Alternatively Spliced Exon B of Myosin Va Is Essential for Binding the Tail-Associated Light Chain Shared by Dynein ² Zsuzsa Ho ´di, ‡ Attila L. Ne ´meth, ‡ La ´szlo ´ Radnai, ‡ Csaba Hete ´nyi, ‡ Katalin Schlett, § Andrea Bodor, | Andra ´s Perczel, ⊥ and La ´szlo ´ Nyitray* ,‡ Departments of Biochemistry, Physiology and Neurobiology, Theoretical Chemistry, and Organic Chemistry, Eo ¨tVo ¨s Lora ´ nd UniVersity, Budapest, Hungary ReceiVed May 18, 2006; ReVised Manuscript ReceiVed July 31, 2006 ABSTRACT: A 10 kDa dynein light chain (DLC), previously identified as a tail light chain of myosin Va, may function as a cargo-binding and/or regulatory subunit of both myosin and dynein. Here, we identify and characterize the binding site of DLC on myosin Va. Fragments of the human myosin Va tail and the DLC2 isoform were expressed, and their complex formation was analyzed by pull-down assays, gel filtration, and spectroscopic methods. DLC2 was found to bind as a homodimer to a ∼15 residue segment (Ile1280-Ile1294) localized between the medial and distal coiled-coil domains of the tail. The binding region contains the three residues coded by the alternatively spliced exon B (Asp1284-Lys1286). Removal of exon B eliminates DLC2 binding. Co-localization experiments in a transfected mammalian cell line confirm our finding that exon B is essential for DLC2 binding. Using circular dichroism, we demonstrate that binding of DLC2 to a ∼85 residue disordered domain (Pro1235-Arg1320) induces some helical structure and stabilizes both flanking coiled-coil domains (melting temperature increases by ∼7 °C). This result shows that DLC2 promotes the assembly of the coiled-coil domains of myosin Va. Nuclear magnetic resonance spectroscopy and docking simulations show that a 15 residue peptide (Ile1280-Ile1294) binds to the surface grooves on DLC2 similarly to other known binding partners of DLCs. When our data are taken together, they suggest that exon B and its associated DLC2 have a significant effect on the structure of parts of the coiled-coil tail domains and such a way could influence the regulation and cargo-binding function of myosin Va. Class V myosins are involved in the short-range intracel- lular transport of vesicles and molecules mostly in the actin- rich cortical region of the cell (for a review, see ref 1). They are processive actin-based motors moving by a “hand-over- hand” mechanism along actin filaments (2, 3). Mammalian genomes contain three myosin V heavy-chain genes: MYO5A, MYO5B, and MYO5C. Mutations of MYO5A are respon- sible for the dilute phenotype and Griscelli syndrome type 1 in mice and humans, respectively (4, 5). Myosin Va (myoVa) 1 is composed of two heavy chains, six calmodulins (one or two could be substituted by essential light chains) associated with the neck region, and a 10 kDa light chain, originally identified as a light chain of dynein (called DLC; alternative names are LC8, DLC8, and PIN) bound to the tail region (6, 7). The tail region contains coiled-coil dimerizing domains interrupted with noncoiled-coil regions and a C-terminal globular tail domain (GTD) involved in cargo binding (8, 9). At least six isoforms of myoVa are expressed, partially tissue specifically, by alternative splicing of three exons (exons B, D, and F) in the tail region. An isoform that is expressed abundantly in the brain includes only exon B, while a mainly melanocyte-specific isoform includes exons D and F (10-12). Exon F together with GTD was found to bind to melanosomes via the adaptor protein melanophilin (13-15). An isoform containing only exon D associates with vesicles near the Golgi area (16). No function for exon B has been assigned thus far. The regulation of the ATPase and the motor activity of myoVa are thought to be achieved by Ca +2 binding to the calmodulin (CaM) subunits and folding back the globular tail domain toward the heads (17-21). DLC was first described as a light-chain component of the Chlamydomonas outer dynein arm and later a component of mammalian flagellar and cytoplasmic dyneins (22, 23). In the multisubunit cargo-binding tail region of the dynein motor, it is associated with the intermediate chain (24). DLC is a highly conserved protein, with >90% sequence identity between any two orthologues (7, 25). Mammalian species contain two DLC genes (DYNLL1 and DYNLL2; the proteins referred to here as DLC1 and DLC2). DLCs were found to bind to a diverse array of proteins and even to RNAs ² This work was supported by OTKA (Hungarian Scientific Research Fund) Grants T43746, K61784, TS049812, TS044711 (to L.N.), and T46994 (to A.P.). * To whom correspondence should be addressed: Department of Biochemistry, Eo ¨tvo ¨s Lora ´nd University, Pa ´zma ´ny P.s. 1/c, Budapest, Hungary, H-1117. Telephone: 36-1381-2171. Fax: 36-1381-2172. E-mail: nyitray@cerberus.elte.hu. ‡ Department of Biochemistry. § Department of Physiology and Neurobiology. | Department of Theoretical Chemistry. ⊥ Department of Organic Chemistry. 1 Abbreviations: CaM, calmodulin; CD, circular dichroism; DBD, DLC2 binding domain of myosin Va; DSS, sodium 4,4-dimethyl-4- silapentane-1-sulphonate; GFP, green-fluorescent protein; GST, glu- tathione-S-transferase; myoVa, myosin Va; nNOS, neuronal nitric oxide synthase; PBS, phosphate-buffered saline; rmsd, root-mean-square deviation; TFE, trifluoroethanol; TCEP, tris(2-carboxyethyl)phosphine; -ME, 2-mercaptoethanol. 12582 Biochemistry 2006, 45, 12582-12595 10.1021/bi060991e CCC: $33.50 © 2006 American Chemical Society Published on Web 09/22/2006