Mapping the tropomyosin isoform 5 binding site on human erythrocyte tropomodulin: Further insights into E-Tmod/TM5 interaction

doi:10.1016/j.abb.2005.10.002

Archives of Biochemistry and Biophysics

Volume 444, Issue 2, 15 December 2005, Pages 130-138

https://doi.org/10.1016/j.abb.2005.10.002 Get rights and content

Abstract

Actin protofilaments in the erythrocyte membrane skeleton are uniformly ∼37 nm. This length may be in part attributed to a “molecular ruler” made of erythrocyte tropomodulin (E-Tmod) and tropomyosin (TM) isoforms 5 or 5b. We previously mapped the E-Tmod binding site to TM5 N-terminal heptad repeat residues “a” (I⁷, I¹⁴), “d” (V¹⁰) and “f” (R¹²). We now map the TM5 binding site to E-Tmod residues at L¹¹⁶, E¹¹⁷ and/or E¹¹⁸ by identifying among 35 deletion clones and a series of point mutations that no longer bind to human TM5 and rat TM5b. Upstream residues 71–104 contain an actin binding site. The N-terminal “KRK ring” may participate in balancing electrostatic force with hydrophobic interaction in dimerization of TM and its binding to E-Tmod.

Section snippets

Generation of human E-Tmod deletion clones

Plasmid pMALc2-MBP/Tmod [24], which encodes a maltose-binding protein (MBP) and a full-length human E-Tmod cDNA was digested with EcoRI followed by nuclease S1 to create blunt ends. The bidirectional exonuclease III digestion was stopped by adding 5 mM EDTA at different time points (10 s to 3 min). The cDNAs were filled in with Klenow fragment and religated to generate a nested set of deletion clones. Two additional clones were obtained by BamHI and PstI digestion, respectively, followed by

hTM5 binding to truncated E-Tmod

To map the hTM5 binding site, 35 human E-Tmod deletion clones truncated from the C-terminus were generated. They contain E-Tmod residues 1–334, -329, -325, -319, -318, -311, -306, -300, -299, -297, -294, -288, -279, -277, -276, -274, -271, -266, -263, -257, -249, -232, -221, -212, -197, -193, -179, -176, -170, -161, -127, -104, -70, -46, and -0, respectively. Recombinant proteins in E. coli were induced and affinity purified, and their ability to bind to hTM5 was determined by

Discussion

In this study, we used recombinant E-Tmod clones truncated from the C-terminus to identify a small region (23 residues from 105 and 127 or 6% of 359 residues) that is essential to interact with the recombinant hTM5 as detected by immunoprecipitation. This region is narrower than the region (100 residues from 39 to 138, or 28%) previously mapped using E-Tmod clones truncated from the N-terminus that interacted with TMs purified from human erythrocyte membranes as detected by solid phase binding

Acknowledgments

This work was supported by NIH Research Grant PO1HL-43026-6 from the National Heart, Lung, and Blood Institute (Project 2, LAS). Carlos Vera was supported by a pre-doctoral fellowship from UCMEXUS-CONACYT and a postdoctoral fellowship from Sung’s Molecular Bioengineering Laboratory. We used the Biotech Core in the Department of Bioengineering established with the support by the Whitaker Foundation. We would like to express our appreciation for Dr. Russell Doolittle’s advice on hTM5 and E-Tmod

