The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor

J Biol Chem. 2014 Jul 25;289(30):20727-39. doi: 10.1074/jbc.M114.576041. Epub 2014 Jun 11.


Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ~34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis.

Keywords: Centriole; Centrosome; Cytoskeleton; Dynein; Isothermal Titration Calorimetry (ITC); Protein Complex; Protein Structure; Structural Biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Cell Cycle Proteins
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster
  • Dyneins / chemistry*
  • Dyneins / genetics
  • Dyneins / metabolism
  • Hydrogen Bonding
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Protein Binding
  • Protein Multimerization / physiology*
  • Protein Structure, Quaternary


  • Ana2 protein, Drosophila
  • Cell Cycle Proteins
  • Drosophila Proteins
  • Multiprotein Complexes
  • ctp protein, Drosophila
  • Dyneins

Associated data

  • PDB/2PG1
  • PDB/3DVP
  • PDB/3ZKE