Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening.

Abstract

Abstract. Mechanical strength Of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function.T HE erythrocyte is among the simplest and best characterized of cellular systems within which the chemistry of composition can be intricately related to structural function. Indeed, most of the red cell's mechanical behavior, except for limited aspects of cell shape, appears to depend little on the complexities of intracellular metabolism (14). However, to withstand the forces of blood flow over a several-month lifespan, the erythrocyte's membrane components must assemble into a highly durable structure. Physical limits to this durability are set by the system's mechanochemistry, which is defined here as those molecular interactions that permit a pliable, yet resilient structure.Spectrin, actin, and protein 4.1 are the principal structural proteins of the thin, cohesive skeletal network that underlies the red cell's lipid bilayer (3,5,19). The network formed by these highly conserved and pervasive proteins confers resilience and durability to the red cell's composite membrane (14,16,36,44,46). Mixtures of these network proteins also show that they can form very stable complexes in vitro (37) which at approximately micromolar protein concentrations can undergo a phase transition to a three-dimensional gel (7,15,49). In the formation of the basic ternary complex, protein 4.1 appears to play a crucial role in stabilizing spectrin cross-links between actin filaments. Consistent with cross-link reinforcement, red cells deficient in protein 4.1 possess membranes that fragment more...

Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton

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