Intrinsic disorder in dynein intermediate chain modulates its interactions with NudE and dynactin

The functional diversity of cytoplasmic dynein is in part attributed to multiple interactions between noncatalytic dynein subunits and an array of regulatory proteins. This study focuses on the interaction between the dynein intermediate chain subunit (IC) and a dynein regulator protein (NudE). We use isothermal titration calorimetry and NMR spectroscopy to map their interacting sections to their respective N-terminal domains, which are predicted to form dimeric coiled-coils. Interestingly, the specific residues within IC that interact with NudE are a subset of the bi-segmental binding region reported for p150(Glued), a subunit of the dynein activator protein dynactin. Although the IC binding domains of both NudE and p150(Glued) form dimeric coiled-coils and bind IC at a common site, we observe distinct binding modes for each regulatory protein: 1) NudE binds region 1 of the bi-segmental binding footprint of p150(Glued), whereas p150(Glued) requires regions 1 and 2 to match the binding affinity of NudE with region 1 alone. 2) Compared with unbound IC, NudE-bound IC shows a slight increase in flexibility in region 2, in contrast to the increase in ordered structure observed for p150(Glued)-bound IC (Morgan, J. L., Song, Y., and Barbar, E. (2011) J. Biol. Chem. 286, 39349-39359). 3) Although NudE has a higher affinity for the common binding segment on IC, when all three proteins are in solution, IC preferentially binds p150(Glued). These results underscore the importance of a bi-segmental binding region of IC and disorder in region 2 and flanking linkers in selecting which regulatory protein binds IC.     

Identification of a novel dynein binding domain in nudel essential for spindle pole organization in Xenopus egg extract

The nuclear distribution protein E (NudE) and nuclear distribution protein E-like (Nudel or Ndel1) interact with both lissencephaly 1 (Lis1) and dynein. These interactions are thought to be essential for dynein function. Previous studies have shown that the highly conserved N terminus of NudE/Nudel directly binds to Lis1, and such binding is critical for dynein activity. By contrast, although the C terminus of NudE/Nudel was reported to bind to dynein, the functional significance of this binding has remained unclear. Using the sperm-mediated spindle assembly assay in Xenopus egg extracts and extensive mutagenesis studies, we have identified a highly conserved dynein binding domain within the first 80 amino acids of Nudel. We further demonstrate that the dynein intermediate chain in the dynein complex is directly involved in this interaction. Importantly, we show that both the dynein and Lis1 binding domains of Nudel are required for spindle pole organization. Finally, we report that spindle defects caused by immuno-depletion of Nudel could be rescued by a 1-fold increase of Lis1 concentration in Xenopus egg extracts. This suggests that an important function of the N terminus of Nudel is to facilitate the interaction between Lis1 and dynein during spindle assembly. Together, our findings open up new avenues to further decipher the mechanism of dynein regulation by Nudel and Lis1.     

Structure and dynamics of LC8 complexes with KXTQT-motif peptides: swallow and dynein intermediate chain compete for a common site

The dynein light chain LC8 is an integral subunit of the cytoplasmic dynein motor complex that binds directly to and promotes assembly of the dynein intermediate chain (IC). LC8 interacts also with a variety of putative dynein cargo molecules such as Bim, a proapoptotic Bcl2 family protein, which have the KXTQT recognition sequence and neuronal nitric oxide synthase (nNOS), which has the GIQVD fingerprint but shares the same binding grooves at the LC8 dimer interface. The work reported here investigates the interaction of LC8 with IC and a putative cargo, Swallow, which share the KXTQT recognition sequence, and addresses the apparent paradox of how LC8, as part of dynein, mediates binding to cargo. The structures of Drosophila LC8 bound to peptides from IC and Swallow solved by X-ray diffraction show that the IC and Swallow peptides bind in the same grooves at the dimer interface. Differences in flexibility between bound and free LC8 were evaluated from hydrogen isotope exchange experiments using heteronuclear NMR spectroscopy. Peptide binding causes an increase in protection from exchange primarily in residues that interact directly with the peptide, such as the beta-strand intertwined at the interface and the N-terminal end of helix alpha2. There is considerably more protection upon Swallow binding, consistent with tighter binding relative to IC. Comparison with the LC8/nNOS complex shows how both the GIQVD and KXTQT fingerprints are recognized in the same groove. The similar structures of LC8/IC and LC8/Swa and the tighter binding of Swallow call into question the role for LC8 as a cargo adaptor protein, and suggest that binding of LC8 to Swallow serves another function, possibly that of a dimerization engine, which is independent of its role in dynein.     

