title: The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor.
abstract: 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.
authors.affiliation: From the Departments of Biology and.
authors.forename: Lauren K
authors.initials: LK
authors.lastname: Slevin
authors.affiliation: Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Romes
authors.affiliation: From the Departments of Biology and.
authors.forename: Mary G
authors.initials: MG
authors.lastname: Dandulakis
authors.affiliation: From the Departments of Biology and kslep@bio.unc.edu.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Slep
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary
chemical_list: C547835:Ana2 protein, Drosophila; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D046912:Multiprotein Complexes; C542608:ctp protein, Drosophila; D004398:Dyneins
keywords: Centriole; Centrosome; Cytoskeleton; Dynein; Isothermal Titration Calorimetry (ITC); Protein Complex; Protein Structure; Structural Biology