The six AAA domains can be easily identified in the electron density map (overview in ). AAA proteins possess an α/β large domain, which contains the nucleotide binding Walker A (P-loop) and B motifs, and a helical bundle small domain that extends from the C terminus of the large domain (). The large domain (AAAL) is characterized by a parallel five-strand β sheet surrounded by two α helices (H0 and H1) on one face and three helices (H2, H3, and H4) on the opposite face, although different subclasses of AAA proteins contain unique insertions to this core structure (4
). In our map of both motor domains, the AAA domains show a nearly identical pattern of electron density (fig. S3A
), with five helices (H0 to H4) in similar positions to other AAA proteins ( and fig. S3, B and C
). H0 and H1 in the six AAA domains are on the exterior of the ring, as in other hexameric AAA proteins. Although individual β strands are difficult to identify, a diffuse electron density in the position and orientation expected for the AAA β sheet is observed ( and fig. S1
). When viewed from the side, the large domains form the top layer of the linker face of the ring.
Fig. 2 The dynein AAA domains. (A) The six individual AAA domains are highlighted in colors. The linker spanning over the center of the ring is magenta. (B) Topology of the dynein large and small domains. (C) The electron density map [experimental (Fo) map contoured (more ...)
The six dynein AAA small domains (AAAs), which form the C-terminal face of the ring, all show a similar pattern of five helices (H5 to H9) ( and fig S4A
), whose connectivity can be established by well-ordered loops. The general placement of the small domains in the ring and connectivity of H5 to H8 are similar to NSF (N-ethylmaleimide–sensitive fusion protein) (fig. S4C
) and several other AAA proteins. Dynein has evolved a fifth helix (H9) at the bottom of the small domain, which helps to connect the small domain of one AAA domain to the large domain of the neighboring AAA in the poly-peptide chain. In contrast to an earlier suggestion (4
), the extra helix in dynein is at the C terminus rather than the N terminus of the small domain; thus, the small-domain architecture is not similar to the BchI subclass of AAA proteins. AAA5s and AAA6s contain additional helices beyond H9, although their connectivity is not well established in our model. The stalk and buttress coiled coils are extensions of small-domain helices (fig. S4A
Several pieces of information enabled us to assign AAA1 to 6 within the electron density map. First, we could use information from previous EM studies. The apo form of dynein seen by EM is strikingly similar to our crystal structure (fig. S2, A and B
); the N terminus of the linker and the stalk base create fiducial markers that allow the x-ray and EM structures to be aligned with one another. The order of the AAA domains around the ring has been determined by EM after tagging particular domains with engineered fluorescent protein markers (8
). When thus aligned, the order of the AAA domains is clockwise around the ring when viewed from the linker face, with AAA1 positioned close to the linker C terminus based on sequence (). In the model presented in this EM study, however, the linker was assigned to the opposite face of the AAA ring, possibly because of insufficient three-dimensional discrimination.
Further evidence for assigning the individual AAA domains came from the inspection of the electron density maps for characteristic insertions to the canonical large domain AAA core structure. AAA1, AAA2, AAA3, and AAA5 have β-hairpin insertions between H3 and β4, a feature that is found in a subset of other AAA proteins (4
) (, and fig. S5, A and B
). AAA2 has an additional loop insertion within H2 (fig. S5A
); a similar combination of H2 and H3 inserts is found in NtrC (23
) (fig. S3B
) and BchI (24
) but few other AAA proteins. AAA4 also has an insertion of a pair of helices that extend from H3 and β4. Furthermore, AAA2, 4, and 5 have an extra N-terminal helix that packs against H0 and H1, as observed in certain other AAA proteins such as ClpX (fig. S3C
). All of these features in the dynein sequence (fig. S5B
) could be observed in the electron density maps (fig. S3
) and helped to make our domain assignments.
The AAA small domains also have unique characteristics that could be matched to the electron density maps. Our AAA domain assignment places the stalk and buttress coiled coils within the small domains of AAA4 and AAA5, respectively, in agreement with their position in the sequence (fig. S5B
). In addition, AAAs H5 to H8 are predicted to be longer than in other AAA domains, which is seen in the electron density map (fig. S4A
) and agrees with an alignment of our crystal structure to the EM of Chlamydomonas
axonemal dynein (7
) (fig. S2B
). When viewed from the linker face, the small domains are located clockwise with respect to each large domain, as is true of many other AAA ATPases such as ClpX (25
) and HslU (26
). Of the six AAA domains, AAA1 and 3 small and large domains are the most similar to one another in structure, as is true of their sequences as well.