Light chain-dependent self-association of dynein intermediate chain

Dynein light chains are bivalent dimers that bind two copies of dynein intermediate chain IC to form a cargo attachment subcomplex. The interaction of light chain LC8 with the natively disordered N-terminal domain of IC induces helix formation at distant IC sites in or near a region predicted to form a coiled-coil. This fostered the hypothesis that LC8 binding promotes IC self-association to form a coiled-coil or other interchain helical structure. However, recent studies show that the predicted coiled-coil sequence partially overlaps the light chain LC7 recognition sequence on IC, raising questions about the apparently contradictory effects of LC8 and LC7. Here, we use NMR and fluorescence quenching to localize IC self-association to residues within the predicted coiled-coil that also correspond to helix 1 of the LC7 recognition sequence. LC8 binding promotes IC self-association of helix 1 from each of two IC chains, whereas LC7 binding reverses self-association by incorporating the same residues into two symmetrical, but distant, helices of the LC7-IC complex. Isothermal titration experiments confirm the distinction of LC8 enhancement of IC self-association and LC7 binding effects. When all three light chains are bound, IC self-association is shifted to another region. Such flexibility in association modes may function in maintaining a stable and versatile light chain-intermediate chain assembly under changing cellular conditions.     

Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.     

The roadblock light chain binds a novel region of the cytoplasmic Dynein intermediate chain

Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, ROBL-1 and ROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.     

The Sireviruses, a plant-specific lineage of the Ty1/copia retrotransposons, interact with a family of proteins related to dynein light chain 8

Plant genomes are rich in long terminal repeat retrotransposons, and here we describe a plant-specific lineage of Ty1/copia elements called the Sireviruses. The Sireviruses vary greatly in their genomic organization, and many have acquired additional coding information in the form of an envelope-like open reading frame and an extended gag gene. Two-hybrid screens were conducted with the novel domain of Gag (the Gag extension) encoded by a representative Sirevirus from maize (Zea mays) called Hopie. The Hopie Gag extension interacts with a protein related to dynein light chain 8 (LC8). LC8 also interacts with the Gag extension from a Hopie homolog from rice (Oryza sativa). Amino acid motifs were identified in both Hopie Gag and LC8 that are responsible for the interaction. Two amino acids critical for Gag recognition map within the predicted LC8-binding cleft. Two-hybrid screens were also conducted with the Gag extension encoded by the soybean (Glycine max) SIRE1 element, and an interaction was found with light chain 6 (LC6), a member of the LC8 protein family. LC8 and LC6 proteins are components of the dynein microtubule motor, with LC8 being a versatile adapter that can bind many unrelated cellular proteins and viruses. Plant LC8 and LC6 genes are abundant and divergent, yet flowering plants do not encode other components of the dynein motor. Although, to our knowledge, no cellular roles for plant LC8 family members have been proposed, we hypothesize that binding of LC8 proteins to Gag aids in the movement of retrotransposon virus-like particles within the plant cell or possibly induces important conformational changes in the Gag protein.     

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150^Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150^Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150^Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150^Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.     

Multivalency in the Assembly of Intrinsically Disordered Dynein Intermediate Chain

Dynein light chains are thought to increase binding efficiency of dynein intermediate chain to both dynein heavy chain and dynactin, but their exact role is not clear. Isothermal titration calorimetry and x-ray crystallography reported herein indicate that multivalency effects underlie efficient dynein assembly and regulation. For a ternary complex of a 60-amino acid segment of dynein intermediate chain (IC) bound to two homodimeric dynein light chains Tctex1 and LC8, there is a 50-fold affinity enhancement for the second light chain binding. For a designed IC construct containing two LC8 sites, observed the 1000-fold enhancement reflects a remarkably pure entropic chelate effect of a magnitude commensurate with theoretical predictions. The lower enhancement in wild-type IC is attributed to unfavorable free energy changes associated with incremental interactions of IC with Tctex1. Our results show assembled dynein IC as an elongated, flexible polybivalent duplex, and suggest that polybivalency is an important general mechanism for constructing stable yet reversible and functionally versatile complexes.     

NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores

NudE and NudEL are related proteins that interact with cytoplasmic dynein and LIS1. Their functional relationship and involvement in LIS1 and dynein regulation are not completely understood. We find that NudE and NudEL each localize to mitotic kinetochores before dynein, dynactin, ZW10, and LIS1 and exhibit additional temporal and spatial differences in distribution from the motor protein. Inhibition of NudE and NudEL caused metaphase arrest with misoriented chromosomes and defective microtubule attachment. Dynein and dynactin were both displaced from kinetochores by the injection of an anti-NudE/NudEL antibody. Dynein but not dynactin interacted with NudE surprisingly through the dynein intermediate and light chains but not the motor domain. Together, these results identify a common function for NudE and NudEL in mitotic progression and identify an alternative mechanism for dynein recruitment to and regulation at kinetochores.     

Dynein light chain LC8 is a dimerization hub essential in diverse protein networks

The operations within a living cell depend on the collective activity of networks of proteins, sometimes termed "interactomes". Within these networks, most proteins interact with few partners, while a small proportion of proteins, called hubs, participate in a large number of interactions and play a central role in organizing these interactomes. LC8 was first discovered as an essential component of the microtubule-based molecular motor dynein and as such is involved in fundamental processes, including retrograde vesicular trafficking, ciliary/flagellar motility, and cell division. More recently, evidence has accumulated that LC8 also interacts with proteins that are not clearly connected with dynein or microtubule-based transport, including some with roles in apoptosis, viral pathogenesis, enzyme regulation, and kidney development. Here, we introduce the idea that LC8 is a hub protein essential in diverse protein networks, and its function as a dynein light chain is but one of many. We further propose that the crucial regulatory roles of LC8 in various systems are due to its ability to promote dimerization of partially disordered proteins.     

Recognition of novel viral sequences that associate with the dynein light chain LC8 identified through a pepscan technique

Recent data from multiple laboratories indicate that upon infection, many different families of viruses hijack the dynein motor machinery and become transported in a retrograde manner towards the cell nucleus. In certain cases, one of the dynein light chains, LC8, is involved in this interaction. Using a library of overlapping dodecapeptides synthesized on a cellulose membrane (pepscan technique) we have analyzed the interaction of the dynein light chain LC8 with 17 polypeptides of viral origin. We demonstrate the strong binding of two herpesvirus polypeptides, the human adenovirus protease, vaccinia virus polymerase, human papillomavirus E4 protein, yam mosaic virus polyprotein, human respiratory syncytial virus attachment glycoprotein, human coxsackievirus capsid protein and the product of the AMV179 gene of an insect poxvirus to LC8. Our data corroborate the manipulation of the dynein macromolecular complex of the cell during viral infection and point towards the light chain LC8 as one of the most frequently used targets of virus manipulation.     

Potential role for phosphorylation in differential regulation of the assembly of dynein light chains

The homodimeric light chains LC8 and Tctex-1 are integral parts of the microtubule motor cytoplasmic dynein, as they directly associate with dynein intermediate chain IC and various cellular cargoes. These light chains appear to regulate assembly of the dynein complex by binding to and promoting dimerization of IC. In addition, both LC8 and Tctex-1 play roles in signaling, apoptosis, and neuronal development that are independent of their function in dynein, but it is unclear how these various activities are modulated. Both light chains undergo specific phosphorylation, and here we present biochemical and NMR analyses of phosphomimetic mutants that indicate how phosphorylation may regulate light chain function. For both LC8 and Tctex-1, phosphorylation promotes dissociation from IC while retaining their binding activity with other non-dynein proteins. Although LC8 and Tctex-1 are homologs having a common fold, their reduced affinity for IC upon phosphorylation arises by different mechanisms. In the case of Tctex-1, phosphorylation directly masks the IC binding site at the dimer interface, whereas for LC8, phosphorylation dissociates the dimer and indirectly eliminates the binding site. This modulation of the monomer-dimer equilibrium by phosphorylation provides a novel mechanism for discrimination among LC8 binding partners.     

The intermediate chain of cytoplasmic dynein is partially disordered and gains structure upon binding to light-chain LC8

The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.     

