2023 年 20 巻 1 号 論文ID: e200008
Ciliary bending movements are powered by motor protein axonemal dyneins. They are largely classified into two groups, inner-arm dynein and outer-arm dynein. Outer-arm dynein, which is important for the elevation of ciliary beat frequency, has three heavy chains (α, β, and γ), two intermediate chains, and more than 10 light chains in green algae, Chlamydomonas. Most of intermediate chains and light chains bind to the tail regions of heavy chains. In contrast, the light chain LC1 was found to bind to the ATP-dependent microtubule-binding domain of outer-arm dynein γ-heavy chain. Interestingly, LC1 was also found to interact with microtubules directly, but it reduces the affinity of the microtubule-binding domain of γ-heavy chain for microtubules, suggesting the possibility that LC1 may control ciliary movement by regulating the affinity of outer-arm dyneins for microtubules. This hypothesis is supported by the LC1 mutant studies in Chlamydomonas and Planaria showing that ciliary movements in LC1 mutants were disordered with low coordination of beating and low beat frequency. To understand the molecular mechanism of the regulation of outer-arm dynein motor activity by LC1, X-ray crystallography and cryo-electron microscopy have been used to determine the structure of the light chain bound to the microtubule-binding domain of γ-heavy chain. In this review article, we show the recent progress of structural studies of LC1, and suggest the regulatory role of LC1 in the motor activity of outer-arm dyneins. This review article is an extended version of the Japanese article, The Complex of Outer-arm Dynein Light Chain-1 and the Microtubule-binding Domain of the Heavy Chain Shows How Axonemal Dynein Tunes Ciliary Beating, published in SEIBUTSU BUTSURI Vol. 61, p. 20–22 (2021).
To produce ciliary bending movements, the motor activity of more than thousands of ciliary dynein molecules should be spatially and temporally regulated. However, the regulatory mechanism of dyneins has not been understood. The dynein light chain 1 is important for the regulation of outer-arm dyneins, since it binds to motor domain of one of outer-arm dynein heavy chains. Recent structural studies clearly showed that the light chain directly binds to the microtubule-binding domain of the heavy chain, suggesting that the light chain regulates the microtubule-affinity of outer-arm dynein.
Cilia and flagella (here we simply call them cilia) are hair-like organelles that extend from eukaryotic cells and are important for cell motility of lower eukaryotes like Chlamydomonas and Paramecium, and are also indispensable in higher eukaryotes including mammals for sperm motility, generation of fluid flow in the trachea, and left–right asymmetry in embryonic development [1–3]. Defects in various kinds of cilia proteins result in a variety of human diseases called ciliopathies [4,5].
Cilia have a common architecture, axoneme, within the membrane. The axoneme is composed of ‘9+2’ structure in which two central microtubules (MTs) are surrounded by nine doublet microtubules (dMTs) (Figure 1A) [6]. The periodic bending motion of cilia is driven by axonemal dyneins which are MT minus end–directed motor proteins [7]. The dyneins are largely classified into two groups, inner-arm dynein (IAD) and outer-arm dynein (OAD) (Figure 1A) [6]. Previous studies of Chlamydomonas dynein mutants showed that OAD, which generates 70–80% of the ciliary propulsive force [8,9], is important for elevation of the beat frequency [10]. In contrast, IAD is important for maintenance of the normal bend angle [11].
Schematic diagram of axonemal dynein.
(A) Cross section of Chlamydomonas flagella and outer-arm dynein (OAD). Flagella internal structure, axoneme, have ‘9+2’ structure which is composed of nine outer-doublet microtubules (dMTs) surrounding two singlet microtubules (central pair). The dMTs have two arms, inner- and outer-arms, both of which contain dynein molecules with MT motor and ATPase activities. An OAD molecule comprises of three heavy chains (HCs: α, β, and γ), two intermediate chains (ICs: IC1 and IC2), and more than ten light chains (LCs: LC1-LC10). LCs other than LC1, which binds to ‘head’ region of γ HC, bind to ‘tail’ regions of HCs. (B) Dynein heavy chain (DHC) is largely divided into two, tail and motor regions. The tail region with associated ICs/LCs are important for targeting each dynein molecule to a specific site on dMT. The head region containing MT motor activity has several functional domains, linker, six AAA (AAA1-6), stalk, and C domains. The stalk domain has two anti-parallel coiled-coil sequences (CC1 and CC2). MT binding domain (MTBD) is located at the top of the stalk CC. Dynein interacts with a MT through the MTBD in an ATP-dependent manner.
