Analysis of the Phasing of Four Spectrin‐like Repeats in α‐actinin

Gilmore, Andrew; Parr, Tim; Patel, Bipin; Gratzer, Walter; Critchley, David R.

doi:10.1111/j.1432-1033.1994.00235.x

Cited by 29 publications

(29 citation statements)

References 34 publications

(19 reference statements)

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“…2B. In addition, although various start sites have been reported for the ␣-actinin motif based on alignments of sequences, the revised start site presented here for the first motif of spectrin and ␣-actinin agrees with the recent phasing analysis of ␣-actinin using recombinant peptides reported by Gilmore et al (1994).…”

supporting

confidence: 86%

Mapping the Human Erythrocyte β-Spectrin Dimer Initiation Site Using Recombinant Peptides and Correlation of Its Phasing with the α-Actinin Dimer Site

Ursitti¹,

Kotula²,

DeSilva

et al. 1996

Journal of Biological Chemistry

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Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of ␤-spectrin with a complementary region near the C-terminal of ␣-spectrin (Speicher, D. W., Weglarz, L., and DeSilva, T. M. (1992) J. Biol. Chem. 267, 14775-14782). Both spectrin subunits consist primarily of tandem, 106-residue long, homologous, triple-helical motifs. In this study, the minimal region of ␤-spectrin required for association with ␣-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent ␤-spectrin peptides was particularly critical. The beginning of the first homologous motif for both ␤-spectrin and the related dimerization site of ␣-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif ␤-spectrin peptide (␤1-4 ؉ ) with this earlier starting point bound to full-length ␣-spectrin with a K d of about 10 nM, while deletion of these first 8 residues reduced binding nearly 10-fold. N-and C-terminal truncations of one or more motifs from ␤1-4 ؉ showed that the first motif was essential for dimerization since its deletion abolished binding, but ␤1؉ alone could not associate with ␣-monomers. The first two motifs (␤1-2 ؉ ) represented the minimum lateral dimer assembly site with a K d of about 230 nM for interaction with full-length ␣-spectrin or an ␣-spectrin nucleation site recombinant peptide, ␣18 -21. Each additional motif increased the dimerization affinity by approximately 5-fold. In addition to this strong inter-subunit dimer association, interactions between the helices of a single triple-helical motif are frequently strong enough to maintain a noncovalent complex after internal protease cleavage similar to the interactions thought to be involved in tetramer formation. Analysis of hydrodynamic radii of recombinant peptides containing differing numbers of motifs showed that a single motif had a Stokes radius of 2.35 nm, while each additional motif added only 0.85 nm to the Stokes radius. This is the first direct demonstration that spectrin's flexibility arises from regions between each triple helical motif rather than from within the segment itself and suggests that current models of inter-motif connections may need to be revised.The membrane skeleton of the human erythrocyte consists of a network of proteins that associates with the inner surface of the cell membrane and imparts remarkable structural integrity and flexibility to circulating erythrocytes. Spectrin is the major structural component of this specialized submembranous protein network. The basic functional unit of spectrin is a heterodimer formed by side-to-side, antiparallel association of a 280-kDa ␣ subunit with a 246-kDa ␤ subunit. Spectrin dimers associate head-to-head to form tetramers, the predominant form of spectrin in the membrane skeleton. These tetramers cross-link short actin oligomers, an association modulated by band 4

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supporting

confidence: 86%

Mapping the Human Erythrocyte β-Spectrin Dimer Initiation Site Using Recombinant Peptides and Correlation of Its Phasing with the α-Actinin Dimer Site

Ursitti¹,

Kotula²,

DeSilva

et al. 1996

Journal of Biological Chemistry

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“…These experiments clearly showed that these single spectrinlike sequence motifs could fold into a compact structure, which might be true domains. However, one hint that these structures exhibit some cooperativity comes from the observation that when two sequence motifs of actinin are expressed in tandem, they are more resistant to proteolysis than either of the isolated constituent motifs, a terminal fractional motif, or improperly phased motifs (17).…”

