A Fused α-β “Mini-spectrin” Mimics the Intact Erythrocyte Spectrin Head-to-head Tetramer

Sandra L Harper,Donghai Li,Yelena Maksimova,Patrick G Gallagher,David W Speicher

doi:10.1074/jbc.m109.083048

Sandra L Harper, Donghai Li + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.083048

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Abstract

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.

Highlights

Erythrocyte spectrin is a major component of the red cell membrane cytoskeleton, a two-dimensional latticework of proteins associated with the cytoplasmic face of the red cell membrane
We recently showed that most hereditary elliptocytosis/hereditary pyropoikilocytosis mutations located in the ␣0 domain exhibited either decreased tetramer assembly or no detectable tetramer formation when a tetramer binding assay involving formation of univalent head-to-head complexes was used [15]
Design and Expression of a Minispectrin Molecule—Fig. 1A shows a model of human red cell spectrin in its closed dimer, open dimer, and tetramer forms and the dynamic equilibrium between the two dimer states and tetramers

Summary

EXPERIMENTAL PROCEDURES

Construction and Expression of the Mini-spectrin Expression Plasmid—The following fused ␣-␤ mini-spectrin recombinant protein was constructed for use in this study: ␣0 –5 (repeats 0 –5 of the human red cell ␣-spectrin subunit, residues 1–584) followed by a flexible glycine linker with a factor Xa cleavage site inserted in the middle consisting of the sequence GGGGGGIEGRGGGGGG followed by ␤16 –17 (repeats 16 and 17 of the human red cell ␤-spectrin subunit, residues 1902–2080). Sample was concentrated and purified by HPLC gel filtration on a HiLoad 200 column (GE Healthcare) in 10 mM sodium phosphate, 130 mM NaCl, 1 mM EDTA, 0.15 mM PMSF, 1 mM ␤-mercaptoethanol, pH 7.4. Analytical Gel Filtration—Mini-spectrin oligomers, tetramers, open dimers, and closed dimers were evaluated using analytical gel filtration using two BioAssist G4000SWXL (Tosoh Corp., Tokyo, Japan) columns connected in series, equilibrated in 20 mM sodium phosphate, 130 mM NaCl, 1 mM EDTA, 0.15 mM PMSF, 1 mM ␤-mercaptoethanol pH 7.4. Cross-linking Reactions—Cross-linking reactions using 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/sulfoN-hydroxysulfosuccinimide (sulfo-NHS) were performed using 5 ␮l of a freshly prepared aqueous cross-linker solution added to 300 ␮l of mini-spectrin dimer (0.38 mg/ml) in phosphate-buffered saline. With manual de novo sequencing were used to identify the crosslinked peptides and the amino acid residues involved in forming the cross-link

RESULTS

DISCUSSION

Mass error