ABSTRACT
The dynein family of microtubule motors includes cytoplasmic, intraflagella transport (IFT) and axonemal isoforms. Cytoplasmic and IFT dyneins are processive transporters that carry cargo. Axonemal dyneins drive the sliding motion of microtubules responsible for the beating of cilia and flagella. All the dyneins are built around related heavy chains and share a similar architecture (Fig. 6.1-inset). The N-terminus of the heavy chain forms the tail domain which binds to accessory proteins and cargos. In dimeric and trimeric dyneins the tail is also the site of multimerization. The heavy chain C-terminus makes up the motor domain, consisting of a ring of six AAA domains, an N-terminal linker and a long thin structure, called the stalk, which protrudes 15 nm out of the AAA ring [26]. The stalk consists of almost one complete turn of antiparallel coiled coil and has the microtubule-binding Handbook of Dynein Edited by Keiko Hirose and Linda A. Amos Copyright © 2012 Pan Stanford Publishing Pte. Ltd. www.panstanford.com
domain (MTBD) at its tip. This separation of the track binding site from the rest of the motor domain is a very different arrangement from that found in other cytoskeletal motor proteins such as myosin and kinesin where the nucleotide and track (actin or microtubule) binding sites are both part of the same domain. In particular it raises a number of questions about the role of the stalk in dynein’s mechanism. This chapter will give an overview of the stalk’s structure and then deal with the questions of how the MTBD communicates with the rest of the motor and what role the stalk plays in dynein’s power stroke and directionality. 6.2 STRUCTURAL FEATURES OF THE DYNEIN STALK
The stalk was first identified by “deep etch” electron microscopy (EM) of axonemes as a narrow projection that linked the body of dynein (itself bound to the A-microtubule) to the adjacent B-microtubule [11] (Fig. 1.2). Later work on isolated dynein also showed a thin stalk projecting out of the globular dynein “head” [10] (Fig. 1.3). Work by Gee et al. assigned this stalk structure as a stretch of antiparallel coiled coil with the MTBD at its tip [6], by showing that the microtubule-binding activity in dynein was localized to a 131 amino acid domain flanked on either side by stretches of predicted coiled coil [9, 23]. This coiled coil sequence lies between the predicted AAA4 and AAA5 domains (Fig. 6.1), consistent with the stalk protruding from the globular dynein AAA ring. Furthermore, when Gee et al. [6] expressed just this region, they were able to show, by EM, that it formed a rod-like structure of similar dimensions to the stalk projection observed in the earlier studies of axonemes and isolated dynein. More recently, the stalk has been directly visualized by a combination of negative stain EM and advanced image processing in both a monomeric axonemal inner-arm dynein (dynein-c) [1] and a cytoplasmic dynein [26] (Chapter 4). There is also now an X-ray crystal structure of the microtubule-binding domain together with about a quarter of the stalk coiled coil as a fusion to another antiparallel coiled coil protein, seryl tRNA synthetase (SRS) [2] (Fig. 6.2A-inset). The consensus model that emerges from this work (Fig. 6.1) is that the stalk consists of an antiparallel coiled coil of two alpha helices; CC1 coming out of AAA4 and CC2 returning back toward AAA5, with the MTBD at its distal tip. The whole stalk extends out of the globular AAA ring for ~15 nm; after the first 10.5 nm of coiled coil, there is a kink followed by a ~4 nm long globular MTBD [26].