Section: Discussionmentioning

confidence: 99%

“…This places the start of the first structural domain after the onset of the homologous sequence motif (6), indicating that there is a fractional motif at the amino termi-nus in this protein. Proteolysis experiments to delimit compact structural domains have also been done by others on invertebrate spectrin (16), as well as on actinin (17) and dystrophin (18). These studies have shown that the domain boundaries in these spectrin or spectrin-like proteins are similar and may be identified by a pair of highly conserved tryptophan residues at positions 17 and 90 into the domain.…”

mentioning

confidence: 98%

Peptides with More than One 106-amino Acid Sequence Motif Are Needed to Mimic the Structural Stability of Spectrin

Menhart

Mitchell²,

Lusitani³

et al. 1996

Journal of Biological Chemistry

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The primary sequence of human erythrocyte spectrin contains repetitive homologous sequence motifs of approximately 106 amino acids with 22 such motifs in the ␣-subunit and 17 in the ␤-subunit. These homologous sequence motifs have been proposed to form domains with a triple-helical bundle type structure (Speicher, In this study, we show that these sequence motifs, while they do form compact proteolytically resistant units, are not completely independent. Peptides composed of two or three such motifs in tandem are substantially more stable than peptides composed of a single motif, as measured by proteolysis or by fluorescence or circular dichroism studies of urea or thermal denaturation. Circular dichroism and infrared spectroscopy measurements also indicate that these larger, more stable peptides exhibit greater secondary structure. In these respects, the peptides with tandem sequence motifs are more similar to intact spectrin than the peptide with a single sequence motif. Thus, we conclude that peptides with more than one sequence motif model spectrin more adequately than the peptides with one sequence motif, and that these sequence motifs are not completely independent domains.DHuman erythrocytes contain a dense, two-dimensional network of spectrin and other proteins that provides support to the lipid bilayer and maintains erythrocyte deformability (1). Spectrin, comprising ␣-and ␤-subunits, plays a critical role in maintaining the architecture and therefore the integrity of the red cell membrane. Many hereditary hemolytic anemias involve spectrin mutations (2-4). Thus, it is important to understand the structural properties of spectrin. The bulk (about 90%) of the primary structure of spectrin comprises repetitive homologous units of approximately 106 amino acids in length. Several other proteins, including brain spectrin (fodrin), dystrophin, and ␣-actinin, also have similar repetitive amino acid units in their sequences and are known as the spectrin superfamily (5).A triple-helical bundle model has been suggested for the 106-amino acid sequence motif in which the three helices are aligned side by side, with the first and third parallel and the intervening second helix antiparallel (6 -8). X-ray diffraction studies of one such sequence motif unit from a non-erythroid spectrin support this model (9). In these x-ray studies, the peptide used was found to form a homodimer containing two triple-helical structures, in which two of the three helices are contributed by one monomer and the remaining helix by the other monomer. It is thought that this peculiar arrangement may be an artifact of crystallization, and the true structure of a single unit may be similar to the earlier suggested triplehelical bundle with a zigzag arrangement of the helices (9, 10). This arrangement aligns the amino-and carboxyl-terminal residues at opposite ends of this triple-helical bundle, and thus sequential motifs are thought to be linked in tandem in intact spectrin, producing a very long rod-shaped molecule, approximately 100 nm in l...

