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• W2293449161 abstract "•Myosin VI contains a compact structural motif that binds ubiquitin chains•MyUb nestles between ubiquitins of K63-linked chains•Optineurin interaction requires an expanded MyUb and capacity to bind ubiquitin•An isoform-specific helix restricts MyUb binding to ubiquitin chains 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. 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. Myosins are a superfamily of quintessential molecular motors that power movements on actin filaments by converting ATP hydrolysis to mechanical energy and force. A highly conserved N-terminal motor domain undergoes conformational changes during the ATPase cycle that modulate actin affinity (Geeves and Holmes, 1999Geeves M.A. Holmes K.C. Structural mechanism of muscle contraction.Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (634) Google Scholar). These motions are amplified into the myosin powerstroke by a variable calmodulin-binding lever arm causing nanometer-scale movement (Spudich and Sivaramakrishnan, 2010Spudich J.A. Sivaramakrishnan S. Myosin VI: an innovative motor that challenged the swinging lever arm hypothesis.Nat. Rev. Mol. Cell Biol. 2010; 11: 128-137Crossref PubMed Scopus (81) Google Scholar). With the exception of myosin VI, movement is toward the barbed (plus) end of actin filaments (Wells et al., 1999Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Myosin VI is an actin-based motor that moves backwards.Nature. 1999; 401: 505-508Crossref PubMed Scopus (554) Google Scholar). Myosin VI contains an additional calmodulin-binding insertion that redirects the effective lever arm toward the pointed (minus) end of actin filaments (Ménétrey et al., 2005Ménétrey J. Bahloul A. Wells A.L. Yengo C.M. Morris C.A. Sweeney H.L. Houdusse A. The structure of the myosin VI motor reveals the mechanism of directionality reversal.Nature. 2005; 435: 779-785Crossref PubMed Scopus (180) Google Scholar). The C-terminal tail region is divergent among myosins and confers specificity for cargo and distinct interactions that define subcellular localization and specialized functions. Humans express ∼40 known or predicted myosins (Berg et al., 2001Berg J.S. Powell B.C. Cheney R.E. A millennial myosin census.Mol. Biol. Cell. 2001; 12: 780-794Crossref PubMed Scopus (619) Google Scholar) that participate in diverse activities, including conventional skeletal myosin IIs for muscle contraction and unconventional myosins that function in intracellular trafficking, cell division and motility, actin cytoskeletal organization, and cell signaling (Sellers, 2000Sellers J.R. Myosins: a diverse superfamily.Biochim. Biophys. Acta. 2000; 1496: 3-22Crossref PubMed Scopus (617) Google Scholar). Myosin malfunction has been implicated in hypertrophic cardiomyopathy (Mohiddin et al., 2004Mohiddin S.A. Ahmed Z.M. Griffith A.J. Tripodi D. Friedman T.B. Fananapazir L. Morell R.J. Novel association of hypertrophic cardiomyopathy, sensorineural deafness, and a mutation in unconventional myosin VI (MYO6).J. Med. Genet. 2004; 41: 309-314Crossref PubMed Scopus (85) Google Scholar), Usher syndrome (Hasson et al., 1995Hasson T. Heintzelman M.B. Santos-Sacchi J. Corey D.P. Mooseker M.S. Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B.Proc. Natl. Acad. Sci. USA. 1995; 92: 9815-9819Crossref PubMed Scopus (374) Google Scholar, Weil et al., 1995Weil D. Blanchard S. Kaplan J. Guilford P. Gibson F. Walsh J. Mburu P. Varela A. Levilliers J. Weston M.D. et al.Defective myosin VIIA gene responsible for Usher syndrome type 1B.Nature. 1995; 374: 60-61Crossref PubMed Scopus (875) Google Scholar), deafness (Avraham et al., 1995Avraham K.B. Hasson T. Steel K.P. Kingsley D.M. Russell L.B. Mooseker M.S. Copeland N.G. Jenkins N.A. The mouse Snell’s waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells.Nat. Genet. 