Figure 3.1 Structure organization and architecture of the dynein heavy chain. (a) The linear primary sequence maps of the Dictyostelium cytoplasmic dynein heavy chain (above) and the C-terminal 380-kDa motor domain (below). (b) Predicted domain structure of the heavy chain and the motor domain illustrating the ring-like structure of the head, and the elongated stalk and tail domains. Note that the stalk is composed of an antiparallel coiled coil (Stalk-CC) and a distal MTBD. comparable to that of native cytoplasmic dynein [53, 75]. However, the motor activity was noted to be severely impaired even by a small truncation at either the N (9 kDa) or C terminus (46 kDa) of the fragment [11, 69]. Based on these findings, the 380 kDa fragment of the heavy chain has been referred to as the dynein motor domain; however, for yeast cytoplasmic dynein, a larger N-terminal deletion (17 kDa) was achieved without substantially perturbing the motor activity [68].The dynein motor domain, consisting of the N-terminal truncated tail (~the linker) and the entire head containing the AAA+ ring and the C sequence, and the stalk domains, represents a simple model system for studying the dynein mechanism. Because interacting interfaces are lacking, the motor domain exists as a monomeric motor [69, 73] and does not associate with most of the other subunits included in the native cytoplasmic dynein complex [53, 68]. In the following sections of this chapter, we will discuss how each of the three domains works to produce dynein’s motor activity. 3.3 AtPAse cycles In the heAD DoMAInDespite their diverse functions, AAA+ proteins show high sequence similarity within their ATPase sites. Several characteristic motifs in the ATPase sites, such as Walker A, Walker B, Sensor-1, Sensor-2, and arginine finger, are well-conserved among the members of the AAA+ family [52, 56]. The Walker A motif (GXXGXGKT/S) is essential for nucleotide binding to AAA+ modules [74, 91]. The conserved lysine and threonine/serine residues in this motif

appear to interact with b and g phosphates of bound ATP and with Mg2+ ions, respectively. In general, a mutation in the Walker A motif inhibits nucleotide binding of the AAA+ module [23]. The Walker B motif (DEXX) is critical for hydrolyzing ATP. The carboxyl group of the conserved glutamate residue in this motif would act together with a conserved polar amino acid of the Sensor 1 motif to orient a water molecule appropriately for a nucleophilic attack on the g-phosphate of bound ATP [79]. In addition, the conserved aspartate residue in this motif may interact with Mg2+ ions. Thus, a mutation in the Walker B motif generally inhibits ATP hydrolysis without affecting ATP/ADP binding to the mutated site [23]. The arginine finger comprises a part of the nucleotide-binding site in the adjacent AAA+ module and is important for ATP hydrolysis in this adjacent module [55]. Because of this configuration, the arginine finger can play a critical role in intermodular communication within AAA+ proteins.