Investigating the structure, regulation and interactions of KANK proteins

Many cellular activities rely on interactions between cells and the extracellular matrix (ECM) and these attachments are mediated by the integrin family of transmembrane receptors. Talin, a cytoplasmic adapter protein, activates integrin by binding to the cytoplasmic tails and ultimately couples integrin to the actin cytoskeleton. Once the adhesion has formed, talin can act as a mechanosensitive signalling hub and recruit additional proteins in a force-dependent manner.KANK proteins, via direct interaction with talin and with the kinesin KIF21A, mediate the connection of these integrin-based adhesions with dynamic microtubules. A complex of proteins termed the cortical microtubule stabilising complex (CMSC) is recruited upon talin-KANK interaction which stabilises microtubules in the vicinity of adhesions. The talin-KANK connection results in mechanosensitive crosstalk between the actin and microtubule cytoskeletons, contributes to microtubule polarity, and provides a mechanism for turnover of adhesions during cell migration.In this thesis, a combination of structural, biochemical and biophysical approaches were used to broaden our understanding of KANK proteins. With a recurring focus on identifying similarities and differences between the four mammalian KANK isoforms, particularly within specific domains, we discover some key isoform-specific differences in terms of the ankyrin repeat domains including dimeric propensity that is unique to KANK4, characterise a novel interaction between the KN and ankyrin repeat domains of KANKs and narrow down on the required binding surface on the KN domain, and identify a novel interaction between DYNLL2 and an intrinsically disordered region in KANK1. Additionally, as part of this work, two novel crystal structures of KANK ankyrin repeat domains were solved and solution NMR data providing structural information for DYNLL2 was collected. Ultimately, the work in this thesis explores some novel properties of KANKs and provides the foundations for an array of further study into this family of proteins, allowing the deduction of their structures and functions - either related to their role as linkers between actin filaments and microtubules or potentially in other unrelated and yet undiscovered cellular contexts.