Phasing the conformational unit of spectrin.

Abstract

Many proteins contain a repetitive sequence motif, which implies that they contain a repetitive structural motif. Spectrin and the related proteins dystrophin and a-actinin consist largely of repeated motifs of 100-120 residues. But the repeating motif is degenerate and it has been difficult to derme the boundaries of the repeating sequence unit or its corresponding structural unit. We have determined at which residues the structural units that correspond to spectrin's repeating 106lOaano acid motifs begin and end. Drosophila a-spectrin BdNAs were expressed in bacteria to show that single (106 amino acids) and pairs of segments encoded by selected regions of spectrin cDNA can fold into stable conformations whose biophysical and biochemical properties are similar to those of native spectrin. Because such folig was critically dependent on the phasing ofthe expressed sequence with respect to the apparent boundaries of the repeating motifs, our data provide experimental evidence that relates the boundaries of the folded, conformational unit to the chemical sequence of repeating motifs.Spectrins, some of which are also called fodrin, are elongated, flexible molecules whose primary structure is dominated by tandem, homologous, 106-residue motifs (1) that have been referred to as repeating units or segments (2). Dystrophin and a-actinin, other members of the spectrin superfamily (3, 4), contain comparable repetitive sequences. Although secondary-structure predictions suggest that each of the repetitive motifs of spectrin folds as a closely packed, triple a-helical unit, neither spectrin itself, nor proteolytically derived domains of spectrin, have been tractable samples for direct structure determination. Because the 106-residue motifs are generally repeated without interruptions, the residue that corresponds to the beginning (or end) of a structural unit is not self-evident. Consider, for example, the repetitive sequences of letters ABCDABCDABCD .... To produce a 4-letter segment of this sequence that can form a structural unit, it is essential to know whether the structure is ABCD, BCDA, CDAB, or DABC. Similarly, to express a 106-residue segment of spectrin that will fold into a structural unit, one must know at which position in any of the repetitive motifs that are evident in the primary sequence the segment should start and end. Speicher and Marchesi (1) stated clearly that the convention they adopted to illustrate the repetitive sequences was not intended to correlate with the conformational unit of the folded protein.Using cDNA constructs derived from Drosophila a-spectrin and expressed in bacteria, we show that single repetitive motifs (106 amino acids) and pairs of motifs can fold into stable conformations similar to that of native spectrin when their N-terminal ends are 26 residues downstream of the residue that, according to the convention of Speicher and Marchesi (1), has usually been designated to be in the first position of each repetitive motif. Similarly sized polypeptides with other phasing do ...

Molecular Epitopes of the Ankyrin−Spectrin Interaction

“…In view of the finding by Winograd et al (1991) that cloning of well-folded spectrin repeats required their being "phased" or consisting of integral, non-fractional numbers of repeats, the binding activity of "unphased" repeats might be considered to be of dubious physiological significance. Nevertheless, the results presented here on "phased" repeats agree well with those on "unphased" repeats by Kennedy et al (1991) and by HryniewiczJankowska et al (2004), insofar as the former identified the C-terminal third of β-spectrin repeat 14 and the whole of repeat 15 and the latter found a fragment from repeat 13 to the middle of repeat 15 to be the spectrin binding site on ankyrin.…”

“…Only upon later structural determination of spectrin repeats did it become apparent that this "repeat 15" (Kennedy et al, 1991) actually comprised the Cterminal third of repeat 14 and the whole of repeat 15, according to the phasing of start and stop sites of canonical spectrin repeats (37). In order to test well-folded spectrin fragments for binding to the ankyrin subdomain Zu5, re-cloning of different combinations of canonically phased, two and three repeat fragments in the region of the 14 th and 15 th repeating units of human erythroid β-spectrin was undertaken.…”

“…A minimal version of the ankyrin binding subdomain had originally been identified as β-spectrin repeat 15 by cloning and testing of incrementally smaller regions of the C-terminal end of β-spectrin for binding to inside-out red cell vesicles (36). However, it became apparent subsequently that this "repeat 15" was actually the C-terminal third of repeat 14 plus the whole of repeat 15, encompassing the start and stop sites of canonical spectrin repeats defined by surveying the CD spectra and protease sensitivities of differently phased fragments (37). Nevertheless, the identity of this region as the ankyrin binding site is supported by its harboring 1) a sequence in the 15 th repeat, which is well-conserved among β-spectrins I, II and III with ankyrin binding activity, and 2) a 43 residue region of low homology with other repeats (38).…”

Genetic disorders of the red cell membranes

“…In space, the conformation units are described by two models [8,9], that both imply three a-helical segments. The phasing of conformational units is shifted downstream by 26amino acids with respect to that of homologous segments [10]. An a-and aft-chain of spectrin initiate dimerization at two complementary nucleation sites, one on the a-chain (a18 to a21), the other on the ft-chain (ill to ft4) [11].…”

Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains

“…10, 19, and 21-23). An interesting paradigm is the three-helix bundle spectrin domains (24)(25)(26)(27)(28)(29). These have an extended helix, so that helix C of one domain is contiguous with helix A of the next (Fig.…”