Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Mar 22;14(11):2683-94.
doi: 10.1016/j.celrep.2016.01.079. Epub 2016 Mar 10.

Myosin VI Contains a Compact Structural Motif that Binds to Ubiquitin Chains

Fahu He  1 , Hans-Peter Wollscheid  2 , Urszula Nowicka  1 , Matteo Biancospino  2 , Eleonora Valentini  2 , Aaron Ehlinger  3 , Filippo Acconcia  2 , Elisa Magistrati  2 , Simona Polo  4 , Kylie J Walters  5
Affiliations

Myosin VI Contains a Compact Structural Motif that Binds to Ubiquitin Chains

Fahu He et al. Cell Rep. .

Abstract

Myosin VI is critical for cargo trafficking and sorting during early endocytosis and autophagosome maturation, and abnormalities in these processes are linked to cancers, neurodegeneration, deafness, and hypertropic cardiomyopathy. We identify a structured domain in myosin VI, myosin VI ubiquitin-binding domain (MyUb), that binds to ubiquitin chains, especially those linked via K63, K11, and K29. Herein, we solve the solution structure of MyUb and MyUb:K63-linked diubiquitin. MyUb folds as a compact helix-turn-helix-like motif and nestles between the ubiquitins of K63-linked diubiquitin, interacting with distinct surfaces of each. A nine-amino-acid extension at the C-terminal helix (Helix2) of MyUb is required for myosin VI interaction with endocytic and autophagic adaptors. Structure-guided mutations revealed that a functional MyUb is necessary for optineurin interaction. In addition, we found that an isoform-specific helix restricts MyUb binding to ubiquitin chains. This work provides fundamental insights into myosin VI interaction with ubiquitinated cargo and functional adaptors.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Identification and characterization of the MyUb domain
(A) Domain architecture of myosin VI highlighting the motor domain (grey), lever arm (white), MIU (yellow), MyUb (beige), and cargo-binding domain (CBD, light blue). The WWY motif is in the CBD and noted with an asterisk. aa, amino acid. Depicted below are the tail constructs used for the experiment described in (B). (B) GST-tagged myosin VI tail (N835-K1294) wild type and mutant carrying an internal deletion of the MIU domain (S997-I1024) were used to pull-down polyubiquitinated proteins from HEK293T cellular lysates. Immunoblotting (IB) as indicated. Right panel, ponceau showing equal loading of the GST proteins. (C) Sequence alignment of MyUb from Homo sapiens, Mus musculus, Gallus gallus, Rattus vorvegicus, Sus scrofa, Bos Taurus, Xenopus tropicalis, and Drosophila melanogaster with conserved and non-conserved amino acids in black and grey, respectively. The RRL motif is highlighted in magenta. (D) GST pull-down assay with indicated myosin VI constructs or GST as a control. GST-fusion proteins were incubated with purchased diubiquitins linked by M1, K6, K11, K27, K29, K33, K48, or K63 (Boston Biochem, Inc.) and analyzed by immunoblotting with anti-ubiquitin antibody. (E) Fluorescence polarization (FP) assays to determine MyUb binding affinities for K63-, K48- and K11-linked diubiquitin. Results are representative of at least three independent experiments. Dissociation constants with their respective SD are reported. See also Figure S1.
Figure 2
Figure 2. Myosin VI contains a compact ubiquitin-binding domain
(A, B) Ribbon representation of MyUb (G1080-H1122) highlighting the helix boundaries and the interactions that stabilize the fold, including hydrogen bonds from R1117 to S1087 and E1113 (dashed yellow lines) and van der Waals interactions engaging Y1084, L1086, W1089, L1094, and L1106 (pink). (C) GST pull-down assay performed with GST-MyUb (G1080-R1131) wild type (WT) or amino acid substituted-protein as indicated. GST fusion proteins were incubated for 2 hours at 4°C with K63-linked polyUb1-7 and analyzed by immunoblotting (IB) with anti-ubiquitin antibody. Ponceau staining shows comparable loading of GST-tagged proteins (bottom panel). GST was used as a negative control. (D) GST-MyUb (G1080-H1122), GST-MyUb (G1080-R1131), or GST (as a negative control) were incubated with synthetic K63-linked polyubiquitin1-7 or cellular lysates from HEK293T cells transfected with GFP-optineurin, FLAG-T6BP, His-GIPC, or FLAG-NDP52. Immunoblotting (IB) was performed using the specific anti-TAG antibodies. Ponceau staining shows comparable loading of GST-tagged proteins (bottom panel). (E) Ribbon representations of MyUb (G1080-H1122) (left) and MyUb (G1080-R1131) with extended hydrophobic surface highlighted in green formed by addition of A1123-R1131 (right) were shown. The RRL motif was colored magenta. See also Figure S2.