1995; 11: 369-375Crossref PubMed Scopus (421) Google Scholar, Gibson et al., 1995Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. A type VII myosin encoded by the mouse deafness gene shaker-1.Nature. 1995; 374: 62-64Crossref PubMed Scopus (547) Google Scholar), Griscelli syndrome (Kumar et al., 2001Kumar M. Sackey K. Schmalstieg F. Trizna Z. Elghetany M.T. Alter B.P. Griscelli syndrome: rare neonatal syndrome of recurrent hemophagocytosis.J. Pediatr. Hematol. Oncol. 2001; 23: 464-468Crossref PubMed Scopus (25) Google Scholar, Takagishi and Murata, 2006Takagishi Y. Murata Y. Myosin Va mutation in rats is an animal model for the human hereditary neurological disease, Griscelli syndrome type 1.Ann. N Y Acad. Sci. 2006; 1086: 66-80Crossref PubMed Scopus (22) Google Scholar), and cancer (Dunn et al., 2006Dunn T.A. Chen S. Faith D.A. Hicks J.L. Platz E.A. Chen Y. Ewing C.M. Sauvageot J. Isaacs W.B. De Marzo A.M. Luo J. A novel role of myosin VI in human prostate cancer.Am. J. Pathol. 2006; 169: 1843-1854Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Yoshida et al., 2004Yoshida H. Cheng W. Hung J. Montell D. Geisbrecht E. Rosen D. Liu J. Naora H. Lessons from border cell migration in the Drosophila ovary: A role for myosin VI in dissemination of human ovarian cancer.Proc. Natl. Acad. Sci. USA. 2004; 101: 8144-8149Crossref PubMed Scopus (122) Google Scholar), thus prompting the development of small-molecule myosin inhibitors (Bond et al., 2013Bond L.M. Tumbarello D.A. Kendrick-Jones J. Buss F. Small-molecule inhibitors of myosin proteins.Future Med. Chem. 2013; 5: 41-52Crossref PubMed Scopus (65) Google Scholar). The myosin VI cargo-binding tail (Figure 1A) interacts with multiple adaptor proteins, including regulators of clathrin-mediated endocytosis and autophagy (Tumbarello et al., 2013Tumbarello D.A. Kendrick-Jones J. Buss F. Myosin VI and its cargo adaptors - linking endocytosis and autophagy.J. Cell Sci. 2013; 126: 2561-2570Crossref PubMed Scopus (97) Google Scholar). Some of these ligands require a myosin VI Arg-Arg-Leu (RRL) motif (Figure 1C), including nuclear dot protein 52 (NDP52) (Morriswood et al., 2007Morriswood B. Ryzhakov G. Puri C. Arden S.D. Roberts R. Dendrou C. Kendrick-Jones J. Buss F. T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion.J. Cell Sci. 2007; 120: 2574-2585Crossref PubMed Scopus (73) Google Scholar), Traf6-binding protein (T6BP) (Morriswood et al., 2007Morriswood B. Ryzhakov G. Puri C. Arden S.D. Roberts R. Dendrou C. Kendrick-Jones J. Buss F. T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion.J. Cell Sci. 2007; 120: 2574-2585Crossref PubMed Scopus (73) Google Scholar), optineurin (Sahlender et al., 2005Sahlender D.A. Roberts R.C. Arden S.D. Spudich G. Taylor M.J. Luzio J.P. Kendrick-Jones J. Buss F. Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis.J. Cell Biol. 2005; 169: 285-295Crossref PubMed Scopus (326) Google Scholar), and GAIP-interacting protein C terminus (GIPC) (Bunn et al., 1999Bunn R.C. Jensen M.A. Reed B.C. Protein interactions with the glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 and the cytoskeleton.Mol. Biol. Cell. 1999; 10: 819-832Crossref PubMed Scopus (164) Google Scholar, Spudich et al., 2007Spudich G. Chibalina M.V. Au J.S. Arden S.D. Buss F. Kendrick-Jones J. Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2.Nat. Cell Biol. 2007; 9: 176-183Crossref PubMed Scopus (164) Google Scholar). Others engage a Trp-Trp-Tyr (WWY) triplet present in the cargo-binding domain (CBD; Figure 1A), including Tom1/Tom1L2 (Finan et al., 2011Finan D. Hartman M.A. Spudich J.A. Proteomics approach to study the functions of Drosophila myosin VI through identification of multiple cargo-binding proteins.Proc. Natl. Acad. Sci. USA. 2011; 108: 5566-5571Crossref PubMed Scopus (25) Google Scholar, Tumbarello et al., 2012Tumbarello D.A. Waxse B.J. Arden S.D. Bright N.A. Kendrick-Jones J. Buss F. Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome.Nat. Cell Biol. 2012; 14: 1024-1035Crossref PubMed Scopus (206) Google Scholar), Dab2 (Inoue et al., 2002Inoue A. Sato O. Homma K. Ikebe M. DOC-2/DAB2 is the binding partner of myosin VI.Biochem. Biophys. Res. Commun. 2002; 292: 300-307Crossref PubMed Scopus (59) Google Scholar, Morris et al., 2002Morris S.M. Arden S.D. Roberts R.C. Kendrick-Jones J. Cooper J.A. Luzio J.P. Buss F. Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton.Traffic. 2002; 3: 331-341Crossref PubMed Scopus (202) Google Scholar, Spudich et al., 2007Spudich G. Chibalina M.V. Au J.S. Arden S.D. Buss F. Kendrick-Jones J. Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2.Nat. Cell Biol. 2007; 9: 176-183Crossref PubMed Scopus (164) Google Scholar), and lemur tyrosine kinase-2 (LMK2) (Chibalina et al., 2007Chibalina M.V. Seaman M.N. Miller C.C. Kendrick-Jones J. Buss F. Myosin VI and its interacting protein LMTK2 regulate tubule formation and transport to the endocytic recycling compartment.J. Cell Sci. 2007; 120: 4278-4288Crossref PubMed Scopus (110) Google Scholar). We previously reported the existence of a motif interacting with ubiquitin (MIU) domain C-terminal to the myosin VI coiled-coil region (Penengo et al., 2006Penengo L. Mapelli M. Murachelli A.G. Confalonieri S. Magri L. Musacchio A. Di Fiore P.P. Polo S. Schneider T.R. Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin.Cell. 2006; 124: 1183-1195Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) (Figure 1A, yellow). In this article, we identify a second ubiquitin-binding domain (UBD) in myosin VI, which we name MyUb (myosin VI ubiquitin-binding domain), that contains the RRL motif. We use nuclear magnetic resonance (NMR) spectroscopy to find that MyUb adopts a compact protein fold that is required for ubiquitin binding and disrupted by amino acid substitutions in the RRL motif. We evaluate MyUb in the context of myosin VI binding to the autophagy adaptor optineurin and the distinct myosin VI isoforms expressed in humans. In Rabex-5, the MIU domain binds to ubiquitinated epidermal growth factor receptor and promotes coupled monoubiquitination (Penengo et al., 2006Penengo L. Mapelli M. Murachelli A.G. Confalonieri S. Magri L. Musacchio A. Di Fiore P.P. Polo S. Schneider T.R. Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin.Cell. 2006; 124: 1183-1195Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). To obtain insight into the function of the MIU (Q998–S1025) in the context of myosin VI (Figure 1A), we analyzed the myosin VI tail spanning N835–K1294, which recapitulates myosin VI interaction and localization (Buss et al., 2001Buss F. Arden S.D. Lindsay M. Luzio J.P. Kendrick-Jones J. Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis.EMBO J. 2001; 20: 3676-3684Crossref PubMed Scopus (232) Google Scholar). Surprisingly, deletion of the MIU domain (S997–I1024) did not abrogate myosin VI tail binding to ubiquitinated species from cellular lysates (Figure 1B). Deletion analysis led to the identification of a second UBD C-terminal to the identified MIU (Figure 1A), which we narrowed down to a 43-amino-acid fragment (G1080–H1122; Figures S1A–S1C). This region is highly conserved in myosin VI from various species (Figure 1C) and appears to be unique to myosin VI, as bioinformatics analysis was unable to detect its presence in other genes (Kay Hoffman, personal communication). Since this region lacks sequence similarity with any previously described UBD, we henceforth refer to it as MyUb. We used diubiquitin molecules made with the eight possible linkages (M1, K6, K11, K27, K29, K33, K48, and K63) to test whether MyUb or MIU exhibit preference for a specific chain type. glutathione S-transferase (GST)-MIU binding to diubiquitin was barely detectable by this method (Figure 1D); on the contrary, GST-MyUb bound robustly to ubiquitin chains, with preference for K63-, K11-, and K29-linked diubiquitin (Figure 1D). Weak interaction was detected with K48-linked diubiquitin (Figure 1D), a linkage type associated with proteasomal degradation (Ehlinger and Walters, 2013Ehlinger A. Walters K.J. Structural insights into proteasome activation by the 19S regulatory particle.Biochemistry. 