title: Multiple sites in αB-crystallin modulate its interactions with desmin filaments assembled in vitro.
abstract: The β3- and β8-strands and C-terminal residues 155-165 of αB-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 αB-crystallin protein constructs with substituted β3- and β8-strands and with the C-terminal residues 155-165 deleted to demonstrate the importance of these sequences to the interaction of αB-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 αB-crystallin to prevent the self-association of desmin filaments. A high-speed sedimentation assay measured αB-crystallin cosedimentation with desmin filaments. Swapping the β8-strand of αB-crystallin or deleting residues 155-165 increased the cosedimentation of αB-crystallin with desmin filaments, but this coincided with increased filament-filament interactions. In contrast, substitution of the β3-strand with the equivalent αA-crystallin sequences improved the ability of αB-crystallin to prevent desmin filament-filament interactions with no significant change in its cosedimentation properties. These data suggest that all three sequences (β3-strand, β8-strand and C-terminal residues 155-165) contribute to the interaction of αB-crystallin with desmin filaments. The data also suggest that the cosedimentation of αB-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 αB-crystallin and typify desmin related myopathies, but also to the interaction of αB-crystallin with other filamentous protein polymers.
authors.affiliation: Department of Biological Structure, University of Washington, Seattle, Washington, United States of America.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Houck
authors.forename: Andrew
authors.initials: A
authors.lastname: Landsbury
authors.forename: John I
authors.initials: JI
authors.lastname: Clark
authors.forename: Roy A
authors.initials: RA
authors.lastname: Quinlan
mesh_terms: D001665:Binding Sites; D003459:Crystallins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003893:Desmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D046529:Microscopy, Electron, Transmission; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding
chemical_list: D003459:Crystallins; D003893:Desmin
references.citation: PLoS One. 2007 Jun 06;2(6):e498
references.pmid: 17551579
references.citation: J Biol Chem. 2008 Apr 18;283(16):10500-12
references.pmid: 18230612
references.citation: Am J Pathol. 1999 May;154(5):1563-72
references.pmid: 10329608
references.citation: Cell Stress Chaperones. 2006 Summer;11(2):187-97
references.pmid: 16817325
references.citation: J Protein Chem. 2001 Feb;20(2):123-30
references.pmid: 11563692
references.citation: Cell Stress Chaperones. 2007 Summer;12(2):163-71
references.pmid: 17688195
references.citation: Nat Struct Biol. 2001 Dec;8(12):1025-30
references.pmid: 11702068
references.citation: J Mol Biol. 1999 Jun 4;289(2):397-411
references.pmid: 10366513
references.citation: Anal Chem. 2009 Mar 1;81(5):1865-71
references.pmid: 19178288
references.citation: Adv Exp Med Biol. 2008;642:131-64
references.pmid: 19181099
references.citation: J Biol Chem. 2000 Feb 11;275(6):3767-71
references.pmid: 10660525
references.citation: J Clin Invest. 2009 Jul;119(7):1806-13
references.pmid: 19587455
references.citation: Biochemistry. 2006 Nov 21;45(46):13847-54
references.pmid: 17105203
references.citation: Nat Med. 1999 Jan;5(1):25-6
references.pmid: 9883834
references.citation: Int J Biochem Cell Biol. 2007;39(10):1804-15
references.pmid: 17590381
references.citation: Mol Biol Cell. 2004 May;15(5):2335-46
references.pmid: 15004226
references.citation: J Cell Sci. 1999 Jul;112 ( Pt 13):2099-112
references.pmid: 10362540
references.citation: Prog Mol Subcell Biol. 2002;28:219-33
references.pmid: 11908062
references.citation: Int J Biochem Cell Biol. 2008;40(5):954-67
references.pmid: 18162431
references.citation: J Mol Biol. 2008 Jan 25;375(4):1026-39
references.pmid: 18062989
references.citation: Nat Struct Mol Biol. 2010 Sep;17(9):1037-42
references.pmid: 20802487
references.citation: Protein Sci. 2005 Mar;14(3):684-95
references.pmid: 15722445
references.citation: Circulation. 2007 Mar 13;115(10):1244-51
references.pmid: 17325244
references.citation: Biochemistry. 2007 May 29;46(21):6308-17
references.pmid: 17487982
references.citation: Nat Genet. 1998 Sep;20(1):92-5
references.pmid: 9731540
references.citation: Protein Sci. 2010 May;19(5):1031-43
references.pmid: 20440841
references.citation: Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13272-7
references.pmid: 19651604
references.citation: Int J Biol Macromol. 1998 May-Jun;22(3-4):197-209
references.pmid: 9650074
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11312-7
references.pmid: 9736733
references.citation: J Biol Chem. 1999 Nov 19;274(47):33235-43
references.pmid: 10559197
references.citation: Biochemistry. 2006 Aug 15;45(32):9878-86
references.pmid: 16893188
references.citation: J Mol Biol. 1998 Mar 20;277(1):27-35
references.pmid: 9514758
references.citation: J Cell Biol. 2001 Nov 26;155(5):747-54
references.pmid: 11724817
references.citation: Biochemistry. 2003 Oct 14;42(40):11857-63
references.pmid: 14529298
references.citation: EMBO J. 1994 Feb 15;13(4):945-53
references.pmid: 7906647