SUPPLEMENTARY INFORMATION

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1 doi: /nature10955 Supplementary Figures Supplementary Figure 1. Electron-density maps and crystallographic dimer structures of the motor domain. (a f) Stereo views of the final electron-density maps of the AAA+ ring (a), stalk/strut coiled coils (b), MTBD (c), AAA5-extension (d), nucleotide binding site of AAA3 (e) and interacting sites between the linker and AAA2 (f). The final coordinates are represented in the density map. Ribbon models in the panels c and d show the corresponding regions in the molecule. An omit map, depicted as a red wire, of the ADP molecule is also shown in the panel e. (g and h) Crystallographic dimer structures of the MTBD (g) and WT (h) motor domains. The stalk of MTBD-B is kinked compared to that of MTBD-A, and MTBD of WT-B is invisible possibly due to its relatively high mobility. The stalk region including MTBD is potentially flexible, so that the obvious structural differences are found in this region between two molecules. W W W. N A T U R E. C O M / N A T U R E 1

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4 Supplementary Figure 2. Secondary structure assignments of the dynein motor domain. The secondary structure elements of the 380-kDa dynein motor domain are indicated above the amino acid sequence with α-helices and β-strands represented by helical coils and arrows, respectively. Hydrogen bonded turns are indicated by T. The regions, where the electron density for the motor domain is not interpretable, are indicated by dotted lines. The assignments are based on the ΔMTBD-A structure, except for MTBD. The ATPase motifs, Walker-A, Walker-B, Sensor-I, Sensor-II, and arginine-finger are also indicated by abbreviated names, WA, WB, S1, S2, and R, respectively. The figure was created using ESPript

5 Supplementary Figure 3. Structure of the functional unit constituting the dynein motor domain. Structures of the ring (a), linker (b), stalk and strut in WT-A (c), and C-sequence (d). Subdomain definitions in the linker and C-sequence are indicated below the structure. The outward and inward helices of the stalk are denoted as CC1 and CC2, respectively. Dotted lines indicate the regions, where the electron density for the motor domain is missing or is not interpretable. 5

6 Supplementary Figure 4. Modular structure of the ATP-hydrolyzing ring. (a) Hexameric arrangement of the six AAA + modules in the ring (left) and a topological diagram of each AAA + module (right). The ATPase motifs characteristic of AAA + proteins are marked as red dots and labeled in the diagram. (b) Ribbon representation of the six AAA + modules with bound ADP in stick form. Unique insertions are colored in red. Dotted lines indicate the regions, where the electron density for the loop region is missing or is not interpretable. 6

7 Supplementary Figure 5. Comparison of the current atomic structure and the previous 4.5-Å model. Compared to the previous helical model at 4.5-Å resolution 17, three portions have been remodeled in the current structure. Helices in the first half of the C-sequence have been now identified as the AAA5-extension, as indicated by the cyan dotted circle. MTBD is reassigned to the previously unassigned electron density located at the black dotted circle with an angle different from the previously assigned structure (~90 ). The current C-sequence corresponds to the last half of the previous C-sequence and the old MTBD in the crystallographic next molecule, as indicated by the red dotted circle. The comparison of present and previous structures suggests that there seems to be a conformational bias in the previous WT structure due to phase calculation. In the previous analysis, we did not distinguish the heavy atom derivatives from ΔMTBD and WT because of their similar unit cell dimensions and similar molecular weights. Since the Ta 6 Br 12 derivative of the ΔMTBD crystal diffracted better than that of the WT crystal, we used it for phase calculation of WT at the early stage. Now we found that the kinked conformation of the previous WT structure is likely to reflect the bias from the phase calculation based on ΔMTBD. However, the other parts of the previous WT model show good agreement with the present WT model. 7

8 Supplementary Figure 6. Residues interacting with bound ADP in the four nucleotide-binding sites. Magnified view of ADP molecules in the nucleotide-binding sites of AAA1 (a), AAA2 (b), AAA3 (c), and AAA4 (d) in ΔMTBD-A. The ATPase motifs, i.e. Walker-A, Walker-B, Sensor-I, Sensor-II and arginine-finger, and residues interacting with ADP are shown as stick models. The ATPase motifs are labeled in black letters, and the interacting residues are in the standard colors for the motor domain. Hydrogen bonds to the ADP molecules are drawn with grey dotted lines. In the AAA2 nucleotide-binding site, there is well-defined electron density for a magnesium ion, which is coordinated to β-phosphate of bound ADP. In ΔMTBD-B, two magnesium ions are identified in AAA1 and AAA2 with similar coordination patterns (not shown). In the AAA4 nucleotide-binding site, the side chain of Sensor-II R3258 looks improperly oriented. However, in the WT structure, the corresponding side chain is arranged in a suitable position for ATP binding and hydrolysis (not shown). 8

