) and classified according to NOE cross-peak intensities. Upper bounds were 2.8, 3.5, 4.5, and 5.5 Å. The lower bound was always 1.8 Å. For all NOE restraints, r⁻⁶ sum averaging was used. Backbone dihedral angle restraints for φ and ψ as well as hydrogen bond restraints for helical regions were derived based on the determined ¹³C^α chemical shifts, observed helix typical NOE restraints, and initial structure calculations.

MD simulations

Molecular dynamics simulations were performed with Gromacs 2016.3 software (74–76). The FATC domain was initially placed randomly near the preequilibrated micelle (50 DPC molecules) and solvated with ∼54,000 water molecules (box size ∼11 nm). Na⁺ and Cl⁻ ions were added to the system to reach 0.1 m salt concentration. The CHARMM36m force field was used for the protein and DPC (77). A 2-fs time step was used. All bonds were constrained with the LINCS algorithm (78), and water bond lengths and angles were kept constant using the SETTLE algorithm (79). Initial velocities were taken from the Maxwell distribution for 303.15 K. A constant temperature of 303.15 K was maintained with the V-rescale thermostat with a 0.1-ps coupling constant (80). The DPC micelle with the protein and the water molecules with the ions were coupled to two separate thermostats with the same parameters. Constant pressure of 1 bar was maintained by the isotropic Parrinello–Rahman barostat with a 5.0-ps coupling constant and 4.5 × 10⁻⁵ bar⁻¹ compressibility (81). The particle mesh Ewald algorithm was used for long-range contributions to electrostatic interactions (82, 83). Lennard–Jones interactions were cut off at 1.2 nm with a force-switch modifier from 1.0 to 1.2 nm.

Three independent simulations were performed using as starting structures the three lowest-energy NMR structures of the micelle-associated FATC domain of human ATM. Each simulation was 1.3 ms long. For the analysis of the secondary structure based on DSSP calculations (84), the clustering, the immersion depth, and the fraction of solvent-accessible surface area covered by the micelle, the first 300 ns of each simulation were discarded as equilibration time.

RMSD and RMSF calculations were performed on the last 500 ns of each trajectory (800–1300 ns). RMSF analysis with the local fit of the protein structure was performed in addition to the calculations with a whole protein as a reference. The RMSD/RMSF is dominated by the contributions from a change in relative orientation of the helices, and we wanted to highlight local fluctuations in particular regions that are not the result of the change in helix-helix orientation. RMSF graphs that are marked as “local 72–92” or “local 86–98” (Fig. S4) were calculated with the use of a short part of the protein as a reference for the progressive trajectory fitting and subsequent RMSF calculation (residues 72–92 and 86–98; 5-ns windows with the following averaging over all 5-ns intervals). Consequently, the RMSF values will be rather small for the region used for the fit.

The analysis of the angles between the helices in Fig. 3C (Fig. S5) starts also from 800 ns, because the relative orientation of the helices changes slowly compared with the local structure of the protein. Thus, we let the system equilibrate for a longer period of time to have less bias from the initial structure.

The distance between the micelle COM and the whole peptide COM, as well as separate peptide residue COMs, was computed using the simulation period from 300 to 1300 ns. To analyze peptide stability, the secondary structure of each peptide was computed as a function of time with the gmx do_dssp analysis program, which is part of the Gromacs package. The micelle surface for the images of the micelle-associated FATC domain was defined as an iso-surface of averaged DPC density (contour density of 0.250 atoms/ų). The pictures of the FATC domain alone are representative conformations of the protein as defined by cluster analysis of the protein RMSD performed by the gmx cluster program with the GROMOS clustering algorithm and 0.1 nm cutoff. All three trajectories (excluding the first 300 ns of each run) were combined into a single set of structures, and cluster analysis was performed on this combined trajectory. Subsequently, 10 structures, one from each of the first 10 most populated clusters, were selected as representative structures of the micelle-associated ATM FATC domain. The central structures of each cluster (in terms of RMSD distance) were selected as representative for a given cluster. The combined size of the 10 most populated clusters was equal to 69.3% of the complete set of structures sampled. For the picture of a representative structure of micelle-associated hATMfatc for each run, a clustering analysis for reach run was performed. From the most populated cluster, a structure with an RMSD corresponding to the average for the whole cluster, thus being in the middle, was selected. To estimate whether there are interhelical contacts below about 0.5 nm that could give rise to NOEs, interresidue distances were calculated between the centers of mass for pairs of residues that belong to different helices (excluding adjacent residue pairs; Fig. S4D).

Author contributions

S. A. D. designed the study. M. S. A. R. performed the NMR experiments, analyzed the results, and calculated the structures supported by S. A. D. D. P. T. and Y. K. C. designed the MD simulations, and Y. K. C. performed the simulations and analyzed the corresponding results. S. A. D. wrote the paper with M. S. A. R., Y. K. C., and D. P. T.