An OAD molecule is a huge complex (about 2 MDa) composed of multiple subunits, heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs harboring both of ATPase and MT motor activities are the most important. In this review, nomenclature of the components is based on the Chlamydomonas OAD, unless otherwise noted. The OAD molecule is composed of three HCs (α, β, and γ HCs), two ICs (IC1 and IC2), and 11 LCs (Figure 1A) [12,13]. Each of the HCs comprises two regions, a tail region (corresponding to the N-terminal one-third of entire sequence) and a head region (the C-terminal two-thirds) (Figure 1B) [12,13]. Structural studies of cytoplasmic dynein HC by X-ray crystallography showed that the head region can be further divided into several functional units, such as linker, AAA+ ring, stalk, microtubule-binding domain (MTBD), and C-sequence (Figure 1B) [14–17]. The stalk domain, emerging from the AAA+ ring, is composed of a 15-nm-long antiparallel coiled-coil structure (consisting of coiled-coil 1 (CC1) and coiled-coil 2 (CC2) α-helices) with the MTBD at the tip (Figure 1B) [18,19]. The MTBD interacts with B-tubule of dMT in an ATP-sensitive manner [20,21]. Registry shift of two α-helices in the coiled-coil would induce the structural changes in the MTBD, which are considered to regulate its binding affinity for MTs [22–24]. Most of ICs and LCs bind to the N-terminal tail region of HCs (Figure 1A) [13]. Chlamydomonas mutants lacking one of IC or LC subunits swam slower than wild type (WT) owing to lack or reduction of OAD [10,13,25]. Recent structural studies suggest that IC/LC subunits are important to stabilize OAD molecules bound to A-tubule of a dMT [26–28]. In contrast, light chain 1 (LC1), which is a member of a leucin-rich repeat (LRR) protein family and is highly conserved in organisms with cilia [29], was found to associate with the globular head region of OAD γ-HC in Chlamydomonas [30].
To consider the in vivo function, LC1 mutants have been isolated and characterized in various organisms (Table1). RNAi knockdown of LC1 gene expression reduced the number of OAD molecules in Paramecium and Trypanosome; the Paramecium knockdown cells displayed slower swimming than control cells [31], while the Trypanosome knockdown cells did not display forward swimming which requires OAD, but swam backwards only [32]. Reduction of ciliary OAD molecules has been also observed in human Primary Ciliary Dyskinesia (PCD) patients with a point mutation in the LC1 gene [33]. The patients had typical PCD symptoms, situs inversus (the mirror-image transposition of internal organs) and bronchiectasis (the condition where the airways of the lungs are widened, leading to chronic airway diseases) with reduction of the motility in airway cilia [4]. These results suggest that LC1 helps the stability of OAD molecules. In Chlamydomonas, no knockout or knockdown mutant has been obtained and examined, but the effect of various LC1 mutant proteins on cell motility has been analyzed in cells expressing the mutant proteins in a wild-type background. Expression of the mutant forms of LC1 leads to dominant-negative effects on swimming velocity, as the cilia continuously beat out of phase and stalled near the power/recovery stroke switch point [34]. This suggests that LC1 is important for the control of OAD activity during the transition between the two stroke states. Requirement of LC1 for regulation of cilia motility was also observed in an RNAi study in Planaria. Planaria glides on solid substratum in water by coordinated movement of multiple cilia on the epithelial cells. Knockdown animals could not coordinate the ciliary oscillation and form methachronal waves, and in addition, the beat frequency was lower than control animals. As a result, they glided significantly slower than control animals [35]. Interestingly, EM analysis showed that there is no difference between knockdown and control cells in the OAD number and structure. Thus, regulation of OAD activity would be disturbed due to the absence of LC1. These suggest that LC1 acts as a part of a switch controlling OAD motor function during the ciliary beat cycle [34,35]. To understand the molecular mechanism how LC1 stabilizes and regulates OAD, structural studies of LC1 has been carried out. Here we review recent progress in such structural studies.