Figure 3
Figure 3. MyUb nestles between the moieties of K63-linked diubiquitin
(A, B) Stoichiometry of the complex formed by MyUb and K63-linked (A) diubiquitin or (B) triubiquitin, as indicated. 100 μM of purified K63-linked diubiquitin or K63-linked triubiquitin were incubated (1 hr, 20°C) with either 200 or 400 μM MyUb, followed by fractionation on a Superdex75 column. Selected fractions were separated by SDS-PAGE gel and stained with Coomassie. (C) Ribbon diagram of MyUb (G1080-H1122):K63-linked diubiquitin with MyUb (beige) nestled between the proximal ubiquitin (light grey) and distal ubiquitin (dark grey) with interactions to a I44-centered surface on distal ubiquitin (green) and a F45-centered surface of proximal ubiquitin (blue). Interacting MyUb amino acids are displayed in yellow. Labels for MyUb amino acids are underlined. (D) Expanded view of (C) showing critical interactions of I1104 from MyUb with L8 and H68 of the distal K63-linked diubiquitin. (E) GST pull-down assay performed with GST-MyUb (G1080-R1131) wild type (WT) or I1104A. GST fusion proteins were incubated for 2 hours at 4°C with K63-linked polyUb1-7 and analyzed by immunoblotting (IB) with anti-ubiquitin antibody. Ponceau staining shows comparable loading of GST-tagged proteins (bottom panel). GST was used as a negative control. (F) Selected regions that include A1092 and T1100 signals from 1H-15N HSQC spectra acquired on 15N-labeled MyUb wild type (top) or MyUb I1104A (bottom) with K63-linked diubiquitin at the indicated molar ratio. Arrows point to new peak positions caused by addition of diubiquitin. All spectra were recorded at 850 MHz on MyUb sample concentrations of 0.1 mM. (G) Ribbon diagrams of the MyUb:K63-linked diubiquitin complex showing the opposite surface compared to (C), rotated 145 degrees along the x-axis, to highlight hydrogen bond (yellow dashed line) interactions involving MyUb and K63-linked diubiquitin. An expanded region (boxed in red) is displayed to the right. See also Figure S3.
Figure 4
Figure 4. MyUb binds to K11-linked diubiquitin through the hydrophobic patch of the proximal ubiquitin moiety
(A) Selected regions showing methyl groups from L8 and I44 from a 3D 13C half-filtered NOESY experiment acquired on ~0.5 mM unlabeled MyUb (G1080-H1122) mixed with equimolar K63- (left) or K11-linked (right) diubiquitin with the proximal or distal ubiquitin moiety 15N and 13C labeled, respectively. NOEs assigned uniquely to MyUb atoms are labeled. D-K63, K63-linked diubiquitin with the distal moiety 15N and 13C labeled; P-K11, K11-linked diubiquitin with the proximal moiety 15N and 13C labeled. (B) Selected regions that include A46 and I44 amide signals from 1H-15N HSQC spectra acquired on 13C, 15N-labeled proximal (left) or distal (right) K11-linked diubiquitin (black) and with equimolar unlabeled MyUb (orange). (C) Model structure of MyUb bound to a signal ubiquitin based on Figure 3C, illustrating the amino acids involved in complex formation as revealed in (A–B).
Figure 5
Figure 5. Ubiquitin binding by MyUb contributes to myosin VI;optineurin interaction
(A) Lysate from HEK293T cells expressing GFP-optineurin and HA-ubiquitin was immunoprecipitated and immunoblotted as indicated. (B) 2 μM of the indicated GST-fusion proteins were incubated with the same cellular lysate (2 mg) shown in (A). Immunoblotting was performed using anti-GFP or anti-ubiquitin antibodies. Ponceau staining shows comparable loading of GST-tagged proteins (bottom panel). (C) GFP-Trap beads were used to purify GFP-optineurin from cellular lysates of HEK293T transfected cells. Washed beads were incubated with the indicated eluted GST proteins and immunoblotting was performed using anti-GST. Ponceau staining shows comparable loading of GFP-optineurin (upper panel).
