The Mechanism of Dynein Light Chain LC8-mediated Oligomerization of the Ana2 Centriole Duplication Factor [Protein Structure and Folding]

June 11th, 2014 by Slevin, L. K., Romes, E. M., Dandulakis, M. G., Slep, K. C.

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. While recent work 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 Ana2′s central region, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μM) while 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 Ana2′s first LC8 binding site, forming extensive van der Waals contacts and hydrogen bonding with the peptide, while 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 multi-angle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization likely enhances Ana2′s avidity for centriole binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis.