Structures of Two Repeats of Spectrin Suggest Models of Flexibility

Grum, Valerie L; Li, Dongning; MacDonald, Robert C.; Mondragón, Alfonso

doi:10.1016/s0092-8674(00)81980-7

Cited by 229 publications

(308 citation statements)

References 70 publications

(12 reference statements)

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“…7). Tworepeat fragments, HEβ89 (25) and CBα1617 (33), previously characterized by X-ray crystallography are also shown in Fig. 7 for comparison.…”

Section: Zu5 Forms a Complex With Heβ1315 And With Heβ1415 But Not Wimentioning

confidence: 99%

“…This reasoning also applies, of course, to the other prominent members of the spectrin family, α-actinin and dystrophin (30). It remains a mystery, however, as to what dictates this structural versatility of the signature spectrin repeating unit motif (31) in spite of the availability of Xray crystal structures of two (25,32,33) and three (34) repeat fragments of spectrin and α-actinin. An exception is the structure of four repeat fragments of α-actinin (35) with a lengthwise twist which enables interaction of α-actinin with actin at each end.…”

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confidence: 99%

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Molecular Epitopes of the Ankyrin−Spectrin Interaction

et al. 2008

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Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for β-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin−spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30−60% unstructured. In contrast, human erythroid β-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and β-spectrin repeats 14−15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with β-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of β-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th β-spectrin repeats comprise the minimal, phased region of β-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.

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“…7). Tworepeat fragments, HEβ89 (25) and CBα1617 (33), previously characterized by X-ray crystallography are also shown in Fig. 7 for comparison.…”

Section: Zu5 Forms a Complex With Heβ1315 And With Heβ1415 But Not Wimentioning

confidence: 99%

mentioning

confidence: 99%

Molecular Epitopes of the Ankyrin−Spectrin Interaction

et al. 2008

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“…These repeats might have been generated by gene duplication [10]. Spectrin repeats form a triple-helix supercoil connected by an  helical linker, thus allowing spectrin to expand and contract [11]. Furthermore, spectrin comprises numerous structural motifs, including actin-binding, pleckstrin homology, and Src homology 3 domains that contribute to spectrin function.…”

Section: Figurementioning

confidence: 99%

Spectrin: Structure, function and disease

Zhang

Zhao

et al. 2013

Sci. China Life Sci.

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Spectrin is a large, cytoskeletal, and heterodimeric protein composed of modular structure of and subunits, it typically contains 106 contiguous amino acid sequence motifs called "spectrin repeats". Spectrin is crucial for maintaining the stability and structure of the cell membrane and the shape of a cell. Moreover, it contributes to diverse cell functions such as cell adhesion, cell spreading, and the cell cycle. Mutations of spectrin lead to various human diseases such as hereditary hemolytic anemia, type 5 spinocerebellar ataxia, cancer, as well as others. This review focuses on recent advances in determining the structure and function of spectrin as well as its role in disease.erythrocyte, spectrin, cell cycle, mass spectrometry, disease Citation:Zhang R, Zhang C Y, Zhao Q, et al. Spectrin: Structure, function and disease.

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“…34 Therefore, we used homology modeling methods to predict the free form of bI-C1 and bII-C1. The modeling program (see ''Materials and Methods'' Section) selected the protein with a Protein Data Bank (PDB) code of 1CUN 35 as the template for predicting the structures for both bI-C1 (residues 1898-2083). This template protein (1CUN) is a recombinant protein of domains 16-17 of chicken a-spectrin.…”

Section: Predicted Structures Of Bi-c1 and Bii-c1mentioning

confidence: 99%

“…This template protein (1CUN) is a recombinant protein of domains 16-17 of chicken a-spectrin. 35 With this template, a predicted structure of bI-C1 was obtained, with a triple helical bundle as the full structural domain, followed by two helices as the partial domain [ Fig. 6(A)].…”

Section: Predicted Structures Of Bi-c1 and Bii-c1mentioning

confidence: 99%

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

et al. 2011

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We have screened a human immunoglobulin single-chain variable fragment (scFv) phage library against the C-terminal tetramerization regions of erythroid and nonerythroid beta spectrin (bI-C1 and bII-C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid b-spectrin isoforms. We have identified interacting scFvs, with clones ''G5'' and ''A2'' binding only to bI-C1, and clone ''F11'' binding only to bII-C1. The K d values, estimated by competitive enzyme-linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1-0.3 lM. A more quantitative K d value from isothermal titration calorimetry experiments with the recombinant G5 and bI-C1 was 0.15 lM. The a-spectrin fragments (model proteins), aI-N1 and aII-N1, competed with the bI-C1, or bII-C1, binding scFvs, with inhibitory concentration (IC 50 ) values of~50 lM for aI-N1, and 0.5 lM for aII-N1. Our predicted structures of bI-C1 and bII-C1 suggest that the Helix B 0 of the Cterminal partial domain of bI differs from that of bII. Consequently, an unstructured region downstream of Helix B 0 in bI may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in bII was helical, and bII did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C-termini of different b-spectrin isoforms to regulate a-and b-spectrin association to form functional spectrin tetramers, and may sort b-spectrin isoforms to their specific cellular localizations.Keywords: beta spectrin; erythroid and nonerythroid; scFv; structural difference; affinity difference Abbreviations: aI-N1, a recombinant protein of aI-spectrin segment with residues 1-156; aI-spectrin, erythroid a-spectrin; aII-N1, a GST fusion protein of aII-spectrin segment with residues 1-147; aII-spectrin, nonerythroid a-spectrin; bI-C1, a GST fusion protein of bI-spectrin segment with residues 1898-2083; bI-C1D, a GST fusion protein of bI-spectrin segment with residues 1898-2070, that is, the residues 2071-2083 in bI-C1 were deleted; bI-spectrin, erythroid b-spectrin; bII-C1, a GST fusion protein of bII-spectrin segment with residues 1906-2093; bII-spectrin, nonerythroid b-spectrin; CD, circular dichroism; CDRs, complementarity determining regions; ELISA, enzyme-linked immunosorbent assay; EPR, electron paramagnetic resonance; GST, glutathione S-transferase; HRP, horseradish peroxidase; ITC, isothermal titration calorimetry; PBS, phosphate buffered saline at pH 7.4; rG5, recombinant G5, a single-chain variable fragment found to bind bI-C1; scFv, single-chain variable fragment.Yuanli Song's current address is

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Structures of Two Repeats of Spectrin Suggest Models of Flexibility

Cited by 229 publications

References 70 publications

Molecular Epitopes of the Ankyrin−Spectrin Interaction

Molecular Epitopes of the Ankyrin−Spectrin Interaction

Spectrin: Structure, function and disease

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

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