Structural dynamism and analysis of functional domain of membrane proteins in signal transduction.

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(1)In vitro protein binding assays by the resonant mirror detection method (IAsys^<TM>) identified two distinct calmodulin (CaM) binding sites within the NH_2-terminal 30kDa domain of erythrocyte protein 4.1 (4.1R^<80>) : a Ca^<2+>-sensitive and -insensitive binding sites. CaM bound to 4.1R^<80> at a stoichiometry of 1:1 both in the presence and absence of Ca^<2+>. Ca^<2+> and CaM regulated interactions of 4.1R^<80> with membrane proteins, Glycophorin C (GPC), Band 3 and p55. This regulation required binding of CaM to both Ca^<2+>-sensitive and -insensitive sites in 4.1R^<80>.(2)Little is known regarding the molecular basis for the interaction of 4.1R^<80> with either GPC or p55 and regarding the role of 4.1R^<80> in regulating the various protein-protein interactions that constitute the GPC-4.1R^<80>-p55 ternary complex. We showed that 4.1R^<80> increases the affinity of p55 binding to GPC by an order of magnitude, implying that4.1R^<80> modulates the interaction between p55 and GPC.(3)The crystal structure of 30kDa domain of 4.1R^<80> has been determined and shows a cloverleaf-like architecture. Each lobe of the cloverleaf contains a specific binding site for Band 3, GPC or p55. At a central region of the molecule near where the three lobes are joined are two separate CaM binding regions.(4)Another isoform of 4.1R, 135kDa 4.1R (4.1R^<135>) is expressed in erythroblasts. 4.1R^<135> has an additional 209 amino acids polypeptide, referred to as head-piece (HP), upstream of the NH_2-terminal end of the 30kDa domain. Our results support that HP in 4.1R^<135> may regulate the 30kDa domain binding to membrane in erythroblasts.(5)The protein 4.1G, which is an isoform of 4.1R, is also expressed in erythroblasts. Our experiments support that the membrane binding properties of 4.1G are different from those of 4.1R^<135>, suggesting that 4.1G and 4.1R^<135> may share different functions in erythroblasts.