2013; 52: 3618-3628Crossref PubMed Scopus (35) Google Scholar). This difference in affinity was confirmed by fluorescence polarization (FP) assays. MyUb bound to K11-, K48-, and K63-linked diubiquitin with low micromolar affinity, with the K63- and K11-linked diubiquitin exhibiting 10- and 4-fold greater affinity compared to K48-linked diubiquitin, respectively (Figure 1E). We used NMR techniques (described in Experimental Procedures) to solve the MyUb (G1080–H1122) structure. The 20 lowest-energy structures calculated from 100 extended starting ones converged to fit recorded NMR data (Table S1), with a backbone root mean square deviation (rmsd) of 0.20 Å (Figure S2A). From these data, we concluded that MyUb adopts a compact helix-turn-helix-like fold with two helices spanning Y1091 to T1100 (Helix1) and I1104 to K1119 (Helix2) (Figure 2A). Importantly, the structural fold is stabilized by numerous interactions with an N-terminal region (Figure 2B). Y1084 and L1086 from the N-terminal region pack against hydrophobic amino acids (L1094 and L1106) from Helix2, while W1089 is partially buried by L1094 and L1086 (Figure 2B, pink). Alanine substitution of L1086 or L1106 causes misfolding of the MyUb domain (Figures S2B and S2C) and significantly decreased binding to K63-linked ubiquitin chains (Figure 2C). A W1089A mutation was instead tolerated, as it did not fully disrupt the MyUb structure (Figure S2D) and did not prevent MyUb binding to K63-linked diubiquitin (Figures 2C and S2E). Changing the temperature and salt conditions did not alter MyUb structure (Figures S2F–S2H) or interaction with K63-linked diubiquitin (Figure S2I). Several myosin VI adaptor proteins, namely optineurin, GIPC, T6BP, and NDP52, were shown to require an intact 1116RRL1118 motif for interaction with myosin VI (Bunn et al., 1999Bunn R.C. Jensen M.A. Reed B.C. Protein interactions with the glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 and the cytoskeleton.Mol. Biol. Cell. 1999; 10: 819-832Crossref PubMed Scopus (164) Google Scholar, Morriswood et al., 2007Morriswood B. Ryzhakov G. Puri C. Arden S.D. Roberts R. Dendrou C. Kendrick-Jones J. Buss F. T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion.J. Cell Sci. 2007; 120: 2574-2585Crossref PubMed Scopus (73) Google Scholar, Sahlender et al., 2005Sahlender D.A. Roberts R.C. Arden S.D. Spudich G. Taylor M.J. Luzio J.P. Kendrick-Jones J. Buss F. Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis.J. Cell Biol. 2005; 169: 285-295Crossref PubMed Scopus (326) Google Scholar, Spudich et al., 2007Spudich G. Chibalina M.V. Au J.S. Arden S.D. Buss F. Kendrick-Jones J. Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2.Nat. Cell Biol. 2007; 9: 176-183Crossref PubMed Scopus (164) Google Scholar). In particular, amino acid substitution of this motif with the alanine triple AAA was reported to abolish myosin VI interaction with these autophagy adaptor proteins (Morriswood et al., 2007Morriswood B. Ryzhakov G. Puri C. Arden S.D. Roberts R. Dendrou C. Kendrick-Jones J. Buss F. T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion.J. Cell Sci. 2007; 120: 2574-2585Crossref PubMed Scopus (73) Google Scholar). The RRL motif resides in Helix2 where R1117 forms hydrogen bonds to S1087 and E1113 (Figure 