A fused alpha-beta "mini-spectrin" mimics the intact erythrocyte spectrin head-to-head tetramer.
Head-to-head assembly of two spectrin heterodimers to form an actin-cross-linking tetramer is a physiologically dynamic interaction that contributes to red cell membrane integrity. Recombinant beta-spectrin C-terminal and alpha-spectrin N-terminal peptides can form tetramer-like univalent complexes, but they cannot evaluate effects of the open-closed dimer interactions or lateral associations of the two-spectrin strands on tetramer formation. In this study we produced and characterized a fused "mini-spectrin dimer" containing the beta-spectrin C-terminal region linked to the alpha-spectrin N-terminal region. This fused mini-spectrin mimics structural and functional properties of intact, full-length dimers and tetramers, including lateral association of the alpha and beta subunits in the dimer and formation of a closed dimer. High performance liquid chromatography gel filtration analyses of this mini-spectrin provide the first direct non-imaging experimental evidence for open and closed spectrin dimers and show that dimer-tetramer-oligomer interconversion is slow at low temperatures and accelerated at 30 degrees C, analogous to full-length spectrin. This protein exhibits wild type dimer-tetramer dissociation constants of approximately 1 mum at 30 degrees C, independent of initial oligomeric state. Conformational states of the mini-spectrin dimer were probed further using chemical cross-linking, which identified distinct groups of cross-links for "open" and "closed" dimers and confirmed the N-terminal region of alpha-spectrin remains highly flexible in the complex, exhibiting closely analogous structures to those observed for the isolated alpha-spectrin N-terminal using NMR (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844). This fusion protein should serve as a useful template for structural and functional studies of the divalent tetramer site.
Harper
Sandra L
SL
Center for Systems and Computational Biology, The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104, USA. speicher@wistar.org
Li
Donghai
D
Maksimova
Yelena
Y
Gallagher
Patrick G
PG
Speicher
David W
DW
D032701:Biomimetics; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D055503:Protein Multimerization; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D013049:Spectrin