title: 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.
authors.affiliation: Department of Biochemistry, Tokyo Women's Medical University, Kawada 8-1, Shinjuku, Tokyo 162-8666, Japan. nunomura@gipc.akita-u.ac.jp
authors.forename: Wataru
authors.initials: W
authors.lastname: Nunomura
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sasakura
authors.forename: Kohei
authors.initials: K
authors.lastname: Shiba
authors.forename: Shigeyoshi
authors.initials: S
authors.lastname: Nakamura
authors.forename: Shun-Ichi
authors.initials: S
authors.lastname: Kidokoro
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Takakuwa
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D006801:Humans; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D001059:Apoproteins; D002147:Calmodulin; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C028841:erythrocyte membrane band 4.1 protein; D002118:Calcium