To further validate our assignment, we obtained information on methionine positions using selenomethionine (SeMet). SeMet was incorporated into our yeast-expressed dynein to 64% occupancy, using a modification of previously described methods (20
). Although our 7.5 Å SeMet data was not sufficient to calculate phases, we were able to combine it with our experimentally determined phases to generate an anomalous difference Fourier density map in which we could locate approximate positions of 64 of the 126 methionines (peaks at >3 σ) in the GST-dynein dimer (table S1
and fig. S6
). Although only four sites (out of 18 total methionines) were observed in the poorly ordered GST molecule, their locations corresponded to known methionine positions (fig. S6
). Within the dynein motor domain, 42 sites were located in similar positions in each motor domain (21 in each monomer), whereas the remaining 18 sites were only observed in one of the two motor domains. Some of these peaks corresponded to predicted Met locations based on our model (fig. S6B
). However, due to the low-resolution SeMet map, it was not possible to use this information to establish a registry of the polypeptide chain. Thus, although the secondary structure assignments in our model (fig. S5B
) are likely to be mostly correct, the precise registry of the residues in our model will not be accurate (20
). However, by counting the number of SeMet peaks in each of the large or small AAA domains, we could determine whether these numbers were consistent with our assignment of the six AAA domains. In the assignment shown in , the number of SeMet peaks was equal to or less than that expected from the dynein sequence (fig. S6C
). On the other hand, if the AAA assignment was shifted by one in either the clockwise or counterclockwise direction, then mismatches occurred, in which more SeMet peaks occurred in several domains than would be predicted from the sequence (fig. S6C
). Thus, the SeMet data, the positions of large and small domain insertions, and agreement with previous EM mapping all support the AAA assignment shown in .
Distinct conformations of the AAA domains produce an asymmetry of ring
The arrangement of AAA domains around the motor domain ring is more asymmetric than any AAA hexamer structure solved to date. Viewed from the side, the AAA large domains occupy different planes within the ring (). Viewed from the linker face, larger gaps are evident between the large domains of AAA1 and AAA2 and between AAA5 and AAA6 (). Two major factors determine the positions of the AAA domains. First, as noted for other AAA proteins (25
), a flexible linker between β5 of the large domain and H5 of the small domain allows for rigid body rotations between these domains ( and fig. S7
). Second, the small domains all pack rigidly and in a similar orientation against the large domains of the neighboring AAA (e.g., AAA1s against AAA2L) ( and fig. S7
), as was noted for ClpX (25
). Thus, AAA(n
+1)large may behave as rigid units that can move relative to one another by rotations about the peptide linker that connects the large and small subdomains within a AAA domain.
Fig. 3 Asymmetry of the dynein ring. (A) Side view of AAA ring showing the different planes occupied by the AAA1 to 3 large domains. (B) View of the linker face of the AAA ring (as in ), showing just the large domains. Note the large gaps between AAA1 (more ...)
Hexameric AAA enzymes bind ATP between adjacent AAA subunits, with the clockwise neighbor [viewed from the linker face ()] contributing residues that promote nucleotide hydrolysis (4
). In other AAA proteins, the close apposition of large domains enables nucleotide binding and hydrolysis (28
), whereas a greater separation is associated with a nucleotide-free or apo state (25
). In dynein, mutagenesis studies suggest that AAA3 can bind and hydrolyze ATP (13
). Although we cannot ascertain whether nucleotide is bound to the AAA domains at our current resolution, AAA3 and AAA4 are positioned in a similar closed conformation to the interface of the nucleotide-bound subunits in the ClpX hexameric ring () (25
). On the other hand, AAA1, the main hydrolytic site of dynein, is separated by a large distance from AAA2, which is more similar to the interface of the nucleotide-free subunits in ClpX () (25
). The conformation of AAA1 and AAA2, combined with our crystallization in a nucleotide-free buffer and the observed position of the linker domain, makes it likely that AAA1 is in an apo state. It seems highly probable that ATP binding will close the gap between AAA1 and AAA2 and help to trigger other conformational changes in the dynein ring.