Thermal Stabilities of Brain Spectrin and the Constituent Repeats of Subunits

Abstract

The different genes that encode mammalian spectrins give rise to proteins differing in their apparent stiffness. To explore this, we have compared the thermal stabilities of the structural repeats of brain spectrin subunits (αII-and βII) with those of erythrocyte spectrin (αI-and βI). The unfolding transition mid-points (T m ) of the 36 αII-and βII-spectrin repeats extend between 24 and 82°C, with an average higher by some 10°C than that of the αI-and βI-spectrin repeats. This difference is reflected in the T m -s of the intact brain and erythrocyte spectrins. Two of three tandem-repeat constructs from brain spectrin showed strong cooperative coupling, with elevation of the T m of the less stable partner corresponding to coupling free energies of about −4.4 and −3.5 kcal mol −1 . The third tandem-repeat construct, by contrast, showed negligible cooperativity. Tandem-repeat mutants, in which a part of the 'linker' helix that connects the two domains was replaced by a corresponding helical segment from erythroid spectrin, showed only minor perturbation of the thermal melting profiles, without breakdown of cooperativity. Thus the linker regions, which tolerate few point-mutations without loss of cooperative function, have evidently evolved to permit conformational coupling in specified regions. The greater structural stability of the repeats in αII-and βII-spectrin may account, at least in part, for the higher rigidity of brain compared to erythrocyte spectrin.Spectrin arose in evolution with the metazoa to meet the need for structures that strengthen cell adhesions and stabilize the plasma membrane against the forces of animal movement (1). The protein also plays a part in organizing plasma membrane signalling complexes (1, 2). Spectrin occurs as an (αβ) 2 tetramer, specialized for cross-linking actin filaments to membrane constituents. Both the α and β chains are largely made up of consecutive triplehelical repeating units of about 106 amino acids (3, 4); these act in ensemble as spacers between actin-binding domains in the β-subunits at opposite ends of the tetramers, and some contain binding sites for proteins such as ankyrin or for aminophospholipids (5, 6). *Author for correspondence: Xiuli An, Red Cell Physiology Laboratory, 310 E 67 th St, New York, NY10021, Tel: 212-570-3247; Fax: 212-570-3195. xan@nybloodcenter.org. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe number of spectrin genes increased during evolution with the advent of vertebrates.Invertebrates have one α-spectrin and, one β-spectrin with 16 complete triple-helical repeats and a β Heavy subunit with 30 complete triple helices. Vertebrates have four genes encoding 'conventional' β subunits (βI-IV) that have 16 complete triple helical modules, and one β Heavy subunit that has 30 triple helices (βV-spectrin) (1). Mammals have gained an additional α-spectrin by duplication of the pre-existing α-spectrin gene (7). There is now clear evidence of functional specialization of the two mammalian α-...

The structure of the ankyrin-binding site of β-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties

“…27 Thus, the helix C, linker, and interhelix loops (particularly the B-C loop), are sites that readily accommodate modification without disruption of the structural repeat. Although the linker provides a crucial bridge coupling the folding and unfolding of paired repeats to each other, 42 we propose that the linker has additional roles. Specifically, we propose that the spectrin di-repeat unit represents a novel and here-to-fore unappreciated general protein-protein interaction motif.…”

“…Previously determined spectrin di-repeat structures 36,38 indicate that the side-chains of these 3 residues form a tightly packed hydrophobic core that has been proposed to contribute to peptide folding and stability and possibly to its physiologic role. 23,26,41,42 However, we find that the absence of Trp and the Trp-His couple in ␤I-15 is not critical for stability of this di-repeat, nor does it modify ankyrin recognition (Figure 1). Furthermore, we find in our structure that the hydrophobic core of ␤I-15 is tightly packed, similar to that of other spectrin repeats, regardless of its lack of the invariant Trp residue in helix A.…”

Spectrin Tetramer Formation Is Not Required for Viable Development in Drosophila

“…D-DЈ, ␣wt, weeP␣/TM6; ␣1cx, is weeP␣/P{␣-spec R22S } ␣-spec rg41 . E, overexpression of wild-type ␣-spectrin (MTDGal4ϾUAS-␣-spectrin 37 ). F-FЉ, egg chambers from ␣-spectrin R22S rescued flies expressing 2 or 3 copies of the ␣-spec R22S transgene stained with 1B1 that exhibit gaps in the normally continuous follicle cell monolayer (arrows, other labels as in B).…”

“…We therefore overexpressed wild-type ␣-spectrin in the germline in a wild-type ␣-spectrin background using the Gal4/UAS system (42) with the MTD-Gal4 driver. Staining MTD-Gal4ϾUAS-␣-spectrin 37 ovarioles for 1B1 reveals fusomes of normal complexity (Fig. 6E).…”

Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin

“…33,34,40,41 In addition to their principal role as an elastic module, there is increasing evidence that spectrin repeats may also serve as docking sites for cytoskeletal and signal transduction proteins. For example, the repeats of ␣-actinin (a member of spectrin family) have been shown to interact with cytoplasmic domains of integrins 42 and intercellular adhesion molecules.…”