Heterozygous Tropomodulin 3 mice have improved lung vascularization after chronic hypoxia.

The molecular mechanisms leading to high-altitude pulmonary hypertension (HAPH) remains poorly understood. We previously analyzed the whole genome sequence of Kyrgyz highland population and identified eight genomic intervals having a potential role in HAPH. Tropomodulin 3 gene (TMOD3), which encodes a protein that binds and caps the pointed ends of actin filaments and inhibits cell migration, was one of the top candidates. Here we systematically sought additional evidence to validate the functional role of TMOD3. In-silico analysis reveals that some of the SNPs in HAPH associated genomic intervals were positioned in a regulatory region that could result in alternative splicing of TMOD3. In order to functionally validate the role of TMOD3 in HAPH, we exposed Tmod3-/+ mice to 4 weeks of constant hypoxia, i.e. 10% O2 and analyzed both functional (hemodynamic measurements) and structural (angiography) parameters related to HAPH. The hemodynamic measurements, such as right ventricular systolic pressure, a surrogate measure for pulmonary arterial systolic pressure, and right ventricular contractility (RV- ± dP/dt), increases with hypoxia did not separate between Tmod3-/+ and control mice. Remarkably, there was a significant increase in the number of lung vascular branches and total length of pulmonary vascular branches (P < 0.001) in Tmod3-/+ after 4 weeks of constant hypoxia as compared with controls. Notably, the Tmod3-/+ endothelial cells migration was also significantly higher than that from the wild-type littermates. Our results indicate that, under chronic hypoxia, lower levels of Tmod3 play an important role in the maintenance or neo-vascularization of pulmonary arteries.

Force-history dependence and cyclic mechanical reinforcement of actin filaments at the single molecular level.

The actin cytoskeleton is subjected to dynamic mechanical forces over time and the history of force loading may serve as mechanical preconditioning. While the actin cytoskeleton is known to be mechanosensitive, the mechanisms underlying force regulation of actin dynamics still need to be elucidated. Here, we investigated actin depolymerization under a range of dynamic tensile forces using atomic force microscopy. Mechanical loading by cyclic tensile forces induced significantly enhanced bond lifetimes and different force-loading histories resulted in different dissociation kinetics in G-actin-G-actin and G-actin-F-actin interactions. Actin subunits at the two ends of filaments formed bonds with distinct kinetics under dynamic force, with cyclic mechanical reinforcement more effective at the pointed end compared to that at the barbed end. Our data demonstrate force-history dependent reinforcement in actin-actin bonds and polarity of the actin depolymerization kinetics under cyclic tensile forces. These properties of actin may be important clues to understanding regulatory mechanisms underlying actin-dependent mechanotransduction and mechanosensitive cytoskeletal dynamics.This article has an associated First Person interview with the first author of the paper.

Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles.

Cytoskeletal structures characterized by actin filaments with uniform lengths, including the thin filaments of striated muscles and the spectrin-based membrane skeleton, use barbed and pointed-end capping proteins to control subunit addition/dissociation at filament ends. While several proteins cap the barbed end, tropomodulins (Tmods), a family of four closely related isoforms in vertebrates, are the only proteins known to specifically cap the pointed end. Tmods are ∼350 amino acids in length, and comprise alternating tropomyosin- and actin-binding sites (TMBS1, ABS1, TMBS2, and ABS2). Leiomodins (Lmods) are related in sequence to Tmods, but display important differences, including most notably the lack of TMBS2 and the presence of a C-terminal extension featuring a proline-rich domain and an actin-binding WASP-Homology 2 domain. The Lmod subfamily comprises three somewhat divergent isoforms expressed predominantly in muscle cells. Biochemically, Lmods differ from Tmods, acting as powerful nucleators of actin polymerization, not capping proteins. Structurally, Lmods and Tmods display crucial differences that correlate well with their different biochemical activities. Physiologically, loss of Lmods in striated muscle results in cardiomyopathy or nemaline myopathy, whereas complete loss of Tmods leads to failure of myofibril assembly and developmental defects. Yet, interpretation of some of the in vivo data has led to the idea that Tmods and Lmods are interchangeable or, at best, different variants of two subfamilies of pointed-end capping proteins. Here, we review and contrast the existing literature on Tmods and Lmods, and propose a model of Lmod function that attempts to reconcile the in vitro and in vivo data, whereby Lmods nucleate actin filaments that are subsequently capped by Tmods during sarcomere assembly, turnover, and repair.