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An actin protofilament wrapped around by 6 spectrin dimers forms a six-armed junctional complex (Sche et al., 2011; Sung and Vera, 2003). Such basic repeating units interact to construct a protein network with “spoked” hexagons to support the mechanical stability of the associated lipid bilayer and provide the elastic deformability for erythrocytes (de Oliveira et al., 2010; Vera et al., 2005; Zhu et al., 2007), preparing them for circulation. We investigated the lacZ expression in the kidneys of postnatal and adult mice (Fig. 4), which yielded similar findings.
Full-length erythrocyte tropomodulin (E-Tmod or Tmod1) isoform of 41 kDa is an actin nucleation protein and caps the pointed end of tropomyosin-coated actin filaments. It participates in the length control of short actin protofilaments in the erythrocyte membrane skeletal network as well as the organization of microfilaments in non-erythroid cells. Recently we discovered and characterized a truncated isoform of 29 kDa, which lacks the N-terminal sequence encoded by exons 1 and 2 required for nucleation and capping. Thus, it is important to study the expression pattern of solely the E-Tmod41 isoform in tissues. We utilized our exon 1 knockout (KO) mouse model with a knock-in lacZ reporter gene which reports the expression of E-Tmod41, but not E-Tmod29. Because this homozygous isoform-specific KO is an embryonic lethal mutation, we used heterozygous mice. X-gal staining localized specific signals at the single cell level and revealed a timed expression during embryonic development and restricted expression in adult mice. Our results showed that E-Tmod41 expressing cells include developing and young erythroid cells, developing somites, young fiber cells in the lens, certain subtype(s) of tubular cells in the kidney, smooth muscle cells in various tissues, and horizontal cells in the retina. A comparison with previous studies revealed that most if not all tissues known to express E-Tmod contained lacZ-expressing cells. Interestingly, some tubular cells were lacZ-positive while others in the same renal tubule were not, indicating heterogeneity within the tubular cells. Combined with double immunocytochemistry, we further localized E-Tmod41 to dendritic spines of horizontal cells. These timed and cell-type restricted expressions of E-Tmod41 suggest a role of actin nucleation and/or short actin protofilaments in these cell types and sub-cellular structures.
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pAb 2644 was made in a rabbit against a human synthetic peptide corresponding to residues 70–84. Because this peptide is located within the N-terminal ABD and encoded by exon 2 (E2), it is also called anti-E2 pAb (23, 38). Mouse mAb 204 was made against recombinant human E-Tmod, and its epitope was mapped to include residues 212–249.
Erythrocyte tropomodulin (E-Tmod or Tmod1) of 41 kDa is a tropomyosin (TM)-binding protein that caps the slow-growing end of the actin filaments. Its N-terminal half is flexible, whereas the C-terminal half has a single domain structure. E-Tmod/TM5 complex may function as a “molecular ruler” generating actin protofilaments of ∼37 nm. Here we report the discovery of a short isoform of 29 kDa that lacks the N-terminal actin-binding domain (N-ABD) but retains the C-terminal actin-binding domain (C-ABD). E-Tmod29 can be generated by alternative splicing from an upstream promoter or by multiple transcriptional start sites from a downstream promoter. Promoter switching leads to a surge of E-Tmod41 in reticulocytes, which degrades quickly in the cytosol. We expressed recombinant isoforms in Escherichia coli and tested their binding toward TM5, G-actin, and F-actin. Solid-phase binding assays show that, without the N-terminal 102 residues, E-Tmod29 binds to TM5 or G-actin more strongly than E-Tmod41 does, but barely binds to F-actin after TM5 binding. Differential bindings explain the distinct localizations of E-Tmod29 in the cytosol and E-Tmod41 on the membrane. Sequential bindings and immunofluorescent staining further suggest that 1) TM5 binding to E-Tmod41 may open up the flexible N-terminal half, exposing N-ABD and unblocking C-ABD; 2) N-ABD binds to F-actin and C-ABD binds to G-actin; and 3) F-actin binding to N-ABD may prevent G-actin from binding to C-ABD. E-Tmod29 may thus modulate the availability of TM5 and G-actin for E-Tmod41 to construct the protofilament-based membrane skeletal network for circulating erythrocytes.
Molecular Basis of Tropomyosin Binding to Tropomodulin, an Actin-capping Protein
2007, Journal of Molecular Biology