Figure 6
Figure 6. The presence of an isoform-specific helix in the MIU-MyUb region of myosin VI affects its affinity for ubiquitin chains
(A) Sequence alignment of myosin VI isoforms spanning MIU (yellow box) to MyUb (beige box). Linker-α1 (boxed in blue) and linker-α2 (boxed in purple) are indicated. The RRL motif is highlighted in magenta. Amino acids from the LI region are boxed in gray. (B) Expanded region of 1H-15N HSQC spectra recorded on 0.1 mM 15N MIU (Q998-S1025) with unlabeled monoubiquitin added at the indicated molar ratio. This dataset was acquired at 700 MHz. (C) GST-tagged myosin VI MIU-MyUb constructs from the different isoforms were used to pull-down polyubiquitinated proteins from HEK293T cellular lysates (1 μg). GST was used as a control, immunoblotting (IB) was done with anti-ubiquitin antibody, and ponceau staining is included below to indicate loading of GST-tagged proteins. (D) GST pull-down assay for 2 μM indicated myosin VI GST-MIU-MyUb or GST (as a control) with 2 hour incubation with K63-linked ubiquitin chains at 4°C. Analysis was done by immunoblotting (IB) with anti-ubiquitin antibody. All six isoforms bind longer chains, but only iso2 MIU-MyUb retains binding to K63-linked diubiquitin. Bottom panel, ponceau staining showing equal loading of the GST proteins. (E) Binding affinities (± SD) determined by FP between K63-, K11-, and K48-linked diubiquitin and the indicated myosin VI constructs. (F) Snapshot ribbon representation of the solution structure of iso3 Q998-A1071. MIU is part of a 6.5-turn helix (yellow), which is followed by a 3-residue kink and 2.5-turn helix (linker-α1, blue). A long, flexible linker region separates linker-α1 from isoform-specific linker-α2 (pink) and the orientation of these two helices is not defined. Amino acids from the LI region are boxed in gray. See also Figure S4.
Figure 7
Figure 7. MyUb interaction with K63-linked ubiquitin chains is unique
(A–D) Structures of (A) myosin VI MyUb: K63-linked diubiquitin, (B) Rap80 UIM: K63-linked diubiquitin (3A1Q), (C) TAB2 NZF: K63-linked diubiquitin (3A9J), and (D) AMSH-LP DUB: K63-linked diubiquitin (2ZNV) displayed with their proximal ubiquitin in the same orientation. The proximal ubiquitin is depicted in light blue and the distal ubiquitin in light green. Amino acids in proximal and distal ubiquitin at the contact surface are displayed in blue and green, respectively, and labeled. (E) Structure of monomeric ubiquitin highlighting the amino acids involved in binding to K63-linked chain specific receptors, as assessed by coordinates deposited in the Protein Data Bank (3A1Q, 3A9J, 2ZNV) and our structure (A). A legend is included to the right illustrating the color coding with corresponding amino acids on monoubiquitin for the listed binding domains. Amino acids in yellow are unique to MyUb binding.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Acconcia F, Sigismund S, Polo S. Ubiquitin in trafficking: the network at work. Experimental cell research. 2009;315:1610–1618. - PubMed
    1. Au JS, Puri C, Ihrke G, Kendrick-Jones J, Buss F. Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells. The Journal of cell biology. 2007;177:103–114. - PMC - PubMed
    1. Avraham KB, Hasson T, Steel KP, Kingsley DM, Russell LB, Mooseker MS, Copeland NG, Jenkins NA. The mouse Snell’s waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells. Nature genetics. 1995;11:369–375. - PubMed
    1. Berg JS, Powell BC, Cheney RE. A millennial myosin census. Molecular biology of the cell. 2001;12:780–794. - PMC - PubMed
    1. Boname JM, Thomas M, Stagg HR, Xu P, Peng J, Lehner PJ. Efficient internalization of MHC I requires lysine-11 and lysine-63 mixed linkage polyubiquitin chains. Traffic. 2010;11:210–220. - PMC - PubMed

Publication types