Localization of the binding interface between leiomodin-2 and α-tropomyosin.

The development of some familial dilated cardiomyopathies (DCM) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Harmful effects of some mutations might be caused by disruption of yet uncharacterized protein-protein interactions. We used nuclear magnetic resonance spectroscopy to localize the region of striated muscle α-tropomyosin (Tpm1.1) that interacts with leiomodin-2 (Lmod2), a member of tropomodulin (Tmod) family of actin-binding proteins. We found that 21 N-terminal residues of Tpm1.1 are involved in interactions with residues 7-41 of Lmod2. The K15N mutation in Tpm1.1, known to be associated with familial DCM, is located within the newly identified Lmod2 binding site of Tpm1.1. We studied the effect of this mutation on binding Lmod2 and Tmod1. The mutation reduced binding affinity for both Lmod2 and Tmod1, which are responsible for correct lengths of thin filaments. The effect of the K15N mutation on Tpm1.1 binding to Lmod2 and Tmod1 provides a molecular rationale for the development of familial DCM.

The cardiomyopathy-associated K15N mutation in tropomyosin alters actin filament pointed end dynamics.

Correct assembly of thin filaments composed of actin and actin-binding proteins is of crucial importance for properly functioning muscle cells. Tropomyosin (Tpm) mediates the binding of tropomodulin (Tmod) and leiomodin (Lmod) at the slow-growing, or pointed, ends of the thin filaments. Together these proteins regulate thin filament lengths and actin dynamics in cardiac muscle. The K15N mutation in the TPM1 gene is associated with familial dilated cardiomyopathy (DCM) but the effect of this mutation on Tpm's function is unknown. In this study, we introduced the K15N mutation in striated muscle α-Tpm (Tpm1.1) and investigated its interaction with actin, Tmod and Lmod. The mutation caused a ∼3-fold decrease in the affinity of Tpm1.1 for actin. The binding of Lmod and Tmod to Tpm1.1-covered actin filaments also decreased in the presence of the K15N mutation. Furthermore, the K15N mutation in Tpm1.1 disrupted the inhibition of actin polymerization and affected the competition between Tmod1 and Lmod2 for binding at the pointed ends. Our data demonstrate that the K15N mutation alters pointed end dynamics by affecting molecular interactions between Tpm1.1, Lmod2 and Tmod1.

Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities.

Role of tropomodulin's leucine rich repeat domain in the formation of neurite-like processes.

Actin dynamics is fundamental for neurite development; monomer depolymerization from pointed ends is rate-limiting in actin treadmilling. Tropomodulins (Tmod) make up a family of actin pointed end-capping proteins. Of the four known isoforms, Tmod1-Tmod3 are expressed in brain cells. We investigated the role of Tmod's C-terminal (LRR) domain in the formation of neurite-like processes by overexpressing Tmod1 and Tmod2 with deleted or mutated LRR domains in PC12 cells, a model system used to study neuritogenesis. Tmod1 overexpression results in a normal quantity and a normal length of processes, while Tmod2 overexpression reduces both measures. The Tmod2 overexpression phenotype is mimicked by overexpression of Tmod1 with the LRR domain removed or with three point mutations in the LRR domain that disrupt exposed clusters of conserved residues. Removal of Tmod2's LRR domain does not significantly alter the outgrowth of neurite-like processes compared to that of Tmod2. Overexpression of chimeras with the N-terminal and C-terminal domains switched between Tmod1 and Tmod2 reinforces the idea that Tmod1's LRR domain counteracts the reductive effect of the Tmod N-terminal domain upon formation of processes while Tmod2's LRR domain does not. We suggest that the TM-dependent actin capping ability of both Tmods inhibits the formation of processes, but in Tmod1, this inhibition can be controlled via its LRR domain. Circular dichroism, limited proteolysis, and molecular dynamics demonstrate structural differences in the C-terminal region of the LRR domains of Tmod1, Tmod2, and the Tmod1 mutant.

