LC8 Motif Recognition: Insights from the LC8-Chica Complex

Abstract

LC8 is a dimeric hub protein involved in a large number of interactions central to cell function. It binds short linear motifs - usually containing a Thr-Gln-Thr (TQT) triplet - in intrinsically disordered regions of its binding partners, some of which have several LC8 recognition motifs in tandem. Hallmarks of the motif are a high variability of sequence and of LC8 binding affinity. Human Chica, a spindle adaptor protein, contains a 70-residue segment with four putative recognition motifs but only three reported LC8 binding sites. Here, NMR-derived secondary chemical shifts and relaxation properties show that the Chica LC8 binding domain is essentially disordered with a dynamically restricted segment in one linker between motifs. Calorimetry of LC8 binding to synthetic motif-mimicking peptides shows that the first motif dominates LC8 recruitment. Crystal structures of LC8 bound to each of two motif peptides show highly ordered and invariant TQT-LC8 interactions and more flexible and conformationally variable non-TQT-LC8 interactions. These data add a new and complex LC8 binding partner to the LC8 knowledgebase and provide new insights into the determinants of motif specificity. We propose that LC8 recognition is based on inflexible interactions between LC8 and TQT residues that act as an anchor, coupled with inherent flexibility of interactions between LC8 and non-TQT residues. The ‘anchored flexibility’ model combines highly rigid as well as highly flexible regions of the same binding groove on the surface of LC8.