2B), suggesting that it is critical for MyUb (G1080–H1122) structural integrity. As expected, replacement of R1117 with alanine resulted in misfolding, as measured by a 2D NMR experiment (Figure S2J, red compared to black) and abolished interaction with ubiquitin (Figure 2C). This result prompted us to re-examine the interaction of myosin VI with previously characterized autophagy adaptor proteins using various RRL-containing fragments. The minimal binding region was found to be a MyUb construct spanning G1080–R1131 (Figure 2D). The shorter construct spanning G1080–H1122 was unable to bind optineurin, GIPC, T6BP, and NDP52, even though it was competent for binding to ubiquitin (Figures 2D and S2K) and adopted a stable protein fold (Figure 2A). Thus, the interaction surface between myosin VI and its known partners extends beyond the previously identified 1116RRL1118 motif toward the C-terminal part of Helix2 of the MyUb domain. We next used NMR to solve the structure of this longer construct (Figure S2L; Table S1). The backbone rmsd of the extended MyUb (G1080–R1131) to MyUb (G1080–H1122) was 0.46 Å for the overlapping region. Helix 2 was extended, however, by six amino acids forming an additional 1.5 helical turns, with an expanded hydrophobic surface contributed by Y1121, H1122, and W1124 (Figure 2E, right). We speculate that this extended hydrophobic surface is involved in myosin VI binding to autophagy adaptor proteins. The MyUb bound well to K11-, K29, and K63-linked ubiquitin chains (Figure 1D). We therefore used K63-linked diubiquitin as a model to dissect the molecular mechanism of MyUb binding to ubiquitin chains. Initially, we determined the binding stoichiometry of the MyUb:K63-linked ubiquitin chain complex by using size exclusion chromatography. 2- or 4-fold molar excess MyUb incubated with 100 μM diubiquitin or triubiquitin was loaded on a size exclusion column (Figures 3A and 3B ). At 2-fold molar excess, MyUb and K63-linked diubiquitin co-eluted at the expected molecular weight for a 1:1 complex with the excess MyUb eluting separately (Figure 3A, blue). By contrast, very little MyUb eluted separately from K63-linked triubiquitin (Figure 3B, blue), and the presence of free MyUb in the mixture with triubiquitin increased significantly when MyUb was at 4-fold molar excess (Figure 3B, purple). Altogether, these data indicate a 1:1 stoichiometry for the MyUb:diubiquitin complex and a 2:1 stoichiometry for the MyUb:triubiquitin complex. To explore the molecular basis of MyUb binding to ubiquitin, we used NMR to solve the structure of MyUb (G1080–H1122) in complex with K63-linked diubiquitin. The MyUb structure was unchanged upon ubiquitin binding, as demonstrated by an almost identical interaction network for free and K63-linked diubiquitin-bound MyUb (Figure S3A). We isotopically labeled either the proximal (defined by a free G76 that can be in principle conjugated to a substrate protein) or the distal ubiquitin of K63-linked diubiquitin and used 13C half-filtered nuclear Overhauser effect spectroscopy (NOESY) experiments to detect interactions between MyUb and each ubiquitin (Figures S3B–S3D). We were able to assign 59 intermolecular interactions between MyUb and the proximal ubiquitin and 47 intermolecular interactions between MyUb and the distal ubiquitin; these were used to solve the structure of the MyUb:K63-linked diubiquitin complex (Figures S3E and S3F; Table S1). We found that MyUb nestles between the two ubiquitins, making extensive contacts to both ubiquitin moieties (Figure 3C). On the distal ubiquitin, an exposed MyUb hydrophobic surface formed by the two helices contacts the classic hydrophobic patch centered on I44 (Liu and Walters, 2010Liu F. Walters K.J. Multitasking with ubiquitin through multivalent interactions.Trends Biochem. Sci. 2010; 35: 