Coiled-coil Dimers Research Articles

TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly.

Tightly controlled duplication of centrosomes, the major microtubule-organizing centers of animal cells, ensures bipolarity of the mitotic spindle and accurate chromosome segregation. The RBCC (RING-B-box-coiled coil) ubiquitin ligase TRIM37, whose loss is associated with elevated chromosome missegregation and the tumor-prone developmental human disorder Mulibrey nanism, prevents the formation of ectopic spindle poles that assemble around structured condensates containing the centrosomal protein centrobin. Here, we show that TRIM37's TRAF domain, unique in the extended TRIM family, engages peptide motifs in centrobin to suppress condensate formation. TRIM proteins form anti-parallel coiled-coil dimers with RING-B-box domains on each end. Oligomerization due to RING-RING interactions and conformational regulation by B-box-2-B-box-2 interfaces are critical for TRIM37 to suppress centrobin condensate formation. These results indicate that, analogous to anti-viral TRIM ligases, TRIM37 activation is linked to the detection of oligomerized substrates. Thus, TRIM37 couples peptide motif recognition and substrate-dependent oligomerization to effect ubiquitination-mediated clearance of ectopic centrosomal protein assemblies.

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-β) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-β production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

The Lrs14 family of DNA-binding proteins as nucleoid-associated proteins in the Crenarchaeal order Sulfolobales.

Organization of archaeal chromatin combines bacterial, eukaryotic, and unique characteristics. Many archaeal lineages harbor a wide diversity of small and highly expressed nucleoid-associated proteins, which are involved in DNA structuring. In Sulfolobales, representing model organisms within the Crenarchaeota, Sul7d, Cren7, Sul10a, and Sul12a are well-characterized nucleoid-associated proteins. Here, we combine evidence that the Lrs14 family of DNA binders is part of the repertoire of nucleoid-associated proteins in Sulfolobales. Lrs14-encoding genes are widespread within genomes of different members of the Sulfolobales, typically encoded as four to nine homologs per genome. The Lrs14 proteins harbor a winged helix-turn-helix DNA-binding domain and are typified by a coiled-coil dimerization. They are characterized by distinct sequence- and structure-based features, including redox-sensitive motifs and residues targeted for posttranslational modification, allowing a further classification of the family into five conserved clusters. Lrs14-like proteins have unique DNA-organizing properties. By binding to the DNA nonsequence specifically and in a highly cooperative manner, with a slight preference for AT-rich promoter regions, they introduce DNA kinks and are able to affect transcription of adjacent transcription units either positively or negatively. Genes encoding Lrs14-type proteins display considerable differential expression themselves in response to various stress conditions, with certain homologs being specific to a particular stressor. Taken together, we postulate that members of the Lrs14 family can be considered nucleoid-associated proteins in Sulfolobales, combining a DNA-structuring role with a global gene expression role in response to stress conditions.

Structural and biophysical characterization of PadR family protein Rv1176c of Mycobacterium tuberculosis H37Rv

Rv1176c of Mycobacterium tuberculosis H37Rv belongs to the PadR-s1 subfamily of the PadR family of protein. Rv1176c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 39.4 °C. The crystal structure of Rv1176c was determined at a resolution of 2.94 Å, with two monomers in the asymmetric unit. Each monomer has a characteristic N-terminal winged-helix-turn-helix DNA-binding domain. Rv1176c C-terminal is a coiled-coil dimerization domain formed of α-helices α5 to α7. In the Rv1176c dimer, there is domain-swapping of the C-terminal domain in comparison to other PadR homologs. In the dimer, there is a long inter-subunit tunnel in which different ligands can bind. Rv1176c was found to bind to the promoter region of its own gene with high specificity. M. smegmatis MC2 155 genome lacks homolog of Rv1176c. Therefore, it was used as a surrogate to characterize the functional role of Rv1176c. Expression of Rv1176c in M. smegmatis MC2 155 cells imparted enhanced tolerance towards oxidative stress. Rv1176c expressing M. smegmatis MC2 155 cells exhibited enhanced intracellular survival in J774A.1 murine macrophage cells. Overall, our studies demonstrate Rv1176c to be a PadR-s1 subfamily transcription factor that can moderate the effect of oxidative stress.

