Mapping of a spectrin-binding domain of human erythrocyte membrane protein 4.2

Abstract

Protein 4.2 is a major component of the red blood cell membrane skeleton. Deficiency of protein 4.2 is linked with a variety of hereditary haemolytic anaemias. However, the interactions of protein 4.2 with other proteins of the erythrocyte membrane remain poorly understood. The major membrane-binding site for protein 4.2 resides on the cytoplasmic domain of band 3. Protein 4.2 interacts directly with spectrin in solution, suggesting that it stabilizes interactions between the membrane skeleton and the erythrocyte membrane. A 30 kDa polypeptide, with its N-terminus corresponding to amino acid residue 269, derived by partial proteolysis of protein 4.2, was found to interact with biotinylated spectrin in gel renaturation assays. A series of overlapping glutathione S-transferase fusion peptides were constructed, and an alpha-helical domain encompassing residues 470-492 was found to be instrumental in mediating protein 4.2-spectrin interactions. Direct binding of a synthetic peptide, with the sequence corresponding to residues 470-492, to spectrin and the ability of the peptide to inhibit spectrin binding of protein 4.2 confirmed that these residues are crucial in mediating protein 4.2-spectrin interactions.

The carboxyterminal EF domain of erythroid α-spectrin is necessary for optimal spectrin-actin binding

“…The deletion removes the final ␣-helix of EF 4 . This helix interacts intimately with the initial ␣-helix of EF 3 in the nuclear magnetic resonance structure of EF 34 from ␣-actinin, 3 so it is very probable that the deletion disrupts the overall structure of EF 34 in ␣-spectrin. This is consistent with the CD spectra, which show a significant difference between ␣18-21EF and ␣18-21EF⌬13 ( Figure 1B).…”

“…And, as noted, we find that the EF domain binds protein 4.2. 20 The 22-amino acid segment in protein 4.2 that binds spectrin (amino acids 34 lies on the surface of protein 4.2 35 and is probably helical according to the structure predicting algorithms indicated in "Structure prediction." As shown in Figure 7C, it is similar to the CH2-R1 linker peptide, also predicted to be ␣-helical, along 2 faces of the helix.…”

“…Actin and protein 4.1R bind to calponin homology (CH) domains in the actin-binding domain (ABD) at the N-terminus of the spectrin ␤-chain. 1 The adjacent, C-terminal end of ␣-spectrin, called the EF domain, contains an N-terminal pair of calmodulin-like, Ca 2ϩ -responsive EF hands, termed EF 12 , that bind Ca 2ϩ with affinities in the low millimolar range, 2 and a C-terminal pair of Ca 2ϩ -insensitive EF hands, EF 34 , similar to those in ␣-actinin, 3 which extend to the C-terminus of the ␣-spectrin protein. Because Ca 2ϩ is not bound at the micromolar concentrations that exist inside red cells and because Ca 2ϩ has no apparent effect on the binding of erythrocyte spectrin to actin, the EF domain is generally assumed to be inert and vestigial in red cells.…”

Protein 4.2 Binds to the Carboxyl-terminal EF-hands of Erythroid α-Spectrin in a Calcium- and Calmodulin-dependent Manner

“…However, the binding affinity of GST-␣EF for 4.2-G 3 was about 14-fold less than its affinity for full-length, native protein 4.2 ( Fig. 3) and about 6 times less than the published affinity of the 4.2-G 3 peptide for full-length, native spectrin (18). This suggests that other sites on protein 4.2 and spectrin or unknown post-translational modifications of …”

“…Table 1. Spectrin GST fusion proteins and the G 3 spectrin-binding peptide from protein 4.2 (18) were generated by PCR amplification of cDNA clones from the human spectrin A-I (SPTA1) sequence GST-␣EF 45,182 18,197 6970-7440 2262-2418 pGEX-6p PreScission a GST-␣EF⌬13 43,653 16,668 6970-7402 2262-2405 pGEX-6p PreScission GST-␣18-21EF 97,615 70,630 5603-7440 1806-2418 pGEX-6p PreScission GST-␣18-21EF⌬13 96,090 69,105 5603-7402 1806-2405 pGEX-6p PreScission GST-␤ABD1-4 113 TM accession no. M29399).…”