352-360Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) (Figure 3C, green). This contact surface includes I1098 from Helix1 and I1104, L1107, A1108, R1111, and F1114 from Helix2 (Figure 3C, yellow). In particular, I1104 forms critical interactions with L8 and H68 of distal ubiquitin (Figure 3D). Its replacement with alanine does not affect the overall MyUb fold (Figure S3G) but abolishes binding to K63-linked diubiquitin, as shown in a pull-down experiment (Figure 3E) and by NMR titration experiments (Figure 3F). In the slow exchange regime on the NMR timescale (Walters et al., 2001Walters K.J. Ferentz A.E. Hare B.J. Hidalgo P. Jasanoff A. Matsuo H. Wagner G. Characterizing protein-protein complexes and oligomers by nuclear magnetic resonance spectroscopy.Methods Enzymol. 2001; 339: 238-258Crossref PubMed Scopus (45) Google Scholar), amino acids A1092 and T1100 shift to a new position upon K63-linked diubiquitin addition (Figure 3F, top panels). This effect is lost in the MyUb I1104A mutant (Figure 3F, bottom). These data were confirmed by FP analysis, which demonstrated a Kd >400 μM for the I1104A mutant (Figure S3H). On the proximal ubiquitin, aforementioned A1092 and T1100 from Helix1 of MyUb contact a surface formed by F4, F45, A46, K48, and T66 (Figure 3C, blue). Likely, the involvement of K48 at this location explains the relatively poor affinity of the MyUb for this chain type (Figures 1D and 1E). Some nuclear Overhauser effects (NOEs) were assigned to the L8-I44-V70 hydrophobic patch of the proximal ubiquitin, indicating that MyUb can form a lower affinity interaction with this region (Figures S3C and S3F, orange, and S3I). However, binding to this second, low-affinity site was not retained in the size exclusion chromatography experiment (Figure 3A), indicating that this interaction is weak and possibly not present in the context of the full-length protein. MyUb interaction with K63-linked diubiquitin is stabilized by electrostatic contacts that surround the ubiquitin isopeptide region (Figure 3G). MyUb R1095 forms hydrogen bonds with distal ubiquitin L73 and proximal ubiquitin Q62. Distal ubiquitin R42 and Q49 are spatially close to MyUb N1099 and form hydrogen bonds with its side chain and backbone, respectively. We also explored how MyUb binds to K11-linked diubiquitin taking advantage of K11-linked diubiquitin in which either the proximal or distal ubiquitin was 13C and 15N labeled. We found in a 13C half-filtered NOESY experiment that L8 and I44 from the proximal K11-linked ubiquitin formed similar contacts to MyUb as was observed for the distal K63-linked ubiquitin component (Figure 4A). For example, L8 and I44 from the proximal ubiquitin of K11-linked diubiquitin were similarly shown to directly interact with I1104 from MyUb (Figure 4A). No such intermolecular NOE interactions were observed for L8 or I44 from the distal ubiquitin of K11-linked diubiquitin (data not shown). Furthermore, the I44 and A46 amide signals from the proximal, but not distal, ubiquitin demonstrated significant shifting following addition of equimolar MyUb (Figure 4B, orange versus black). Taken together, our data suggest that MyUb prefers the hydrophobic patch of proximal ubiquitin to distal ubiquitin in the context of K11-linked diubiquitin and that the proximal ubiquitin hydrophobic patch of K11-linked diubiquitin binds to MyUb in a similar mode as that observed for distal ubiquitin of K63-linked diubiquitin (Figures 4C and 3C). Further studies are needed to solve the structure of this MyUb complex, but we concluded that MyUb binds to K11-linked chains in a distinct manner that involves contacts between MyUb I1104 and the L8 and I44 methyl groups (Figures 4A and 4C). Optineurin is a well-characterized myosin VI interactor. Previous work has suggested a direct interaction mediated by the RRL motif in myosin VI and the UBDs of optineurin (Sahlender et al., 2005Sahlender D.A. Roberts R.C. Arden S.D. Spudich G. Taylor M.J. Luzio J.P. Kendrick-Jones J. Buss F. Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis.J. Cell Biol. 2005; 169: 285-295Crossref PubMed Scopus (326) Google Scholar, Shen et al., 2015Shen W.C. Li H.Y. Chen G.C. Chern Y. Tu P.H. Mutations in the ubiquitin-binding domain of OPTN/optineurin interfere with autophagy-mediated degradation of misfolded proteins by a dominant-negative mechanism.Autophagy. 2015; 11: 685-700Crossref PubMed Scopus (99) Google Scholar). Our results suggest that ubiquitin may be part of the myosin VI interaction with this adaptor protein. To investigate this issue, we first analyzed whether optineurin undergoes ubiquitination. HEK293T cells were transfected with GFP-optineurin and HA-ubiquitin and the cell lysate subjected to immunoprecipitation using anti-HA antibody. As visible in Figure 5A, optineurin is poly- or multi-ubiquitinated under these conditions. The lysate was next used for a pull-down assay in which we compared MyUb wild-type (WT) with the ubiquitin-binding-impaired I1104A mutant (Figure 3E). WT MyUb bound strongly to the ubiquitinated form of optineurin, while the I1104A mutant retained only a basal interaction with this autophagic adaptor (Figure 5B). Notably, the I1104A mutant also showed a clear impairment for ubiquitin binding in vivo where ubiquitinated proteins were robustly immunoprecipitated with the WT protein (Figure 5B). Similar results were obtained incubating immunoprecipitated GFP-optineurin with GST proteins eluted from the beads (Figure 5C). Altogether, these results indicate that the ubiquitin-binding surface of the MyUb participates in binding to this RRL interactor, either by direct binding or by binding to ubiquitin that is conjugated to optineurin. We have previously identified an MIU domain that is C-terminal to the myosin VI coiled-coil region (Penengo et al., 2006Penengo L. Mapelli M. Murachelli A.G. Confalonieri S. Magri L. Musacchio A. Di Fiore P.P. Polo S. Schneider T.R. Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin.Cell. 2006; 124: 1183-1195Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) (Figure 6A). We tested whether we could detect binding between a peptide that encompasses the myosin VI MIU (spanning Q998–S1025) and ubiquitin by NMR. Unlabeled ubiquitin was added to 15N-labeled MIU and the effects recorded (Figure 6B). Shifting and disappearance of MIU signals was observed with ubiquitin addition (Figures 6B and S4A), including signals from A1013 and L1014, which were predicted to be at the center of the MIU ubiquitin-binding surface (Penengo et al., 2006Penengo L. Mapelli M. Murachelli A.G. Confalonieri S. Magri L. Musacchio A. Di Fiore P.P. Polo S. Schneider T.R. Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin.Cell. 2006; 124: 1183-1195Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). By contrast, unlabeled MyUb did not affect NMR spectra recorded on 15N MIU (Figure S4B), indicating that these two structural elements do not interact. In support of this finding, the MIU is not significantly different in spectra recorded on the MIU by itself (Q998–S1025) compared to in the context of the MIU-MyUb region (Q998–R1131; Figure S4C), similarly demonstrating that it does not interact with these other regions of the protein. Myosin VI is generated by alternatively spliced isoforms, which are differentially expressed in tissues and cell lines, and associated with specific subcellular compartments (Au et al., 2007Au J.S. Puri C" @default.
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• W2293449161 title "Myosin VI Contains a Compact Structural Motif that Binds to Ubiquitin Chains" @default.
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