Role of GARP family transcription factors in the regulatory network for nitrogen and phosphorus acquisition

The GARP (Golden2, ARR-B, Psr1) family proteins with a conserved DNA-binding domain, called the B-motif, are plant-specific transcription factors involved in the regulation of various physiological processes. The GARP family proteins are divided into members that function as monomeric transcription factors, and members that function as transcription factors in the dimeric form, owing to the presence of a coiled-coil dimerization domain. Recent studies revealed that the dimer-forming GARP family members, which are further divided into the PHR1 and NIGT1 subfamilies, play critical roles in the regulation of phosphorus (P) and nitrogen (N) acquisition. In this review, we present a general overview of the GARP family proteins and discuss how several members of the PHR1 and NIGT1 subfamilies are involved in the coordinated acquisition of P and N in response to changes in environmental nutrient conditions, while mainly focusing on the recent findings that enhance our knowledge of the roles of PHR1 and NIGT1 in phosphate starvation signaling and nitrate signaling.

Crystal Structure of de Novo Designed Coiled-Coil Protein Origami Triangle.

Coiled-coil protein origami (CCPO) uses modular coiled-coil building blocks and topological principles to design polyhedral structures distinct from those of natural globular proteins. While the CCPO strategy has proven successful in designing diverse protein topologies, no high-resolution structural information has been available about these novel protein folds. Here we report the crystal structure of a single-chain CCPO in the shape of a triangle. While neither cyclization nor the addition of nanobodies enabled crystallization, it was ultimately facilitated by the inclusion of a GCN2 homodimer. Triangle edges are formed by the orthogonal parallel coiled-coil dimers P1:P2, P3:P4, and GCN2 connected by short linkers. A triangle has a large central cavity and is additionally stabilized by side-chain interactions between neighboring segments at each vertex. The crystal lattice is densely packed and stabilized by a large number of contacts between triangles. Interestingly, the polypeptide chain folds into a trefoil-type protein knot topology, and AlphaFold2 fails to predict the correct fold. The structure validates the modular CC-based protein design strategy, providing molecular insight underlying CCPO stabilization and new opportunities for the design.

Establishing Fluorine-Containing Amino Acids as an Orthogonal Tool in Coiled Coil Assembly.

The α-helical coiled coil (CC) is one of the best-characterized folding motifs in the protein world. In this context, fluorinated amino acids have been shown to be capable of tuning the properties of CC assemblies, and especially fluorinated derivatives of aliphatic amino acids can significantly increase the stability of this folding motif when placed in the hydrophobic a and d positions. However, it has not been shown yet whether fluorinated amino acids, by means of rational design, can be used as an orthogonal tool to control CC assembly processes. In the current work, we approached this question by creating a combinatorial peptide library based on a VPE/VPK heteromeric CC system previously established and characterized in our group. This CC model allowed us to screen fluorinated amino acids for interaction with different potential binding partners in position a of the VPE/VPK model with a particular emphasis on studying the impact of stereochemistry within the side chain of α-branched aliphatic fluorinated amino acids on CC properties such as oligomerization state, thermodynamic stability, and orientation. 28 combinations of library members were characterized regarding structure, oligomerization, and thermal stability utilizing circular dichroism, size exclusion chromatography, and Förster resonance energy transfer measurements. This detailed approach showed that the stability and oligomerization state of the motif were not only dependent on the steric demand and the fluorination of corresponding amino acids but also on the stereochemistry within the side chain. The results were applied for a rational design of the fluorine-driven orthogonal assembly, and we could show that CC dimer formation occurred based on specific interactions between fluorinated amino acids. These results demonstrate the potential of fluorinated amino acids as an orthogonal tool besides classical electrostatic and hydrophobic interactions for the fine-tuning and direction of peptide-peptide interactions. Furthermore, within the space of fluorinated amino acids, we could demonstrate the specificity of interactions between differently fluorinated side chains.