Previous biochemical analyses suggest that LC1 associates with the head region of γ-HC as well as tubulin [30,36]. Based on the geometrical dimension of LC1, the measured distance between the head region of OADγ and MT, it had been proposed that LC1 links the AAA+ domain of γ-HC with A-tubule of a dMT [36,37].
To determine the precise location of LC1 within OAD superstructure, purified and negatively-stained Chlamydomonas OAD was observed and analyzed by EM [38]. The structural analysis showed that one of the stalk tip in the three heads of OAD was larger than the other two (Figure 2A). A further EM analysis on a recombinant OAD with histidine (His)-tagged LC1 labeled with nickel–nitriloacetic acid (NTA)-nanogold showed that the larger stalk tip holds LC1 (Figure 2A). Thus, it is likely that LC1 binds to the tip region of a dynein stalk, which corresponds to the MTBD. Next, interaction between His-tagged stalk fragments from α, β, and γ HC (His stalk) and LC1 constructs were analyzed by pull-down Ni-NTA beads assay (Figure 2B). The analysis clearly showed that LC1 specifically binds to His-γ stalk. These results indicate that LC1 binds to the stalk MTBD of OAD γ, but not to the AAA+ domain (Figure 3A).
Dynein light chain 1 (LC1) binds to MTBD of OAD γ stalk.
(A) Analysis of LC1 localization on an OAD molecule. Electron microscopy (EM) showed that his-tagged LC1 was labelled with Ni-NTA nanogold (yellow) just beneath the γ stalk MT-binding domain (MTBD) (red), which appeared to be larger than the other two stalk MTBD of α and β HCs. (B) Biochemical analyses of LC1 binding to recombinant stalks. SDS-PAGE analyses of LC1 interacting with his-tagged stalks of three OAD HCs (αβγ) showed that LC1 specifically binds to γ stalk, but not α or β stalks. (C) MT co-pelleting assay of γ stalk in the absence and the presence of LC1. The amount of γ stalk bound to MTs decreased in the presence of LC1, suggesting that LC1 negatively regulates MT-binding affinity of OAD γ-HC.
3D structure of LC1-γ-MTBD complex.
(A) Schematic diagram of OAD γ. (B) Crystal structure of LC1 bound to γ-stalk. LC1 (orange), Coiled-coil (CC) of dynein stalk (cyan), microtubule binding domain (MTBD) of stalk (green), and flap region (purple) (PDB ID: 6L4P). (C) Comparison of stalk structures between OAD γ and IAD c DHCs (PDB ID: 2RR7). Overall structural folds were similar between the two stalks other than the flap regions (arrows); the flap of γ stalk attaches to the MTBD surface, while that of c stalk sticks out from the MTBD.
The direct interaction between LC1 and OAD γ MTBD suggests that LC1 may regulate the binding of the OAD γ-stalk to MT. To verify the possibility, the effect of LC1 on the MT binding of γ-stalk was examined by MT co-pelleting assay (Figure 2C). γ-stalk was found to bind MTs either in the absence and the presence of LC1, but the MT affinity of γ-stalk in the presence of LC1 was approximately 50% lower than that in the absence of LC1. This finding suggests that LC1 negatively regulates the MT binding of γ-stalk.
An NMR study reported that LC1 has a curved structure consisting of an N-terminal α-helices capping βαβ barrel followed by C-terminal helices [37]. To analyze how LC1 interacts with γ-stalk, the three-dimensional structure of LC1: γ-stalk complex has been determined by X-ray crystallography (PDB ID: 6L4P) [39]. In this complex, the MTBD of γ-stalk attached to the concave portion of the LC1 β-barrel through the H5 helix and a flap (Figure 3B). The flap was first observed as a flexible loop extended from the MTBD core in Chlamydomonas IAD c stalk by NMR (PDB ID: 2RR7; Figure 3C) [40]. Sequence comparison of the MTBD in various dynein HCs indicated that specific isoforms of axonemal dynein have the flap structure [38,40]. By comparing the MTBD structure of γ-stalk with that of IAD c stalk, overall structural folds were similar to each other, but the relative positions of the flap to the MTBD core were different between them (Figure 3C). The flap of γ-stalk was shifted toward the MTBD core by LC1.
