Multiple Sites in αB-Crystallin Modulate Its Interactions with Desmin Filaments Assembled In Vitro

The beta 3- and beta 8-strands and C-terminal residues 155-165 of alpha B-crystallin were identified by pin arrays as interaction sites for various client proteins including the intermediate filament protein desmin. Here we present data using 5 well-characterised alpha B-crystallin protein constructs with substituted beta 3- and beta 8-strands and with the C-terminal residues 155-165 deleted to demonstrate the importance of these sequences to the interaction of alpha B-crystallin with desmin filaments. We used electron microscopy of negatively stained samples to visualize increased interactions followed by sedimentation assays to quantify our observations. A low-speed sedimentation assay measured the ability of alpha B-crystallin to prevent the self-association of desmin filaments. A high-speed sedimentation assay measured alpha B-crystallin cosedimentation with desmin filaments. Swapping the beta 8-strand of alpha B-crystallin or deleting residues 155-165 increased the cosedimentation of alpha B-crystallin with desmin filaments, but this coincided with increased filament-filament interactions. In contrast, substitution of the beta 3-strand with the equivalent alpha A-crystallin sequences improved the ability of alpha B-crystallin to prevent desmin filament-filament interactions with no significant change in its cosedimentation properties. These data suggest that all three sequences (beta 3-strand, beta 8-strand and C-terminal residues 155-165) contribute to the interaction of alpha B-crystallin with desmin filaments. The data also suggest that the cosedimentation of alpha B-crystallin with desmin filaments does not necessarily correlate with preventing desmin filament-filament interactions. This important observation is relevant not only to the formation of the protein aggregates that contain both desmin and alpha B-crystallin and typify desmin related myopathies, but also to the interaction of alpha B-crystallin with other filamentous protein polymers.