Intrinsic mesoscale properties of a Polycomb protein underpin heterochromatin fidelity.

Little is understood about how the two major types of heterochromatin domains (HP1 and Polycomb) are kept separate. In the yeast Cryptococcus neoformans, the Polycomb-like protein Ccc1 prevents deposition of H3K27me3 at HP1 domains. Here we show that phase separation propensity underpins Ccc1 function. Mutations of the two basic clusters in the intrinsically disordered region or deletion of the coiled-coil dimerization domain alter phase separation behavior of Ccc1 in vitro and have commensurate effects on formation of Ccc1 condensates in vivo, which are enriched for PRC2. Notably, mutations that alter phase separation trigger ectopic H3K27me3 at HP1 domains. Supporting a direct condensate-driven mechanism for fidelity, Ccc1 droplets efficiently concentrate recombinant C. neoformans PRC2 in vitro whereas HP1 droplets do so only weakly. These studies establish a biochemical basis for chromatin regulation in which mesoscale biophysical properties play a key functional role.

Enhanced Liposomal Drug Delivery Via Membrane Fusion Triggered by Dimeric Coiled-Coil Peptides.

An ideal nanomedicine system improves the therapeutic efficacy of drugs. However, most nanomedicines enter cells via endosomal/lysosomal pathways and only a small fraction of the cargo enters the cytosol inducing therapeutic effects. To circumvent this inefficiency, alternative approaches are desired. Inspired by fusion machinery found in nature, synthetic lipidated peptide pair E4/K4 is used to induce membrane fusion previously. Peptide K4 interacts specifically with E4, and it has a lipid membrane affinity and resulting in membrane remodeling. To design efficient fusogens with multiple interactions, dimeric K4 variants are synthesized to improve fusion with E4-modified liposomes and cells. The secondary structure and self-assembly of dimers are studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The structures and membrane interactions of PK4 are supported by molecular dynamics simulations. Upon addition of E4, PK4 induced the strongest coiled-coil interaction resulting in a higher liposomal delivery compared to linear dimers and monomer. Using a wide spectrum of endocytosis inhibitors, membrane fusion is found to be the main cellular uptake pathway. Doxorubicin delivery results in efficient cellular uptake and concomitant antitumor efficacy. These findings aid the development of efficient delivery systems of drugs into cells using liposome-cell fusion strategies.

Domain tethering impacts dimerization and DNA-mediated allostery in the human transcription factor FoxP1.

Transcription factors are multidomain proteins with specific DNA binding and regulatory domains. In the human FoxP subfamily (FoxP1, FoxP2, FoxP3, and FoxP4) of transcription factors, a 90 residue-long disordered region links a Leucine Zipper (ZIP)-known to form coiled-coil dimers-and a Forkhead (FKH) domain-known to form domain swapping dimers. We used replica exchange discrete molecular dynamics simulations, single-molecule fluorescence experiments, and other biophysical tools to understand how domain tethering in FoxP1 impacts dimerization at ZIP and FKH domains and how DNA binding allosterically regulates their dimerization. We found that domain tethering promotes FoxP1 dimerization but inhibits a FKH domain-swapped structure. Furthermore, our findings indicate that the linker mediates the mutual organization and dynamics of ZIP and FKH domains, forming closed and open states with and without interdomain contacts, thus highlighting the role of the linkers in multidomain proteins. Finally, we found that DNA allosterically promotes structural changes that decrease the dimerization propensity of FoxP1. We postulate that, upon DNA binding, the interdomain linker plays a crucial role in the gene regulatory function of FoxP1.

Lamin Filament Assembly Derived from the Atomic Structure of the Antiparallel Four-Helix Bundle.