9 Supplementary Figure 7. ATP hydrolysis activity of the four nucleotide-binding sites. Time courses of phosphate release from the dynein motor domain after the addition of 1 mm ATP, as monitored by fluorescence enhancement of fluorescently-labeled phosphate-binding protein. MT, in the absence of MT. +MT, in the presence of MT. Each time course is the average of four to ten individual traces. The black curves are the best fits to single-exponential plus steady-state linear functions; the mean apparent phosphate burst (k obs (burst)) and steady-state (k steady ) rate constants are given in Supplementary Table 2. To test the conclusion from the structural analysis described in the main text, we examined the ATPase activity of each of the four nucleotide-binding sites by using the motor domains carrying multiple Walker-B mutations in AAA1, AAA3, and AAA4 (E2027Q, E2745Q, and E3075Q, respectively). The mutations were expected to abolish ATP hydrolysis but not to impair ATP binding at the mutated sites 9. After the addition of ATP, WT showed a fast Pi burst and a slower steady-state Pi release (a), whereas a mutant, carrying triple Walker-B mutations (E2027Q/E2745Q/E3075Q), did not show a detectable Pi release (b). The results indicate that the Walker-B mutation in AAA1, 3, and 4 does, in fact, abolishes ATP hydrolysis at the mutated sites and, furthermore, that the intact AAA2 site does not hydrolyze bound ATP, in agreement with the distinctive structural organization of the ATPase motifs. All double Walker-B mutants (E2745Q/E3075Q, E2027Q/E3075Q, or E2027Q/E2745Q) clearly exhibited the Pi burst and/or steady-state Pi release (c e), indicating that each of the intact AAA1, 3, and 4 sites has the capacity to hydrolyze bound ATP. Similar kinetic experiments performed in the presence of MTs showed that MTs accelerate Pi release only from the double mutant (E2745Q/E3075Q) with the intact AAA1 site (c e). Thus, among the three ATPase sites, AAA1 alone drives the MT-activated ATPase activity essential for dynein function. 9

10 Supplementary Figure 8. Comparison of the ring structure between ΔMTBD and WT. (a) Arrangements of the α (left) and α/β (right) submodules in the ring of ΔMTBD-A (top) and WT-A (bottom). The arginine fingers are represented by red sticks with red labels. ADP molecules are shown as CPK models. The distances between the Nη of arginine-finger and β-phosphate of ADP are indicated by black dashed lines. (b) Front and two different bottom views of the superimposed α/β submodules of ΔMTBD-A and WT-A, based on the least-squares fitting of whole AAA + modules. Right square panels colored in orange and in black show the bottom views from the positions of orange and black arrowheads, respectively. The α/β submodules of WT-A are colored as in the panel a, and those of ΔMTBD are in grey. The maximum Cα-distance and the rotation angle between the WT-A/ΔMTBD-A pair are indicated for each of the AAA + modules. The WT-A ring structure has a more closed conformation than that seen in ΔMTBD-A: the AAA1 AAA2 and AAA4 AAA5 gaps are closed by approximately 10 Å and 5 Å, respectively. 10

11 Supplementary Figure 9. Linker ring interactions in WT-A. Side views of the motor domain showing linker AAA2 interactions in MTBD-A (top) and WT-A (bottom). Interacting residues between linker subdomain 2/3 and H2/PS-1 inserts and between H2 and PS-1 inserts are shown. In both the MTBD-A and WT-A structures, the H2/PS-1 inserts of AAA2 form the second major interaction interface between the linker and the ring even though the interacting sites are slightly different. In WT-A, the tips of the H2 and PS-1 inserts form hydrophobic interactions with the H11 and H13 helices, respectively in the linker subdomain

12 Supplementary Figure 10. Functional analyses of ΔH2I, ΔSD, and ΔC-seq. (a) MT binding activity of ΔH2I in the presence or absence of ATP. Each symbol is mean ± s.d. (n = 3). The ΔH2I mutation, disrupting the ATPase-driven linker swing actions, did not abolish ATP-induced change in the affinity for MTs, although the affinity change was smaller than that of WT. (b) FRET efficiency between BFP and GFP moieties in ΔSD and ΔC-seq in the presence of 200 μm of indicated nucleotide. For ΔSD in the presence of ATP, the FRET efficiency was measured with 2.5 mm ATP to avoid depletion of ATP due to its very high ATPase activity. Mean ± s.d. (n = 3) are shown. The dotted lines indicate the high and low FRET values of WT representing the linker positions at the primed (pre-powerstroke) and unprimed (post-powerstroke) states, respectively 12,15. The ΔSD and ΔC-seq mutations, disrupting the coupling between MTBD and the AAA1 ATPase, did not block ATP-induced changes in FRET but altered the FRET efficiency and nucleotide dependence. The results suggest that the two structural units are not essential for the linker swing, but are relevant to the proper positioning of the linker and/or normal kinetics of the ATPase-driven swing actions. 12

