Invariant tryptophan at a shielded site promotesfolding of the conformational unit of spectrin.

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...

Section: Fig 3 CD Spectra and Cd-monitored Denaturationsupporting

confidence: 79%

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

“…Two buried tryptophans, Trp 2820 in helix A and Trp 2915 in helix C, make up a notable hydrophobic cluster of the R23 coiledcoil structure. These observations fit well with the previously reported interactions between two conserved tryptophan residues, which represent a stabilizing feature through most of the spectrin-like repeats (38,50). Among these canonical con- served tryptophans, the Trp 2915 under a heptad position appears in the model as the residue closest to the mutated area.…”

Section: Discussionsupporting

confidence: 79%

“…Circular dichroism spectra (Fig. 2B) display the typical features of proteins with a predominant ␣-helix folding as expected for dystrophin repeats (16,19,30) as well as spectrin repeats (14,50). A circular dichroism [ 222 ]/[ 208 ] value higher than 1 was observed for all proteins, indicating that the repeats are structured in coiled coils (51,52).…”

Section: Resultsmentioning

confidence: 99%

A Two-amino Acid Mutation Encountered in Duchenne Muscular Dystrophy Decreases Stability of the Rod Domain 23 (R23) Spectrin-like Repeat of Dystrophin

Legardinier

Legrand

Raguénès-Nicol

et al. 2009

Lack of functional dystrophin causes severe Duchenne muscular dystrophy. The subsarcolemmal location of dystrophin, as well as its association with both cytoskeleton and membrane, suggests a role in the mechanical regulation of muscular membrane stress. In particular, phenotype rescue in a Duchenne muscular dystrophy mice model has shown that some parts of the central rod domain of dystrophin, constituted by 24 spectrin-like repeats, are essential. In this study, we made use of rare missense pathogenic mutations in the dystrophin gene and analyzed the biochemical properties of the isolated repeat 23 bearing single or double mutations E2910V and N2912D found in muscle dystrophy with severity grading. No dramatic effect on secondary and tertiary structure of the repeat was found in mutants compared with wild type as revealed by circular dichroism and NMR. Thermal and chemical unfolding data from circular dichroism and tryptophan fluorescence show significant decrease of stability for the mutants, and stopped-flow spectroscopy shows decreased refolding rates. The most deleterious single mutation is the N2912D replacement, although we observe additive effects of the two mutations on repeat stability. Based on threedimensional structures built by homology molecular modeling, we discuss the modifications of the mutation-induced repeat stability. We conclude that the main forces involved in repeat stability are electrostatic inter-helix interactions that are disrupted following mutations. This study represents the first analysis at the protein level of the consequences of missense mutations in the human dystrophin rod domain. Our results suggest that it may participate in mechanical weakening of dystrophin-deficient muscle.Dystrophin-associated muscular dystrophies range from the severe Duchenne muscular dystrophy (DMD) 3 to the milder Becker muscular dystrophy (BMD). Molecular genetic studies indicate that both disorders are the result of mutations in the huge gene that encodes dystrophin (1). Approximately twothirds of the mutations in DMD and BMD are intragenic partial deletions (2). Indeed, deletion of exons disrupting the reading frame leads to premature translational termination and absence of the protein in DMD cases. On the other hand, mutations maintaining the translational reading frame can lead to semi-functional microdystrophins in BMD cases (3, 4). The remaining one-third of DMD/BMD patients have rare point mutations, such as micro-deletions/insertions or substitutions of one or more nucleotides (5-7). Despite the potential importance of this source of information on protein behavior in cells, the consequences of these mutations on biological properties of dystrophin are so far poorly documented, mainly because of the size and complexity of the cellular partnership of the protein.Dystrophin is a 427-kDa protein included within the dystrophin glycoprotein complex (DGC) (8). The N-terminal part of dystrophin is reported as a globular actin binding domain 1 (9). The C-terminal cysteine-rich domain of dystro...

“…Earlier studies have pointed to interactions between the 2 tryptophan residues, conserved through most of the repeats (12,24), as a stabilizing feature. In one instance, reduced stability has been linked to the loss of intrahelical hydrogen bonds in helix B of the three-helix bundle (12).…”

Section: Design and Characterization Of Recombinant Spectrin Fragments-mentioning

confidence: 99%

Conformational Stabilities of the Structural Repeats of Erythroid Spectrin and Their Functional Implications

Guo

Zhang

et al. 2006

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T m ) ranging from 21 to 72°C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol ؊1 . Constructs comprising five successive repeats from the ␤-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.