The nucleoskeletal protein lamin is primarily responsible for the mechanical stability of the nucleus. The lamin assembly process requires the A11, A22, and ACN binding modes of the coiled-coil dimers. Although X-ray crystallography and chemical cross-linking analysis of lamin A/C have provided snapshots of A11 and ACN binding modes, the assembly mechanism of the entire filament remains to be explained. Here, we report a crystal structure of a coil 2 fragment, revealing the A22 interaction at the atomic resolution. The structure showed detailed structural features, indicating that two coiled-coil dimers of the coil 2 subdomain are separated and then re-organized into the antiparallel-four-helix bundle. Furthermore, our findings suggest that the ACN binding mode between coil 1a and the C-terminal part of coil 2 when the A11 tetramers are arranged by the A22 interactions. We propose a full assembly model of lamin A/C with the curvature around the linkers, reconciling the discrepancy between the in situ and in vitro observations. Our model accounts for the balanced elasticity and stiffness of the nuclear envelopes, which is essential in protecting the cellular nucleus from external pressure.

Quantifying cooperative multisite binding in the hub protein LC8 through Bayesian inference.

Multistep protein-protein interactions underlie most biological processes, but their characterization through methods such as isothermal titration calorimetry (ITC) is largely confined to simple models that provide little information on the intermediate, individual steps. In this study, we primarily examine the essential hub protein LC8, a small dimer that binds disordered regions of 100+ client proteins in two symmetrical grooves at the dimer interface. Mechanistic details of LC8 binding have remained elusive, hampered in part by ITC data analyses employing simple models that treat bivalent binding as a single event with a single binding affinity. We build on existing Bayesian ITC approaches to quantify thermodynamic parameters for multi-site binding interactions impacted by significant uncertainty in protein concentration. Using a two-site binding model, we identify positive cooperativity with high confidence for LC8 binding to multiple client peptides. In contrast, application of an identical model to the two-site binding between the coiled-coil NudE dimer and the intermediate chain of dynein reveals little evidence of cooperativity. We propose that cooperativity in the LC8 system drives the formation of saturated induced-dimer structures, the functional units of most LC8 complexes. In addition to these system-specific findings, our work advances general ITC analysis in two ways. First, we describe a previously unrecognized mathematical ambiguity in concentrations in standard binding models and clarify how it impacts the precision with which binding parameters are determinable in cases of high uncertainty in analyte concentrations. Second, building on observations in the LC8 system, we develop a system-agnostic heat map of practical parameter identifiability calculated from synthetic data which demonstrates that the ability to determine microscopic binding parameters is strongly dependent on both the parameters themselves and experimental conditions. The work serves as a foundation for determination of multi-step binding interactions, and we outline best practices for Bayesian analysis of ITC experiments.

Structural Determinants and Functional Significance of Dimerization for Osmosensing Transporter ProP in Escherichia coli.

Osmosensing transporter ProP forestalls cellular dehydration by detecting environments with high osmotic pressure and mediating the accumulation of organic osmolytes by bacterial cells. It is composed of 12 transmembrane helices with cytoplasmic N- and C-termini. In Escherichia coli, dimers form when the C-terminal domains of ProP molecules form homodimeric, antiparallel, α-helical coiled coils. No dominant negative effect was detected when inactive and active ProP molecules formed heterodimers in vivo. Purification of ProP in detergent dodecylmaltoside yielded monomers, which were functional after reconstitution in proteoliposomes. With other evidence, this suggests that ProP monomers function independently whether in the monomeric or dimeric state. Amino acid replacements that disrupted or reversed the coiled coil did not prevent in vivo dimerization of ProP detected with a bacterial two-hybrid system. Maleimide labeling detected no osmolality-dependent variation in the reactivities of cysteine residues introduced to transmembrane helix (TM) XII. In contrast, coarse-grained molecular dynamic simulations detected deformation of the lipid around TMs III and VI, on the lipid-exposed protein surface opposite to TM XII. This suggests that the dimer interface of ProP includes the surfaces of TMs III and VI, not of TM XII as previously suggested by crosslinking data. Homology modeling suggested that coiled-coil formation and dimerization via such an interface are not mutually exclusive. In previous work, alterations to the C-terminal coiled coil blocked co-localization of ProP with phospholipid cardiolipin at E. coli cell poles. Thus, dimerization may contribute to ProP targeting, adjust its lipid environment, and hence indirectly modify its osmotic stress response.