13 Supplementary Figure 11. Heptad register of the stalk and strut coiled coils. The heptad register was analyzed by the knobs-into-holes side-chain packing with the program SOCKET 48. Residues assigned as knobs are shown as red one-letter codes of amino acid sequences. Residue numbers in blue (N3566) and green (L3855) indicates knobs-into-hole interaction between the stalk and strut coiled coils (see also Fig. 4a). All four structures adopt the same stalk-coiled-coil registry known as α ; when fixed in this registration, the dynein motor domain is trapped in an allosteric state showing a high ATPase activity and a strong MT-binding affinity The ΔMTBD-A molecule has a straight stalk made of a canonical antiparallel two-stranded coiled coil with knobs-into-holes packing along its entire length (100 residues: F3263-A3362 and L3504-S3596) and a strut of an antiparallel two-stranded coiled-coil-like structure without any knobs-into-holes packing (see also left, Fig. 4a). In contrast, ΔMTBD-B possesses a kinked stalk with a shorter coiled-coil packing (57 residues: L3292-L3348 and L3511-L3567) and a strut with a canonical coiled-coil region (5 residues: L3830-L3834 and T3854-L3858; see also right, Fig. 4a). 13

14 Supplementary Figure 12. Structural comparison of the MTBD-stalk structures. (a) Comparison of the MTBD structures. Structure of the mouse cytoplasmic dynein MTBD with a portion of the stalk (PDB ID: 3ERR) 31, shown as brown tubes, is superimposed onto the WT-A structure, shown as yellow ribbons. The major structural differences are the melted/folded form of the distal portion of the CC2 helix and the angle between MTBD and the adjacent stalk coiled coil, which may reflect their distinct MT-binding states: the mouse MTBD was fixed in a weak-binding state by fusing onto a stable coiled coil with known registry 31, whereas the ADP-bound WT motor domain is trapped in a strong-binding state 37. There are few structural differences between the backbone of the two MTBDs, suggesting that structural changes within MTBD between strong and weak MT-binding states would be small to be detected at the present resolution, in agreement with the results of previous NMR and computational modeling studies 52,53. (b) Comparison of the stalk structures between WT-A and ΔMTBD-A. In WT-A, the electron density for the CC2 helix of the stalk is missing in the distal region adjacent to MTBD (left panel; D3492 L3511) even though that for MTBD is clearly visible, an indication that the stalk coiled coil of WT-A partially melts while keeping MTBD in place, in contrast to the fully folded stalk in ΔMTBD. The superposition of the two coiled-coil structures based on the outward helices (right panel) shows structural distortion of the WT stalk with the maximum deviation of the inward helices at Q3545. Although the MTBD-stalk structure reported here may be affected by crystal packing, the melting feature of the distal portion of the stalk in the ADP-bound motor domain is in agreement with previous biochemical studies suggesting that the distal portion of the stalk does not adopt a particular coiled-coil registry in the ADP-bound motor domain 32 and that the coiled-coil stalk is a relatively unstable structure in solution

15 Supplementary Figure 13. Back side view of the motor domain showing the interactions through the H1 helix of the C-sequence. The left panel shows the motor domain structure behind the H1 helix. The structure at the reader s side from the H1 helix, the AAA5-extension and the C-sequence, was cut out and shown in the middle panel. A different view of the cutout is also shown in the right panel. The interacting residues between the ring and the C-sequence are drawn in white stick models. The C-sequence is composed of three subdomains, the N-terminal H1 helix, helical region, and C-terminal barrel region as indicated in Supplementary Figure 3d. The H1 helix extends from AAA6α adjacent to AAA1α/β, is held extensively between AAA5α and AAA5-extension, reaching to the base of the strut coiled coil in AAA5. The helical and barrel regions interacts mainly with AAA1α and AAA6α, respectively. Our previous mutational analysis has shown that S4448-I4730 corresponding to the helical and barrel regions is not required for motor activity, and the current study suggests that the H1 helix (and possibly the loop region) is critical for the allosteric communication. Truncation positions (S4416, S4448 and I4730) described in the main text are indicated as red circles in the right panel. For reference, the tube presentation of the yeast motor domain structure 16 is displayed in the lower panel. The structural arrangement around the H1 helix is conserved between the yeast and Dictyostelium cytoplasmic dynein: the short C-terminal helix exists at the same position of our H1 helix, and is surrounded by three helices corresponding to H9, H10, and H16 of our AAA5-extension, although AAA5-extension has relatively low sequence conservation among dynein species. 15

