Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering

Abstract

Myosin VI plays crucial roles in diverse cellular processes. In autophagy, Myosin VI can facilitate the maturation of autophagosomes through interactions with Tom1 and the autophagy receptors, Optineurin, NDP52 and TAX1BP1. Here, we report the high-resolution crystal structure of the C-terminal cargo-binding domain (CBD) of Myosin VI in complex with Tom1, which elucidates the mechanistic basis underpinning the specific interaction between Myosin VI and Tom1, and uncovers that the C-terminal CBD of Myosin VI adopts a unique cargo recognition mode to interact with Tom1 for tethering. Furthermore, we show that Myosin VI can serve as a bridging adaptor to simultaneously interact with Tom1 and autophagy receptors through two distinct interfaces. In all, our findings provide mechanistic insights into the interactions of Myosin VI with Tom1 and relevant autophagy receptors, and are valuable for further understanding the functions of these proteins in autophagy and the cargo recognition modes of Myosin VI.

Fig. 1

Biochemical analyses of the interaction between Myosin VI and Tom1. a A schematic diagram showing the domain arrangements of Myosin VI, Tom1, NDP52, TAX1BP1, and Optineurin. In this drawing, domains involved in the protein–protein interaction are highlighted with black lines, and the relevant interactions between two proteins are indicated by two-way arrows. b Superposition plots of the ¹H-¹⁵N HSQC spectra of Tom1(392–463) titrated with the un-labeled C-terminal CBD of Myosin VI proteins at different molar ratios. For clarity, the insert shows the enlarged view of a unique peak corresponding to the side chain of Tom1 W423 residue in the overlaid ¹H-¹⁵N HSQC spectra. c–e ITC-based measurements of the binding affinities of the C-terminal CBD of Myosin VI with Tom1(392–463) (c), Tom1(392–437) (d), and Tom1(437–463) (e). Kd values are the fitted dissociation constants with standard errors, when using the one-site binding model to fit the ITC data. ‘N.D.’ stands for that the Kd value is not detectable. Source data are provided as a Source Data file. f Overlay plots of the multi-angle light-scattering data of the C-terminal CBD of Myosin VI, Tom1(437–463), and the C-terminal CBD of Myosin VI in complex with Tom1(437–463). The derived molecular masses of the C-terminal CBD of Myosin VI and Tom1(437–463) are shown in red and in blue, respectively, while the derived molecular mass of the C-terminal CBD of Myosin VI and Tom1(437–463) complex is shown in black. The molecular masses errors are the fitted errors obtained from the data analysis software, and are showed in the brackets. The results clearly demonstrate that the C-terminal CBD of Myosin VI and Tom1(437–463) both form a stable monomer and may interact with each other to form a 1:1 stoichiometric complex in solution. Source data are provided as a Source Data file

Fig. 2

The overall structure of Myosin VI/Tom1 complex. a Ribbon diagram showing the overall structure of the C-terminal CBD of Myosin VI and Tom1 MBM complex. In this drawing, the C-terminal CBD of Myosin VI is shown in blue, and Tom1 MBM in magenta. b Surface representations showing the overall architecture of Myosin VI/Tom1 complex (left panel), and the open-book view of the binding interface between Myosin VI and Tom1 (right panel) with the same color scheme as in a

Fig. 3

The molecular interface of Myosin VI and Tom1 complex. a The combined surface representation and the ribbon-stick model showing the hydrophobic binding interface between the C-terminal CBD of Myosin VI and Tom1 MBM. In this presentation, the C-terminal CBD of Myosin VI is shown in the surface model and Tom1 MBM in the ribbon-stick model. Particularly, in the surface model of the C-terminal CBD of Myosin VI, the hydrophobic amino acid residues are drawn in yellow, the positively charged residues in blue, the negatively charged residues in red, and the uncharged polar residues in gray. b The combined surface charge representation and the ribbon-stick model showing the charge-charge interactions between the C-terminal CBD of Myosin VI and Tom1 MBM. c Stereo view of the ribbon-stick model showing the detailed interactions between the C-terminal CBD of Myosin VI and Tom1 MBM. The hydrogen bonds and salt bridges involved in the binding are shown as dotted lines. d Structure-based sequence alignment of Tom1 MBM with the corresponding regions of TomL1 and TomL2. In this structure-based sequence alignment, the conserved hydrophobic residues, polar neutral residues, positively charged residues, and negatively charged residues are colored in orange, green, blue, and magenta, respectively. Interface residues of Tom1 that are involved in the polar interactions and hydrophobic interactions with the C-terminal CBD of Myosin VI in the Myosin VI/Tom1 complex are further labeled with magenta stars and magenta triangles, respectively

Fig. 4

Comparisons of the Myosin VI/Tom1 and Myosin VI/Dab2 complexes. a The comparison of the overall structures of the Myosin VI/Dab2 complex (gray-orange, PDB ID: 3H8D) and the Myosin VI/Tom1 complex (blue-magenta). In this presentation, the positions of site I and site II in the C-terminal CBD of Myosin VI are further indicted. b Stereo view in the ribbon-stick model showing the comparison of the binding interfaces of the Myosin VI/Dab2 complex and the Myosin VI/Tom1 complex with the same color scheme as in a. The hydrogen bonds and salt bridges involved in the interactions are shown as dotted lines. The binding interface residues of Tom1 and Dab2 are labeled with magenta and orange numbers, respectively. While, the interface residues of the C-terminal CBD of Myosin VI that involved in the interactions with both Tom1 and Dab2, only for the interaction with Tom1 or Dab2, are labeled with black, blue, and gray numbers, respectively. c Structure-based sequence alignment of Tom1(437–493) and the Myosin VI-binding region of Dab2. In this structure-based sequence alignment, the conserved hydrophobic residues, polar neutral residues, positively charged residues and negatively charged residues are colored in orange, green, blue, and magenta, respectively. Key interface residues of Tom1 involved in the interaction with the site I of the C-terminal CBD of Myosin VI through the polar interactions and the hydrophobic interactions are further labeled with magenta stars and triangles, respectively, and that of Dab2 are highlighted with orange stars and triangles. Meanwhile, key interface residues of Dab2 that are critical for binding to the site II of the C-terminal CBD of Myosin VI, are labeled with orange dots

Fig. 5

Myosin VI can link Tom1 with autophagy receptors. a A co-immunoprecipitation assay showing that point mutations of key interface residues observed in the Myosin VI/Tom1 complex structure abolish the specific interaction between Myosin VI(1060–1285) and Tom1 in cells. In this assay, cell extracts were prepared from HEK293T cells co-transfected with different combinations of plasmids as indicated, and 5% of each extracts were used as loading controls (bottom panel). b A co-immunoprecipitation assay revealing that Myosin VI(1060–1285), Tom1 and autophagy receptor TAX1BP1, NDP52, or Optineurin, can form ternary complexes in co-transfected cells. c A co-immunoprecipitation assay showing that mutations of Tom1, which can disrupt the interaction between Myosin VI(1060–1285) and Tom1, can also abolish the formation of the Tom1/Myosin VI/autophagy receptor ternary complex in cells. In this assay, cell extracts were prepared from HEK293T cells co-transfected with different combinations of plasmids as indicated, and 5% of each extracts were used as loading controls (bottom panel). Source data are provided as a Source Data file. d A proposed model depicting the tethering of endosome and autophagosome mediated by Myosin VI in cooperate with Tom1, the autophagy receptors, NDP52, TAX1BP1, and Optineurin as well as relevant ubiquitin chains, for facilitating the maturation of autophagosome in autophagy