Alteration of tropomyosin-binding properties of tropomodulin-1 affects its capping ability and localization in skeletal myocytes.

Tropomodulin (Tmod) is an actin-capping protein that binds to the two tropomyosins (TM) at the pointed end of the actin filament to prevent further actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying actin filament dependent processes such as muscle contraction and intracellular transport. The capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1-4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the actin-capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the actin filament depends on its TM-binding affinity.

Recessive TMOD1 mutation causes childhood cardiomyopathy.

Familial cardiomyopathy in pediatric stages is a poorly understood presentation of heart disease in children that is attributed to pathogenic mutations. Through exome sequencing, we report a homozygous variant in tropomodulin 1 (TMOD1; c.565C>T, p.R189W) in three individuals from two unrelated families with childhood-onset dilated and restrictive cardiomyopathy. To decipher the mechanism of pathogenicity of the R189W mutation in TMOD1, we utilized a wide array of methods, including protein analyses, biochemistry and cultured cardiomyocytes. Structural modeling revealed potential defects in the local folding of TMOD1R189W and its affinity for actin. Cardiomyocytes expressing GFP-TMOD1R189W demonstrated longer thin filaments than GFP-TMOD1wt-expressing cells, resulting in compromised filament length regulation. Furthermore, TMOD1R189W showed weakened activity in capping actin filament pointed ends, providing direct evidence for the variant's effect on actin filament length regulation. Our data indicate that the p.R189W variant in TMOD1 has altered biochemical properties and reveals a unique mechanism for childhood-onset cardiomyopathy.

Tropomodulins and tropomyosins: working as a team.

Actin filaments are major components of the cytoskeleton in eukaryotic cells and are involved in vital cellular functions such as cell motility and muscle contraction. Tmod and TM are crucial constituents of the actin filament network, making their presence indispensable in living cells. Tropomyosin (TM) is an alpha-helical, coiled coil protein that covers the grooves of actin filaments and stabilizes them. Actin filament length is optimized by tropomodulin (Tmod), which caps the slow growing (pointed end) of thin filaments to inhibit polymerization or depolymerization. Tmod consists of two structurally distinct regions: the N-terminal and the C-terminal domains. The N-terminal domain contains two TM-binding sites and one TM-dependent actin-binding site, whereas the C-terminal domain contains a TM-independent actin-binding site. Tmod binds to two TM molecules and at least one actin molecule during capping. The interaction of Tmod with TM is a key regulatory factor for actin filament organization. The binding efficacy of Tmod to TM is isoform-dependent. The affinities of Tmod/TM binding influence the proper localization and capping efficiency of Tmod at the pointed end of actin filaments in cells. Here we describe how a small difference in the sequence of the TM-binding sites of Tmod may result in dramatic change in localization of Tmod in muscle cells or morphology of non-muscle cells. We also suggest most promising directions to study and elucidate the role of Tmod-TM interaction in formation and maintenance of sarcomeric and cytoskeletal structure.

The human erythrocyte plasma membrane: a Rosetta Stone for decoding membrane-cytoskeleton structure.

The mammalian erythrocyte, or red blood cell (RBC), is a unique experiment of nature: a cell with no intracellular organelles, nucleus or transcellular cytoskeleton, and a plasma membrane with uniform structure across its entire surface. By virtue of these specialized properties, the RBC membrane has provided a template for discovery of the fundamental actin filament network machine of the membrane skeleton, now known to confer mechanical resilience, anchor membrane proteins, and organize membrane domains in all cells. This chapter provides a historical perspective and critical analysis of the biochemistry, structure, and physiological functions of this actin filament network in RBCs. The core units of this network are nodes of ~35-37 nm-long actin filaments, interconnected by long strands of (α1ß1)2-spectrin tetramers, forming a 2D isotropic lattice with quasi-hexagonal symmetry. Actin filament length and stability is critical for network formation, relying upon filament capping at both ends: tropomodulin-1 at pointed ends and αß-adducin at barbed ends. Tropomodulin-1 capping is essential for precise filament lengths, and is enhanced by tropomyosin, which binds along the short actin filaments. αß-adducin capping recruits spectrins to sites near barbed ends, promoting network formation. Accessory proteins, 4.1R and dematin, also promote spectrin binding to actin and, with αß-adducin, link to membrane proteins, targeting actin nodes to the membrane. Dissection of the molecular organization within the RBC membrane skeleton is one of the paramount achievements of cell biological research in the past century. Future studies will reveal the structure and dynamics of actin filament capping, mechanisms of precise length regulation, and spectrin-actin lattice symmetry.