16 Supplementary Methods The following residues are not included in the current models because the corresponding electron density is missing or is not interpretable. ΔMTBD-A: the N-terminal tag (24 residues), residues , , , , , , , , , , , TG linker, , , , , , , , , , and ΔMTBD-B: the N-terminal tag (24 residues), residues , , , , , , , , , , , , TG linker, , , , , , , , and WT-A: the N-terminal tag (24 residues), residues , , , , , , , , , , , , , , , and WT-B: the N-terminal tag (24 residues), residues , , , , , , , , , , , , , , , , , and

17 Supplementary Table 1. Data collection, phasing and refinement Wild type ΔMTBD Native Ta 6 Br 12 PW 12 O 40 SeMet Data collection Space group P P Cell dimensions a, b, c (Å) , , , , , , , , , , Wavelength (Å) Resolution (Å) ( ) a ( ) ( ) ( ) ( ) Measured reflections Unique reflections (4052) (8629) (5452) (5489) (5497) R merge 7.8 (39.7) 9.2 (77.9) 15.8 (67.1) 14.2 (86.2) 8.7 (27.5) I/σ(I) 31.1 (4.2) 17.6 (9.9) 27.4 (5.8) 21.5 (2.9) 28.7 (8.1) Completeness (%) 99.4 (92.3) 98.3 (81.9) (100.0) (100.0) (99.9) Redundancy 6.0 (5.1) 5.5 (4.2) 15.1 (14.9) 7.1 (7.1) 6.1 (6.1) MIRAS phasing R iso (F) (%) No. of sites Resolution (Å) Phasing power (centric/acentric) 1.07/ / /0.24 Figure of merit (centric/acentric) 0.18/0.26 Refinement Resolution (Å) R work/ R free 22.1/ /32.1 No. of construct residues b /atoms (3367/54204) 2 (3245/52209) 2 No. of residues c in chain A/B 3042/ /2853 No. of atoms in chain A/B 23377/ /22147 No. of ADP residues/atoms 8/216 8/216 No. of spermine residues/atoms - 4/56 No. of Mg ions - 3 No. of waters - 42 Average B-factors (Å 2 ) Protein Ligand Water R.m.s deviations Bond lengths (Å) Bond angles ( ) Ramachandran plot d (%) (favored, allowed, 80.8/17.8/1.4/ /15.3/0.5/0.0 generous, and disallowed) PDB code 3VKH 3VKG a Highest resolution shell is shown in parentheses. b The number of residues includes the N-terminal expression tag (24 amino acids). c Of these, 131 and 90 residues in wild type chain A and B, 238 and 194 residues in ΔMTBD chain A and B were modeled as ALA due to lack of side chain electron density. d Ramachandran plot was calculated by PROCHECK. 17

18 Supplementary Table 2. Phosphate release rates from the wild type and the double Walker-B mutants -MT +MT k obs (burst) k steady k obs (burst) k steady Dynein (s -1 ) (s -1 ) (s -1 ) (s -1 ) Wild type HFB ± ± 0.3 nm 31.6 ±3.1 E2027Q/E2745Q/E3075Q E2745Q/E3075Q E2027Q/E3075Q E2027Q/E2745Q nm nm nm nm 6.0 ± ± ± ± ± ± ± ± ± ± ± ± 0.05 The apparent phosphate burst rate constants (k obs (burst)) and steady-state rate constants (k steady ) in the presence (+MT) or absence (-MT) of 20 μm MTs are shown as mean ± s.d. of three independent measurements. nm, not measurable. Supplementary References 51 Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, (1999). 52 McNaughton, L., Tikhonenko, I., Banavali, N. K., LeMaster, D. M. & Koonce, M. P. A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain. J. Biol. Chem. 285, (2010). 53 Choi, J., Park, H. & Seok, C. How does a registry change in dynein's coiled-coil stalk drive binding of dynein to microtubules? Biochemistry 50, (2011). 54 Hook, P., Yagi, T., Ghosh-Roy, A., Williams, J. & Vallee, R. B. The dynein stalk contains an antiparallel coiled coil with region-specific stability. Biochemistry (2009). 18