Molecular basis of dystrobrevin interaction with kinesin heavy chain: structural determinants of their binding.

Dystrobrevins are a family of widely expressed dystrophin-associated proteins that comprises alpha and beta isoforms and displays significant sequence homology with several protein-binding domains of the dystrophin C-terminal region. The complex distribution of the multiple dystrobrevin isoforms suggests that the variability of their composition may be important in mediating their function. We have recently identified kinesin as a novel dystrobrevin-interacting protein and localized the dystrobrevin-binding site on the cargo-binding domain of neuronal kinesin heavy chain (Kif5A). In the present study, we assessed the kinetics of the dystrobrevin-Kif5A interaction by quantitative pull-down assay and surface plasmon resonance (SPR) analysis and found that beta-dystrobrevin binds to kinesin with high affinity (K(D) approximately 40 nM). Comparison of the sensorgrams obtained with alpha and beta-dystrobrevin at the same concentration of analyte showed a lower affinity of alpha compared to that of beta-dystrobrevin, despite their functional domain homology and about 70% sequence identity. Analysis of the contribution of single dystrobrevin domains to the interaction revealed that the deletion of either the ZZ domain or the coiled-coil region decreased the kinetics of the interaction, suggesting that the tertiary structure of dystrobrevin may play a role in regulating the interaction of dystrobrevin with kinesin. In order to understand if structural changes induced by post-translational modifications could affect dystrobrevin affinity for kinesin, we phosphorylated beta-dystrobrevin in vitro and found that it showed reduced binding capacity towards kinesin. The interaction between the adaptor/scaffolding protein dystrobrevin and the motor protein kinesin may play a role in the transport and targeting of components of the dystrophin-associated protein complex to specific sites in the cell, with the differences in the binding properties of dystrobrevin isoforms reflecting their functional diversity within the same cell type. Phosphorylation events could have a regulatory role in this context.