Anchoring of many cellular cargoes to the motor complex requires the regulatory KLCs that can recognise various adaptor proteins ultimately resulting in relief of autoinhibition and anterograde movement along MTs. A common mechanism of cargo recruitment relies on the recognition of short unstructured regions on cargo adaptor proteins by the tetratricopeptide repeat (TPR) domain of KLCs. Important examples of such ‘recognition tags’ are the internal 'W-acidic' and the C-terminal ‘Y-acidic’ (Trp or Tyr flanked by Asp/Glu residues, respectively) motifs. Whilst W/Y amino acids within their respective motifs play a key role in the recognition process, residues immediately peripheral to the W/Y-acidic core are however also important for affinity modulation and can either potentiate or abolish binding. Furthermore, as W and Y residues do not occupy the same position on the KLC_TPR receptor, the possibility of merging elements of these recognition tags can uncover novel cargo adaptors, as shown by recently developed artificial high-affinity KinTag. In this project the CNR and UniPD units will deploy their complementary expertise in an integrated ML-driven computational and experimental approach aimed at i) rationalizing determinants of sequence variability for ‘W-acidic’ and ‘Y-acidic’ motifs and ii) developing novel small molecule agents that can modulate the interaction of recognition tags with their motor complex KLC_TPR receptors. Overall, this will allow us to develop a complete understanding of the general KLC_TPR-dependent cargo recognition process and to expand the array of putative cargo adaptors by mining, for example, bacterial and viral genomes utilizing a more sophisticated set of sequence determinants. Equally important, we will develop molecular tools that can selectively probe the effects of perturbations in cargo transport.