title: The small heat-shock proteins HSPB2 and HSPB3 form well-defined heterooligomers in a unique 3 to 1 subunit ratio.
abstract: Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from Escherichia coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintaining a strict 3:1 HSPB2/HSPB3 subunit ratio. These complexes are thermally stable up to 40 degrees C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or alphaB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could associate efficiently with HSP20. Taken altogether, this study provides evidence that, despite the high level of sequence homology within the sHSP family the biochemical properties of the HSPB2/B3 complex are distinctly different from those of other sHSPs, indicating that the HSPB2/B3 assembly is likely to possess cellular functions other than those of its family members.
authors.affiliation: Department of Biomolecular Chemistry 271, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: John
authors.initials: J
authors.lastname: den Engelsman
authors.forename: Sandor
authors.initials: S
authors.lastname: Boros
authors.forename: Patricia Y W
authors.initials: PY
authors.lastname: Dankers
authors.forename: Bram
authors.initials: B
authors.lastname: Kamps
authors.forename: Wilma T
authors.initials: WT
authors.lastname: Vree Egberts
authors.forename: Csaba S
authors.initials: CS
authors.lastname: Böde
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lane
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Aquilina
authors.forename: Justin L P
authors.initials: JL
authors.lastname: Benesch
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Wilfried W
authors.initials: WW
authors.lastname: de Jong
authors.forename: Wilbert C
authors.initials: WC
authors.lastname: Boelens
mesh_terms: D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D055551:HSP27 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013499:Surface Properties
chemical_list: D000817:Anilino Naphthalenesulfonates; D055551:HSP27 Heat-Shock Proteins; C545085:HSPB3 protein, rat; D006360:Heat-Shock Proteins; C486700:Hspb2 protein, rat; D021122:Protein Subunits; C027132:1-anilino-8-naphthalenesulfonate