A previous pull-down assay had reported amino acids responsible for binding of LC1 to γ-stalk. Mutants with point mutations in the LC1 β-barrel significantly decreased the affinity for γ-MTBD (Supplementary Table S1) [39]. Positions of the effective residues were located at the interface between the LC1 and γ-MTBD revealed by the structural studies (Figure 4A). Interestingly, a LC1 point mutation in human PCD patients (Table 1) [33] was also located in the same region (N150S in Figure 4A). Therefore, the causative mutation for PCD likely diminishes the interaction between LC1 and γ-MTBD, and greatly reduces the number of OAD in cilia.
LC1 mutations analyzed in the previous studies.
(A) Mutations affecting the interaction between LC1 and γMTBD. Biochemical analyses of binding affinity of LC1 to γ MTBD showed that the specific residues in LC1 are important for the binding to γ MTBD. The mutations were indicated by blue circles in the right figure. A point mutation (N150S), which is causative for a human genetic disorder (primary ciliary diskinecia; PCD), was also indicated by a green circle in this figure (Supplementary Table S1) [33]. (B) LC1 mutations affecting the affinity to MTs. Analyses of MT binding affinity in mutant LC1 showed that the plus charged residues are important for the binding to MTs (Supplementary Table S1, Patel-King and King, 2012). The mutations were indicated by pink circles in the right figure. Cryo-EM structure of γ OAD bound to ciliary microtubule (PDB ID: 7K58 and 7N32) was shown using UCSF chimera.
| Organisms | Mutation | Phenotypes | References |
|---|---|---|---|
| Human | Patients with | Situs inversus | [33] |
| PCD symptoms | Bronchiectasis | ||
| Low airway cilia movement | |||
| Missing OAD structure | |||
| Paramecium | RNAi knock down | Slow swimming | [31] |
| Low ciliary beat frequency | |||
| Reduction of OAD structure | |||
| Trypanosome | RNAi knock down | Fixed to backward swimming | [32] |
| Missing OAD structure | |||
| Chlamydomonas | Co-expression with | Slow swimming | [34] |
| wt and mutated LC1 | Low flagellar beat frequency | ||
| Abnormal swimming track | |||
| Planaria | RNAi knock down | Slow gliding locomotion | [35] |
| Low ciliary beat frequency | |||
| Disordered metachronal wave | |||
| Normal OAD structure |
Recently, the high-resolution structure of Tetrahymena OAD arrays bound to dMTs was determined by cryo-electron microscopy (cryo-EM) (EMDB: EMD-22677; PDB ID: 7K58 and 7N32) [27]. Tetrahymena OAD also has three DHCs (Dyh3, 4, and 5 HCs), and the Dyh3 HC associates with LC1 through the MTBD like Chlamydomonas [38]. MTBDs of all three OAD HCs attached to α-tubulin and β-tubulin at the intradimer interface in distinct manners. By comparing the structure of LC1: γ-stalk complex in MT-unbound state with that in MT-bound state (corresponding to the X-ray structure of Chlamydomonas stalk and the cryo-EM structure of Tetrahymena OAD, respectively), we found that the majority of the complex structure was similar to each other, but that minor structural differences were observed in specific regions (Figure 5). In the MT-bound state, the positions of the N- and C-terminal helices and the β-barrel of LC1 were slightly different from those in the MT-unbound state (Figure 5C), and the H1 and CC1 in stalk moved up into a raised position over the intradimer interface (Figure 5D), which was also observed in the MT-bound forms of cytoplasmic and axonemal dynein stalks [41,42]. Interestingly, the convex surface of LC1 interacted with negatively-charged C-terminal tail of β-tubulin on the adjacent protofilament (Figure 4B and 5B). Thus, OAD γ binds to B-tubule of a dMT at two positions through LC1 as well as the stalk MTBD (Figure 5B).
Comparison of MT-bound and unbound structures of LC1-γMTBD.
(A) Schematic diagram of OAD γ. (B) (Left) Crystal structure of LC1-γ MTBD complex. LC1 (orange) and MTBD (dark blue). (Right) Cryo-EM structure of γ OAD bound to ciliary microtubule. LC1 (green), γ stalk (cyan), and tubulin (gold). Note that MTBD binds to a tubulin molecule by the H1 helix, while LC1 binds to the C-terminal region of the neighboring tubulin molecule through the surface of the LC1 β barrel. Images shown in Figure 5A and 5B are corresponding to the 90 degree counterclockwise rotational view of Figure 3A and B. (C, D) Comparison of structural folds between the two LC1-MTBD structures. Structures of LC1 and MTBD were superimposed using the ‘Machmaker’ function of UCSF chimera, respectively. Overall structural folds were similar to each other. In LC1, however, the positions of N- and C-terminal helices, and a β-sheet in the middle of the molecule were slightly different between the two structures. In γ MTBD, the tubulin-bound H1 helix appears to move upward, as shown in the previous study [41].
Binding interface of LC1 for MTs had been analyzed using several mutant forms of LC1, in which single basic residues on the convex surface of LC1 were changed to Glu. The affinity of the LC1 mutants to axonemes of the outer-armless mutant oda was examined by co-pelleting assay. Most of LC1 mutations with the significantly reduced MT-affinity were located in the specific region on the convex surface, which was identified as the MT binding interface of LC1 in cryo-EM study (Supplementary Table S1, Figure 4B) [34]. The results strongly suggest that the interaction between LC1 and tubulin is mediated by a cluster of positively charged residues on the LC1 β-barrel surface that attract the negatively-charged C-terminal tail of β-tubulin from the adjacent protofilament [27].
Here we reviewed recent structural studies of the outer-arm dynein light chain LC1. X-ray crystallography revealed that LC1 directly interacts with the MTBD of OAD γ stalk through the H5 helix and the flap (Figure 3B). In addition, cryo-EM showed that LC1 also binds to the C-terminal tail of β-tubulin on the adjacent protofilament (Figure 5B). Thus, OAD γ has two binding sites for a dMT; one is directly with the MTBD itself, and the other is through the bound LC1. This is a remarkable contrast to the single interaction site for a MT in cytoplasmic dynein [41]. Two binding sites for a dMT in axonemal dynein stalk have been reported in DNAH7 (a human homologue of Chlamydomonas IAD c) and Tetrahymena Dyh5 (corresponding to Chlamydomonas OAD α) [27,42]. In these dyneins, the second MT binding site is the flap region extending from the MTBD core. Interestingly, the flap appeared to be masked by LC1 in OAD γ (Figure 3B and 3C), and the MT affinity of OAD γ-stalk was found to be reduced by LC1 (Figure 2C). This indicates that LC1 weakens the MT affinity of OAD γ. Previous mutational studies suggest that LC1 controls ciliary movement through the regulation of OAD activity [34,35]. How and when can LC1 regulate OAD activity? LC1 might modulate the MT affinity of OAD γ depending on the beating conditions. Deletion mutant of the LC1 gene has been obtained in Chlamydomonas mutant library project [43]. Further functional and structural studies of the mutant would be helpful to understand the regulatory mechanism of LC1.
The authors declare no conflict of interest.
T. Y., A. T., M. I., and G. K. wrote the manuscript.
The evidence data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
We thank Dr. K. Zhang for sharing the coordinate data for Tetrahymena OAD arrays bound to a dMT. T.Y. is supported by JSPS Grant Number JP19H02566, JP21K0125, JP22H01922 from the Japan Society for the Promotion of Science (JSPS). M.I. is supported by JSPS KAKENHI Grant Number JP21KK0125 and JP22K15075, and PRESTO (JPMJPR20E1) from the Japan Science and Technology Agency (JST). G.K. is grateful to the grant support from JSPS Grant Number JP18H02390.
