αβ Spectrin Coiled Coil Association at the Tetramerization Site

Abstract

On the basis of sequence homology studies, it has been suggested that the association of human erythrocytes alpha and beta spectrin at the tetramerization site involves interactions between helices. However, no empirical details are available, presumably due to the experimental difficulties in studying spectrin molecules because of its size and/or its structural flexibility. It has been speculated that erythrocyte tetramerization involves helical bundling rather than coiled coil association. We have used recombinant spectrin peptides to model alpha and beta spectrin to study their association at the tetramerization site. Two alpha peptides, Sp alpha 1-156 and Sp alpha 1-368, and one beta peptide, Sp beta 1898-2083, were used as model peptides to demonstrate the formation of the alpha beta complex. We also found that the replacement of R28 in Sp alpha 1-368 to give Sp alpha 1-368R28C abolished complex formation with the beta peptide. Circular dichroism techniques were used to monitor the secondary structures of the individual peptides and of the complex, and the results showed that both Sp alpha 1-156 and Sp beta 1898-2083 peptides in solution, separately, included helices that were not paired with other helices in the absence of their binding partners. However, in a mixture of Sp alpha 1-156 and Sp beta 1898-2083 and formation of the alpha beta complex, the unpaired helices associated to form coiled coils. Since the sequences of these two peptides that are involved in the coiled coil association are derived from a native protein, the information obtained from this study also provides insight toward a better understanding of naturally occurring coiled coil subunit-subunit association.

Mutation of a highly conserved isoleucine disrupts hydrophobic interactions in the αβ spectrin self-association binding site

“…2 Refinements and phasing of the model have been made as determination of the primary structure, 3,4 X-ray, nuclear magnetic resonance, and spectroscopic studies of spectrin have been completed. 28,[35][36][37][38][39][40][41][42][43] These studies predict that the first and third helices are parallel and the second helix is antiparallel. The amphipathic repeats are stabilized by hydrophobic interactions of each repeat at the interior core of each repeat.…”

Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin

“…18,20 The association affinity of each mutant peptide with Sp␤ was assessed qualitatively by K d of the Arg28/Arg45-Sp␤ (␣␤) complex obtained from the results of gel-filtration measurements. Although K d values obtained by gel-filtration methods do not provide absolute quantitative values for affinities, they are a rapid and simple means for good comparison of relative affinities.…”

“…Amino acid replacements at positions 28 and 45 of ␣-spectrin were introduced into an extensively studied model peptide, Sp␣1-156, 15,18,19 to give Sp␣1-156Arg28Ser (Arg28Ser), Sp␣1-156Arg45Ser (Arg45Ser), and Sp␣1-156Arg45Thr (Arg45Thr). The association of these ␣-spectrin mutant peptides with a model peptide of ␤-spectrin, Sp␤1898-2083 (Sp␤), 20 was determined, and structural changes due to mutations were monitored by nuclear magnetic resonance (NMR). These 2 ␣-and ␤-peptides were chosen to mimic the N-terminal end of ␣-spectrin and the C-terminal end of ␤-spectrin, respectively.…”

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

“…General structural prediction shows that each of these two proteins consists of the last structural domain and the C-terminal partial domain. 40,41 The K d values for the complexes of aI-N1 with either bI-C1 or bII-C1 are $1 lM, whereas the values for the complexes of aII-N1 with either bI-C1 or bII-C1 are $10 nM. 15,16 Thus, the affinity differences in tetramer formation between spectrin I and II are mostly attributable to the conformational differences between aI and aII.…”