Structural stabilization of protein 4.1R FERM domain upon binding to apo-calmodulin: novel insights into the biological significance of the calcium-independent binding of calmodulin to protein 4.1R

Abstract

In erythrocytes, 4.1R80 (80 kDa isoform of protein 4.1R) binds to the cytoplasmic tail of the transmembrane proteins band 3 and GPC (glycophorin C), and to the membrane-associated protein p55 through the N- (N-terminal), α- (α-helix-rich) and C- (C-terminal) lobes of R30 [N-terminal 30 kDa FERM (4.1/ezrin/radixin/moesin) domain of protein 4.1R] respectively. We have shown previously that R30 binds to CaM (calmodulin) in a Ca2+-independent manner, the equilibrium dissociation constant (Kd) for R30-CaM binding being very similar (in the submicromolar range) in the presence or absence of Ca2+. In the present study, we investigated the consequences of CaM binding on R30's structural stability using resonant mirror detection and FTIR (Fourier-transform IR) spectroscopy. After a 30 min incubation above 40° C, R30 could no longer bind to band 3 or to GPC. In contrast, R30 binding to p55, which could be detected at a temperature as low as 34° C, was maintained up to 44° C in the presence of apo-CaM. Dynamic light scattering measurements indicated that R30, either alone or complexed with apo-CaM, did not aggregate up to 40° C. FTIR spectroscopy revealed that the dramatic variations in the structure of the β-sheet structure of R30 observed at various temperatures were minimized in the presence of apo-CaM. On the basis of Kd values calculated at various temperatures, ΔCp and ΔG° for R30 binding to apo-CaM were determined as -10 kJ · K(-1) · mol-1 and ~ -38 kJ · mol(-1) at 37° C (310.15 K) respectively. These data support the notion that apo-CaM stabilizes R30 through interaction with its β-strand-rich C-lobe and provide a novel function for CaM, i.e. structural stabilization of 4.1R80.

Light Scattering: a Novel Approach to Analyze Protein 4.1R FERM Domain Interaction with Inside-out-vesicles in Solution

“…After enzymatic removal of the GST tag, R30 was further purified on heparin-Sepharose and Sephacryl S-100 as previously described. 15 Concentration of R30 (1.2 μM) was determined by absorption measurement at A280. 15 Fresh human erythrocytes were collected from healthy adult male volunteers at Tokyo Women's Medical University after obtaining informed consent.…”

“…15 Concentration of R30 (1.2 μM) was determined by absorption measurement at A280. 15 Fresh human erythrocytes were collected from healthy adult male volunteers at Tokyo Women's Medical University after obtaining informed consent. IOVs depleted of all peripheral proteins by treatment with a pH 11 solution were prepared as described in our previous report and based on the original method by Danilov et al 7,8,16 The concentration of IOVs (5.5 mg/ml) was determined by Bradford assay using a commercial kit (Pierce, USA).…”

【Original Contribution】 Characterization of Mouse Monoclonal Antibodies to Human Protein 4.1R FERM Domain: Epitope Mapping and Application to FERM Domain Binding to Red Blood Cell Inside-out Vesicles

“…Preparation of full length and various fragments of human 4.1R, including the N-terminal 209 amino acid head-piece region (HP), the FERM domain (R30), HP+R30, the 16-kDa domain, the 10-kDa domain and the C-terminal 22/24-kDa domain (CTD) (Fig. 1A), as well as that of human ezrin FERM domain, was performed as previously described 17) . Concentrations of R30 were determined as previously described 17) and those of IgG were determined by measuring absorbance at 280 nm (A280) with an extinction coefficient of E 1% = 15.…”

“…1A), as well as that of human ezrin FERM domain, was performed as previously described 17) . Concentrations of R30 were determined as previously described 17) and those of IgG were determined by measuring absorbance at 280 nm (A280) with an extinction coefficient of E 1% = 15. Single exon deletion in R30 cDNA was carried out as previously described 9,15) .…”

“…GST was cleaved with thrombin and R30 was further purified sequentially on a heparin Sepharose and a Sephacryl S-100 column 17) . Preparation of full length and various fragments of human 4.1R, including the N-terminal 209 amino acid head-piece region (HP), the FERM domain (R30), HP+R30, the 16-kDa domain, the 10-kDa domain and the C-terminal 22/24-kDa domain (CTD) (Fig.…”