The tropomodulin (Tmod) family of proteins that cap the pointed, slow-growing end of actin filaments require tropomyosin (TM) for optimal function. Earlier studies identified two regions in Tmod1 that bind the N terminus of TM, though the ability of different isoforms to bind the two sites is controversial. We used model peptides to determine the affinity and define the specificity of the highly conserved N termini of three short, non-muscle TMs (α, γ, δ-TM) for the two Tmod1 binding sites using circular dichroism spectroscopy, native gel electrophoresis, and chemical crosslinking. All TM peptides have high affinity for the second Tmod1 binding site (within residues 109–144; α-TM, 2.5 nM; γ-TM, δ-TM, 40–90 nM), but differ >100-fold for the first site (residues 1–38; α-TM, 90 nM; undetectable at 10 μM, γ-TM, δ-TM). Residue 14 (R in α; Q in γ and δ) and, to a lesser extent, residue 4 (S in α; T in γ and δ) are primarily responsible for the differences. The functional consequence of the sequence differences is reflected in more effective inhibition of actin filament elongation by full-length α-TMs than γ-TM in the presence of Tmod1. The binding sites of the two Tmod1 peptides on a model TM peptide differ, as defined by comparing ¹⁵N,¹H HSQC spectra of a ¹⁵N-labeled model TM peptide in both the absence and presence of Tmod1 peptide. The NMR and CD studies show that there is an increase in α-helix upon Tmod1–TM complex formation, indicating that intrinsically disordered regions of the two proteins become ordered upon binding. A model proposed for the binding of Tmod to actin and TM at the pointed end of the filament shows how the Tmod–TM accentuates the asymmetry of the pointed end and suggests how subtle differences among TM isoforms may modulate actin filament dynamics.
Leiomodin/tropomyosin interactions are isoform specific
2007, Archives of Biochemistry and Biophysics
Leiomodins are larger homologs of tropomodulin, a tropomyosin-binding, actin-capping protein. There are several leiomodin isoforms, one of them found in smooth muscles (Lmod1) and another one found in cardiac and skeletal muscles (Lmod2). In this work, the tropomyosin-binding abilities of these two isoforms were studied. The tropomyosin-binding sites were localized in the N-terminal regions of Lmod1 and Lmod2. The affinities of the leiomodin fragments containing the tropomyosin-binding sites for tropomyosin peptides containing N-termini of different tropomyosin isoforms, α, γ and δ, were determined and compared using non-denaturing gel-electrophoresis and circular dichroism. It was shown that leiomodin/tropomyosin binding is isoform-specific and differs almost 100-fold for different tropomyosin isoforms.
Leucine 135 of tropomodulin-1 regulates its association with tropomyosin, its cellular localization the integrity of sarcomeres
2006, Journal of Biological Chemistry
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Closer examination of the effects of mutating residue 134 or 135 revealed that both are important for Tmod-1 to maintain its relatively strong interaction with TM5 (Fig. 2 and 3). In addition, as this manuscript was being submitted, a careful study by Vera et al. (18) was published and revealed that residue 116 and/or 117 is also likely involved in the Tmod-1 and TM5 interaction. Which of these several locations are directly involved in contact with TM and whether they maintain critical ligand structural integrity remain to be determined.
Tropomodulin-1 (Tmod-1) is a well defined actin-capping protein that interacts with tropomyosin (TM) at the pointed end of actin filaments. Previous studies by others have mapped its TM-binding domain to the amino terminus from amino acid 39 to 138. In this study, we have identified several amino acid residues on Tmod-1 that are important for its interaction with TM5 (a nonmuscle TM isoform). Glutathione S-transferase affinity chromatography and immunoprecipitation assays reveal that Tmod sense mutations of either amino acid 134, 135, or 136 causes various degrees of loss of function of Tmod TM-binding ability. The reduction of TM-binding ability was relatively mild (reduced ∼20-40%) from the G136A Tmod mutant but more substantially (reduced ∼50-100%) from the I134D, L135E, and L135V Tmod mutants. In addition, mutation at any of these three sites dramatically alters the subcellular location of Tmod-1 when introduced into mammalian cells. Further analysis of these three mutants uncovered a previously unknown nuclear trafficking function of Tmod-1, and residues 134, 135, and 136 are located within a nuclear export signal motif. As a result, mutation on either residue 134 or residue 135 not only will cause a significant reduction of the Tmod-1 ability to bind to TM5 but also lead to predominant nuclear localization of Tmod-1 by crippling its nuclear export mechanism. The failure of the Tmod mutations to fully associate with TM5 when introduced into neonatal rat cardiomyocytes was also associated with an accelerated and severe fragmentation of sarcomeric structures compared with overexpression of wild type Tmod-1. The multiple losses of function of Tmod engendered by these missense mutations are most severe with the single substitution of residue 135.
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Mapping the tropomyosin isoform 5 binding site on human erythrocyte tropomodulin: Further insights into E-Tmod/TM5 interaction

Abstract

Section snippets

Generation of human E-Tmod deletion clones

hTM5 binding to truncated E-Tmod

Discussion

Acknowledgments

Blood

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biophys. J.

FEBS Lett.

Biophys. J.

J. Biol. Chem.

Biophys. J.

Arch. Biochem. Biophys.

Biophys. J.

J. Biol. Chem.

Gene

Comput. Phys. Commun.

J. Mol. Biol.

Nature

J. Cell Biol.

J. Cell Biol.

Ann. Biomed. Eng.