Structures of the free and capped ends of the actin filament.

The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.

Identification and validation of novel serum markers for early diagnosis of endometriosis.

BACKGROUND: Non-invasive diagnosis of endometriosis is urgently required to prevent the long delay between the onset of symptoms and diagnosis. A biomarker that possesses both high sensitivity and specificity is greatly required. Here, we describe the use of a proteomic approach to identify potential novel endometrial antigens using sera from endometriosis patients and healthy controls, with evaluation of biomarkers for non-invasive diagnosis of endometriosis. METHODS: A cross-sectional study was conducted to identify specific endometrial antigens using 1D and 2D western blots in women with early endometriosis (n = 17), advanced endometriosis (n = 23) and without endometriosis (n = 30). Five immunoreactive spots were analyzed using matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry with MASCOT analysis. ELISAs were established for specific epitopes and autoantibody titres were estimated in an independent cohort comprising women with early endometriosis (n = 18), advanced endometriosis (n = 32) and without endometriosis (n = 27) for validation. RESULTS: The 2D western blot analysis resulted in the identification of three endometrial antigens, tropomyosin 3 (TPM3), stomatin-like protein 2 (SLP2) and tropomodulin 3 (TMOD3). Serum levels of antibodies against the epitopes from the immunodominant region of proteins TPM3, SLP2 and TMOD3 were significantly elevated in endometriosis patients when compared with controls. Sensitivity and specificity of serum anti-TPM3a-autoAb (61%, 93%), anti-TPM3c-autoAb (44%, 93%), anti-TPM3d-autoAb (78%, 89%), anti-SLP2a-autoAb (50%, 96%), anti-SLP2c-autoAb (61%, 93%), anti-TMOD3b-autoAb (61%, 96%), serum anti-TMOD3c-autoAb (78%, 93%) and anti-TMOD3d-autoAb (78%, 96%) were better than those of serum CA125 levels (21%, 89%) in the detection of early stages of endometriosis. CONCLUSIONS: Serum anti-TPM3a-autoAb, anti-TPM3c-autoAb, anti-TPM3d-autoAb, anti-SLP2a-autoAb, anti-SLP2c-autoAb, anti-TMOD3b-autoAb, anti-TMOD3c-autoAb and anti-TMOD3d-autoAb could be new markers for the early diagnosis of endometriosis.

Mammalian tropomodulins nucleate actin polymerization via their actin monomer binding and filament pointed end-capping activities.

Many actin-binding proteins have been shown to possess multiple activities to regulate filament dynamics. Tropomodulins (Tmod1-4) are a conserved family of actin filament pointed end-capping proteins. Our previous work has demonstrated that Tmod3 binds to monomeric actin in addition to capping pointed ends. Here, we show a novel actin-nucleating activity in mammalian Tmods. Comparison of Tmod isoforms revealed that Tmod1-3 but not Tmod4 nucleate actin filament assembly. All Tmods bind to monomeric actin, and Tmod3 forms a 1:1 complex with actin. By truncation and mutagenesis studies, we demonstrated that the second α-helix in the N-terminal domain of Tmod3 is essential for actin monomer binding. Chemical cross-linking and LC-MS/MS further indicated that residues in this second α-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this α-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second α-helix of Tmod3, decreases both its actin monomer-binding and -nucleating activities. On the other hand, point mutations of residues in the C-terminal leucine-rich repeat domain of Tmod3 (Lys-317 in the fifth leucine-rich repeat ß-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal α6-helix) significantly reduced pointed end-capping and nucleation without altering actin monomer binding. Taken together, our data indicate that Tmod3 binds actin monomers over an extended interface and that nucleating activity depends on actin monomer binding and pointed end-capping activities, contributed by N- and C-terminal domains of Tmod3, respectively. Tmod3 nucleation of actin assembly may regulate the cytoskeleton in dynamic cellular contexts.

