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

The b3and b8-strands and C-terminal residues 155–165 of aB-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 aB-crystallin protein constructs with substituted b3and b8-strands and with the C-terminal residues 155–165 deleted to demonstrate the importance of these sequences to the interaction of aB-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 aB-crystallin to prevent the self-association of desmin filaments. A high-speed sedimentation assay measured aB-crystallin cosedimentation with desmin filaments. Swapping the b8-strand of aB-crystallin or deleting residues 155–165 increased the cosedimentation of aB-crystallin with desmin filaments, but this coincided with increased filament-filament interactions. In contrast, substitution of the b3-strand with the equivalent aA-crystallin sequences improved the ability of aB-crystallin to prevent desmin filament-filament interactions with no significant change in its cosedimentation properties. These data suggest that all three sequences (b3strand, b8-strand and C-terminal residues 155–165) contribute to the interaction of aB-crystallin with desmin filaments. The data also suggest that the cosedimentation of aB-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 aB-crystallin and typify desmin related myopathies, but also to the interaction of aB-crystallin with other filamentous protein polymers. Citation: Houck SA, Landsbury A, Clark JI, Quinlan RA (2011) Multiple Sites in aB-Crystallin Modulate Its Interactions with Desmin Filaments Assembled In Vitro. PLoS ONE 6(11): e25859. doi:10.1371/journal.pone.0025859 Editor: Laurent Kreplak, Dalhousie University, Canada Received December 21, 2010; Accepted September 13, 2011; Published November 9, 2011 Copyright: 2011 Houck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Grant EY04542 from the National Eye Institute (http://www.nei.nih.gov/), BBSRC (BB/E527898 http://www.bbsrc.ac.uk/) and Leverhulme Trust (RF0546: http://www.leverhulme.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (RAQ); [email protected] (JIC) . These authors contributed equally to this work.