Solution structure and glycophorin C binding studies of the protein 4.1R FERM α‐lobe domain

Abstract

Listen

Translate article

Red blood cell protein 4.1 (4.1R, an 80 kDa isoform),1 one of the major erythrocyte cytoskeletal proteins, forms two major ternary complexes in red blood cells, 4.1Ractin-spectrin and 4.1R-p55-glycophorin C (GPC), and plays important roles in maintaining cell morphology and membrane mechanical properties.1–7 This protein consists of four functional domains: the FERM (4.1 and ezrin/radixin/moesin)8 domain (also termed the N-terminal 30 kDa membrane-binding domain), the 16 kDa regulatory domain, the spectrin-actin-binding (SAB) domain, and the C-terminal 22/24 kDa domain. The 4.1R FERM domain is a multifunctional domain that forms a cloverleaf-like fold containing three structural and functional domains (N-lobe, a-lobe, and C-lobe domains).9 In the 4.1R-p55-GPC ternary complex, the 4.1R FERM a-lobe domain interacts with the N-terminal cytoplasmic domain of GPC, the 4.1R FERM C-lobe10 and/or N-lobe domain11 interacts with the p55 HOOK domain, and the p55 PDZ domain interacts with the C-terminal cytoplasmic domain of GPC.10,12–14 We previously examined the specific binary interaction between the p55 PDZ domain and the C-terminal cytoplasmic domain of GPC by NMR, as the first step to obtain structural insights into the 4.1R-p55-GPC ternary complex.15,16 Our NMR studies clearly revealed how the p55 PDZ domain interacts with two key C-terminal residues, Tyr126 and Ile128, of GPC at the atomic level.16 We have now focused on the binary interaction between the 4.1R FERM a-lobe domain and the N-terminal cytoplasmic domain of GPC, as the second step toward our goal. In the binary interaction between 4.1R and GPC, the 4.1R-binding site on GPC reportedly corresponds to the Nterminal 12 amino acid residues of its cytoplasmic domain (residues 82–93), and the RHK (residues Arg86-His87Lys88) motif is probably crucial for binding the 4.1R FERM a-lobe domain.13 In contrast, the GPC-binding site on the 4.1R FERM domain reportedly corresponds to the sequence encoded by exon 8 (residues Tyr94-Arg166) in the 4.1R alobe domain, and the ELEE motif (residues Glu153Glu156) is considered to be a good candidate for the GPC-binding site.9,10 However, there is no experimental evidence that the ELEE motif contributes to the interaction with the N-terminal cytoplasmic domain of GPC. To better understand this interaction mode, we prepared the 4.1R FERM a-lobe domain (residues Asp83-Tyr187) and performed an NMR analysis of this domain with the N-terminal cytoplasmic domain of GPC. Here, we report the first NMR-derived structure of the 4.1R FERM a-lobe domain and propose a new GPC-binding site on the 4.1R FERM a-lobe domain, based on our NMR experiments.