Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4 MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60-WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zigzag , which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60-WDR34-light chain subcomplex renders dynein-2 monomeric and relieves autoinhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits control dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into dynein-2's interaction with IFT trains and the origin of diverse functions in the dynein family.
Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is known to keep dynein bound to microtubules; however, how this is accomplished mechanistically remains unknown. We have used three-dimensional electron microscopy, single-molecule imaging, biochemistry, and in vivo assays to help establish this mechanism. The three-dimensional structure of the dynein–Lis1 complex shows that binding of Lis1 to dynein's AAA+ ring sterically prevents dynein's main mechanical element, the ‘linker’, from completing its normal conformational cycle. Single-molecule experiments show that eliminating this block by shortening the linker to a point where it can physically bypass Lis1 renders single dynein motors insensitive to regulation by Lis1. Our data reveal that Lis1 keeps dynein in a persistent microtubule-bound state by directly blocking the progression of its mechanochemical cycle.DOI: http://dx.doi.org/10.7554/eLife.03372.001
. We previously confirmed that UL25 occupies the vertex-distal region of the CVSC density by visualizing a large UL25-specific tag in reconstructions calculated from cryo-electron microscopy (cryo-EM) images. We have pursued the same strategy to determine the capsid location of the UL17 protein. Recombinant viruses were generated that contained either a small tandem affinity purification (TAP) tag or the green fluorescent protein (GFP) attached to the C terminus of UL17. Purification of the TAP-tagged UL17 or a similarly TAP-tagged UL25 protein clearly demonstrated that the two proteins interact. A cryo-EM reconstruction of capsids containing the UL17-GFP protein reveals that UL17 is the second component of the CVSC and suggests that UL17 interfaces with the other CVSC component, UL25, through its C terminus. The portion of UL17 nearest the vertex appears to be poorly constrained, which may provide flexibility in interacting with tegument proteins or the DNA-packaging machinery at the portal vertex. The exposed locations of the UL17 and UL25 proteins on the HSV-1 capsid exterior suggest that they may be attractive targets for highly specific antivirals.Herpesviruses are incurable human and animal pathogens that generally infect their hosts for life, escaping immune surveillance and producing recurrent infections between periods of latency. They have been shown recently to share functional and structural characteristics with double-stranded DNA (dsDNA) bacteriophages, and the recognition of common features in the capsid assembly pathways has been (and remains) instrumental in identifying roles for herpesvirus proteins that are less easily studied and manipulated than their phage analogs. These common features include (i) initial assembly into an icosahedral procapsid, (ii) maturational proteolysis of structural proteins, (iii) ATP-driven packaging of the dsDNA chromosome through a specialized capsid vertex complex called the "portal," (iv) maturation of the procapsid during packaging, and (v) capsid stabilization effected by binding accessory proteins or by the formation of intersubunit bonds (reviewed in reference 5). While much of the herpesvirus capsid structure has been detailed, particularly by cryo-electron microscopy (cryo-EM), a number of minor proteins that interact with the capsid during assembly are still understood mostly by analogy with identifiable counterparts in phages. Several of these proteins are essential for packaging and retaining the viral DNA and are potentially valuable targets for interfering with herpesvirus replication.The herpesvirus virion, ϳ200 nm in diameter, consists of an icosahedral capsid of 125 nm in diameter enclosing the dsDNA chromosome and an amorphous layer of tegument proteins linking the capsid to an exterior lipid envelope in which different viral glycoproteins are embedded (5,7,12,25). The herpes simplex virus 1 (HSV-1) capsid is composed primarily of the major capsid protein, VP5, organized as hexameric and pentameric capsomers that are termed "hexons" and "pentons,"...
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