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“…Elongated multidomain proteins are quite frequent in the microfilament system, as for example spectrin, a-actinin and dystrophin (Gilmore et al, 1994) and many of these proteins have similar repetetive-sequence motifs, but correlations between such sequence repeats to structural and functional domains are mostly unknown. For the talin rod domain, we face an analogous difficulty: 50-60 copies of a repetive motif were recognized by fourier sequence analysis (McLachlan et al, 1994), but their re-lation to the 8-10 subdomains contained in the tail, as described here, remains unclear.…”

Section: Discussionmentioning

confidence: 99%

Energy‐Filtered Electron Microscopy Reveals that Talin is a Highly Flexible Protein Composed of a Series of Globular Domains

Winkler¹,

Lünsdorf²,

Jockusch³

1997

European Journal of Biochemistry

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Talin is a multidoinain cytoskeletal protein containing discrete binding sites for acidic phospholipids, [I-integrin, actin and vinculin. Hence, it is thought to link microfilaments to the cytoplasmic membrane in cell-matrix adhesion sites, and this should critically depend on talin structure. To obtain more information on the latter, we used energy-filtered transmission electron microscopy of negatively stained talin purified from chicken smooth muscle. We show that in buffers of physiological ionic strength. talin adopts an elongated shape (56 +-7 nm in length), consisting of a series of globular masses. While these compact elements, arranged like beads on a string, were of rather uniform dimensions (3.8 nm in diameter), their center-to-center spacings varied, indicating the flexibility of the connecting strands. The ends of the elongated molecules frequently formed loops. The images obtained are consistent with the assumption that, under the conditions used, the majority of the talin molecules are monomeric. A minor fraction appeared as dimers, composed of two chains only partially intertwined, thus giving rise to Y-shaped particles. Electron micrographs revealed that the biochemically defined 50-kDa N-terminal talin head domain is composed of two globular subunits, while chemical cross-linking provided evidence that the Cterminal 220-kDa fragment is solely responsible for dimerization. These results imply that in the dimeric molecules, the polypeptide chains are arranged in parallel, in contrast to what has been described for human-platelet talin. In buffers of low ionic strength (0.02 M instead of 0.15 M KCI), the molecules collapsed into a compact shape. By showing the high flexibility and versatility of its morphology, our data favour the concept of talin as an important resilient link in microfilament-plasma-membrane attachment.Keywcri-ds: cytoskeleton; talin; structure ; electron microscopy; electron-spectroscopic imaging.Talin is a major component of cell-matrix junctions, concentrated at the cytoplasmic face of focal contacts, dense plaques of smooth muscle and myotendinous junctions (Burridge and Connell. 1983a.b;Tidball et al., 1986). The amino acid sequence of mouse talin consists of 2541 amino acids, from which the molecular mass of the protein was determined as 269 854 Da (Rees et al., 1990). The molecule can be cleaved by thrombin between residues 433 and 434, yielding a 47-kDa N-terminal and a 200-kDa C-terminal fragment (Fox et al., 1985 ;Beckerle et al., 1987). The N-terminal fragment, which has sequence similarities to the membrane-binding domains of protein band 4.1 and ezrin (Rees et al.. 1990). can interact with liposomes containing acidic phospholipids (Niggli et al., 1994; Tempe1 et al., 1995), while the C-terminal portion binds to the cytosolic domains of integrin (Horwitz et al., 1986). vinculin (Burridge and Mangeat, 1984;Gilmore et al.. 1993) and actin (Muguruma et al., 1990;Kaufmann et al., 1991: Goldmann and essential element in the establishment of discrete cell-matrix junctio...

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Analysis of the Phasing of Four Spectrin‐like Repeats in α‐actinin

Cited by 29 publications

References 34 publications

Mapping the Human Erythrocyte β-Spectrin Dimer Initiation Site Using Recombinant Peptides and Correlation of Its Phasing with the α-Actinin Dimer Site

Mapping the Human Erythrocyte β-Spectrin Dimer Initiation Site Using Recombinant Peptides and Correlation of Its Phasing with the α-Actinin Dimer Site

Peptides with More than One 106-amino Acid Sequence Motif Are Needed to Mimic the Structural Stability of Spectrin

Energy‐Filtered Electron Microscopy Reveals that Talin is a Highly Flexible Protein Composed of a Series of Globular Domains

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