Differences in dynamic structure of LC8 monomer, dimer, and dimer-peptide complexes

Dimerization of dynein light chain LC8 creates two symmetric grooves at the dimer interface with diverse binding capabilities. In addition to pH and protein concentration, dimerization is affected by phosphorylation, as illustrated by a phosphomimetic mutation that promotes dissociation of LC8 to a monomer and subsequent dissociation from the dynein complex in vitro. In this work we characterize the dynamic structure and unfolding profiles of an LC8 mutant, H55K, as a model for monomeric LC8 at neutral pH. Backbone (15)N relaxation experiments show that the monomer, while primarily ordered, has more heterogeneous dynamics relative to the LC8 dimer, predominantly in residues that ultimately form the binding groove, particularly those in beta 1 and beta 3 strands. This heterogeneity suggests that conformations that are primed for binding are sampled in the inactive monomer and favored in the active dimer. Further changes of LC8 backbone dynamics upon binding to short peptides from Swallow (Swa) and dynein intermediate chain (IC) were elucidated. The conformational heterogeneity apparent in the LC8 dimer is retained in LC8/IC but is lost in LC8/Swa, suggesting that the degree of ordering upon binding is ligand dependent. The reduced complexity of motion in LC8/Swa correlates with the less favorable entropy of binding of LC8 to Swa relative to IC. We propose that the conformational motility of beta 3 has functional significance in dimerization and in ligand binding. In the latter, beta 3 flexibility apparently accommodates different binding modes for different ligands resulting in ligand-specific conformational dynamics of the binding site that may impact other processes such as accessibility to phosphorylation.     

Identification of a novel region of the cytoplasmic Dynein intermediate chain important for dimerization in the absence of the light chains

Cytoplasmic dynein is the multisubunit protein complex responsible for many microtubule-based intracellular movements. Its cargo binding domain consists of dimers of five subunits: the intermediate chains, the light intermediate chains, and the Tctex1, Roadblock, and LC8 light chains. The intermediate chains have a key role in the dynein complex. They bind the three light chains and the heavy chains, which contain the motor domains, but little is known about how the two intermediate chains interact. There are six intermediate chain isoforms, and it has been hypothesized that different isoforms may regulate specific dynein functions. However, there are little data on the potential combinations of the intermediate chain isoforms in the dynein complexes. We used co-immunoprecipitation analyses to demonstrate that all combinations of homo- and heterodimers of the six intermediate chains are possible. Therefore the formation of dynein complexes with different combinations of isoforms is not limited by interaction between the various intermediate chains. We further sought to identify the domain necessary for the dimerization of the intermediate chains. Analysis of a series of truncation and deletion mutants showed that a 61-amino-acid region is necessary for dimerization of the intermediate chain. This region does not include the N-terminal coiled-coil, the C-terminal WD repeat domain, or the three different binding sites for the Tctex1, LC8, and Roadblock light chains. Analytical gel filtration and covalent cross-linking of purified recombinant polypeptides further demonstrated that the intermediate chains can dimerize in vitro in the absence of the light chains.     

Analysis of the dynein-dynactin interaction in vitro and in vivo

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.     

Chlamydomonas outer arm dynein alters conformation in response to Ca2+

We have previously shown that Ca(2+) directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the beta and gamma heavy chains (HCs). The gamma HC-associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca(2+) with K(Ca) = 3 x 10(-5) M in vitro, suggesting it may act as a Ca(2+) sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and gamma HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the gamma heavy chain. In vitro experiments indicate that LC4 undergoes a Ca(2+)-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca(2+). The sedimentation profile of the gamma HC subunit changed subtly upon Ca(2+) addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated gamma subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca(2+) addition. We propose that Ca(2+)-dependent conformational change of LC4 has a direct effect on the stem domain of the gamma HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm.     

Distinct but overlapping sites within the cytoplasmic dynein heavy chain for dimerization and for intermediate chain and light intermediate chain binding

Cytoplasmic dynein is a molecular motor complex consisting of four major classes of polypeptide: the catalytic heavy chains (HC), intermediate chains (IC), light intermediate chains (LIC), and light chains (LC). Previous studies have reported that the ICs bind near the N terminus of the HCs, which is thought to correspond to the base of the dynein complex. In this study, we co-overexpressed cytoplasmic dynein subunits in COS-7 cells to map HC binding sites for the ICs and LICs, as well as HC dimerization. We have found that the LICs bind directly to the N terminus of the HC, adjacent to and overlapping with the IC binding site, consistent with a role for the LICs in cargo binding. Mutation of the LIC P-loop had no detectable effect on HC binding. We detected no direct interaction between the ICs and LICs. Using triple overexpression of HC, IC and LIC, we found that both IC and LIC are present in the same complexes, a result verified by anti-IC immunoprecipitation of endogenous complexes and immunoblotting. Our results indicate that the LICs and ICs must be located on independent surfaces of cytoplasmic dynein to allow each to interact with other proteins without steric interference.