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.
Highlights
anastral spindle-2 (Ana2) is a conserved centriole duplication factor involved in nascent centriole biogenesis
We identified two potential binding sites within Ana2, corresponding to residues 159 – conserved predicted coiled-coil (Fig. 1, E and F) and correlate 168 and 237–246
Ana2 is an integral component of the centriole duplication pathway, but how it works with spindle assembly abnormal protein 6 (Sas-6) and Sas-4 and whether LC8 plays a role in this pathway remain to be determined
Summary
Ana is a conserved centriole duplication factor involved in nascent centriole biogenesis. The function of Ana is poorly understood, recent work has demonstrated that Drosophila Ana interacts with the dynein light chain, LC8 (cut up (Ctp)) [24], a ubiquitous protein that binds diverse targets throughout the cell to confer or potentiate target dimerization (reviewed in Ref. 25). LC8 acts as a processivity factor for the dynein motor by enhancing motor dimerization, it largely plays a dynein motor-independent role throughout the cell to potentiate dimerization of its binding partners (26 –37) It was shown in a yeast two-hybrid screen that LC8 binds two Ana fragments: the first fragment spanning residues 1–200 and the second spanning residues 201–274, which includes a predicted ␣-helix highly conserved across fly species (Fig. 1, E and F) [24, 12]. LC8-potentiated Ana oligomerization has spatial and avidity implications for the Ana N-terminal Sas-4 binding motif and its C-terminal Sas-6-binding STAN domain
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