Nebulin regulation of actin filament lengths: new angles.

The highly organized arrays of thick and thin filaments found in striated muscles continue to be the subject of studies that yield groundbreaking concepts regarding cell motility. One example is the idea that massive, linearly extended polypeptides function as molecular rulers that set the length of polymeric filaments. Actin filaments that are polymerized in vitro exhibit wide variations in length, but many cells can assemble structures that contain actin filaments that are remarkably uniform. In striated muscles, the giant nebulin polypeptide extends the length of the actin filaments, and nebulin size has been correlated with actin filament lengths in muscles from different species. Here, I discuss a recent study by Gregorio and colleagues that demonstrates that nebulin knockdown leads to loss of actin filament-length regulation in cardiomyocytes, providing functional evidence that is consistent with the molecular ruler concept.

Tropomodulin capping of actin filaments in striated muscle development and physiology.

Efficient striated muscle contraction requires precise assembly and regulation of diverse actin filament systems, most notably the sarcomeric thin filaments of the contractile apparatus. By capping the pointed ends of actin filaments, tropomodulins (Tmods) regulate actin filament assembly, lengths, and stability. Here, we explore the current understanding of the expression patterns, localizations, and functions of Tmods in both cardiac and skeletal muscle. We first describe the mechanisms by which Tmods regulate myofibril assembly and thin filament lengths, as well as the roles of closely related Tmod family variants, the leiomodins (Lmods), in these processes. We also discuss emerging functions for Tmods in the sarcoplasmic reticulum. This paper provides abundant evidence that Tmods are key structural regulators of striated muscle cytoarchitecture and physiology.

Structural insights into the tropomodulin assembly at the pointed ends of actin filaments.

Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1-Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three-dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin-binding sites of tropomodulin and generated a three-dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self-consistent three-dimensional model of tropomodulin assembly at the pointed end. The model of the pointed-end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics.

A nebulin ruler does not dictate thin filament lengths.

To generate force, striated muscle requires overlap between uniform-length actin and myosin filaments. The hypothesis that a nebulin ruler mechanism specifies thin filament lengths by targeting where tropomodulin (Tmod) caps the slow-growing, pointed end has not been rigorously tested. Using fluorescent microscopy and quantitative image analysis, we found that nebulin extended 1.01-1.03 mum from the Z-line, but Tmod localized 1.13-1.31 mum from the Z-line, in seven different rabbit skeletal muscles. Because nebulin does not extend to the thin filament pointed ends, it can neither target Tmod capping nor specify thin filament lengths. We found instead a strong correspondence between thin filament lengths and titin isoform sizes for each muscle. Our results suggest the existence of a mechanism whereby nebulin specifies the minimum thin filament length and sarcomere length regulates and coordinates pointed-end dynamics to maintain the relative overlap of the thin and thick filaments during myofibril assembly.

Role of intrinsic disorder in muscle sarcomeres.

The role and utility of intrinsically disordered regions (IDRs) is reviewed for two groups of sarcomeric proteins, such as members of tropomodulin/leiomodin (Tmod/Lmod) protein homology group and myosin binding protein C (MyBP-C). These two types of sarcomeric proteins represent very different but strongly interdependent functions, being responsible for maintaining structure and operation of the muscle sarcomere. The role of IDRs in the formation of complexes between thin filaments and Tmods/Lmods is discussed within the framework of current understanding of the thin filament length regulation. For MyBP-C, the function of IDRs is discussed in the context of MYBP-C-dependent sarcomere contraction and actomyosin activation.

Molecular basis of tropomyosin binding to tropomodulin, an actin-capping protein.

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 (alpha, gamma, delta-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; alpha-TM, 2.5 nM; gamma-TM, delta-TM, 40-90 nM), but differ >100-fold for the first site (residues 1-38; alpha-TM, 90 nM; undetectable at 10 microM, gamma-TM, delta-TM). Residue 14 (R in alpha; Q in gamma and delta) and, to a lesser extent, residue 4 (S in alpha; T in gamma and delta) 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 alpha-TMs than gamma-TM in the presence of Tmod1. The binding sites of the two Tmod1 peptides on a model TM peptide differ, as defined by comparing (15)N,(1)H HSQC spectra of a (15)N-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 alpha-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.