Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein

Zsuzsa Hódi, Attila L. Németh, László Radnai, C. Hetényi, K. Schlett, A. Bodor, A. Perczel, L. Nyitray

Research output: Contribution to journalArticle

47 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)12582-12595
Number of pages14
JournalBiochemistry
Volume45
Issue number41
DOIs
Publication statusPublished - Oct 17 2006

Fingerprint

Nonmuscle Myosin Type IIB
Dyneins
Myosins
Tail
Exons
Myosin Light Chains
Circular Dichroism
Freezing
Nuclear magnetic resonance spectroscopy
Gel Chromatography
Melting point
Assays
Protein Isoforms
Magnetic Resonance Spectroscopy
Gels
Binding Sites
Cells
Cell Line
Peptides
Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein. / Hódi, Zsuzsa; Németh, Attila L.; Radnai, László; Hetényi, C.; Schlett, K.; Bodor, A.; Perczel, A.; Nyitray, L.

In: Biochemistry, Vol. 45, No. 41, 17.10.2006, p. 12582-12595.

Research output: Contribution to journalArticle

@article{4ef9292835f9446d94cfd2f958f91ff9,
title = "Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein",
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.",
author = "Zsuzsa H{\'o}di and N{\'e}meth, {Attila L.} and L{\'a}szl{\'o} Radnai and C. Het{\'e}nyi and K. Schlett and A. Bodor and A. Perczel and L. Nyitray",
year = "2006",
month = "10",
day = "17",
doi = "10.1021/bi060991e",
language = "English",
volume = "45",
pages = "12582--12595",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "41",

}

TY - JOUR

T1 - Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein

AU - Hódi, Zsuzsa

AU - Németh, Attila L.

AU - Radnai, László

AU - Hetényi, C.

AU - Schlett, K.

AU - Bodor, A.

AU - Perczel, A.

AU - Nyitray, L.

PY - 2006/10/17

Y1 - 2006/10/17

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33750054653&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33750054653&partnerID=8YFLogxK

U2 - 10.1021/bi060991e

DO - 10.1021/bi060991e

M3 - Article

VL - 45

SP - 12582

EP - 12595

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 41

ER -