Dual-wavelength stopped-flow analysis of the lateral and longitudinal assembly kinetics of vimentin

Vimentin is a highly charged intermediate filament protein that inherently forms extended dimeric coiled coils, which serve as the basic building blocks of intermediate filaments. Under low ionic strength conditions, vimentin filaments dissociate into uniform tetrameric complexes of two anti-parallel-oriented, half-staggered coiled-coil dimers. By addition of salt, vimentin tetramers spontaneously reassemble into filaments in a time-dependent process: 1) lateral assembly of tetramers into unit-length filaments, 2) longitudinal annealing of unit-length filaments, and 3) longitudinal assembly of filaments coupled with subsequent radial compaction. To independently determine the lateral and longitudinal assembly kinetics, we measure with a stopped-flow instrument the static light scattering signal at two different wavelengths (405 and 594 nm) with a temporal resolution of 3 ms and analyze the signals based on Rayleigh-Gans theory. This theory considers that the intensity of the scattered light depends not only on the molecular weight of the scattering object but also on its shape. This shape dependence is more pronounced at shorter wavelengths, allowing us to decompose the scattered light signal into its components arising from lateral and longitudinal filament assembly. We demonstrate that both the lateral and longitudinal filament assembly kinetics increase with salt concentration.

Cyclin-dependent kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain

Nuclear lamins maintain the nuclear envelope structure by forming long linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The A11 binding mode is characterized by the antiparallel interactions between coil 1b domains, whereas the A22 binding mode is facilitated by interactions between the coil 2 domains of lamin. The junction between A11- and A22-interacting dimers in the lamin tetramer produces another parallel head–tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers depolymerization of lamin filaments, but the associated mechanisms remain unknown at the molecular level. In this study, we revealed using the purified proteins that phosphorylation by the CDK1 complex promotes disassembly of lamin filaments by directly abolishing the ACN interaction between coil 1a and the C-terminal portion of coil 2. We further observed that this interaction was disrupted as a result of alteration of the ionic interactions between coil 1a and coil 2. Combined with molecular modeling, we propose a mechanism for CDK1-dependent disassembly of the lamin filaments. Our results will help to elucidate the cell cycle–dependent regulation of nuclear morphology at the molecular level.

Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear. Here, we uncover DNA mimicry by the ciliary kinesin KIF7 as a mechanism for the recruitment of GLI to microtubules, wherein the coiled-coil dimerization domain of KIF7, characterized by its striking shape, size and charge similarity to DNA, forms a complex with the DNA-binding zinc fingers in GLI, thus revealing a mode of tethering a DNA-binding protein to the cytoskeleton. GLI increases KIF7 microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive GLI tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We retooled this coiled-coil-based GLI-KIF7 interaction to inhibit the nuclear and cilium localization of GLI. This strategy can potentially be exploited to downregulate erroneously activated GLI in human cancers.

Metal ion-regulated assembly of designed modular protein cages.

Coiled-coil (CC) dimers are versatile, customizable building modules for the design of diverse protein architectures unknown in nature. Incorporation of dynamic self-assembly, regulated by a selected chemical signal, represents an important challenge in the construction of functional polypeptide nanostructures. Here, we engineered metal binding sites to render an orthogonal set of CC heterodimers Zn(II)-responsive as a generally applicable principle. The designed peptides assemble into CC heterodimers only in the presence of Zn(II) ions, reversibly dissociate by metal ion sequestration, and additionally act as pH switches, with low pH triggering disassembly. The developed Zn(II)-responsive CC set is used to construct programmable folding of CC-based nanostructures, from protein triangles to a two-chain bipyramidal protein cage that closes and opens depending on the metal ion. This demonstrates that dynamic self-assembly can be designed into CC-based protein cages by incorporation of metal ion–responsive CC building modules that act as conformational switches and that could also be used in other contexts.

Equipping Coiled-Coil Peptide Dimers With Furan Warheads Reveals Novel Cross-Link Partners.

Using a coiled-coil peptide dimer as a model system to explore furan reactivity, we describe novel cross-link partners of furan warheads for site-specific cross-linking. We demonstrate that replacement of weak interhelical ionic contacts with a furan moiety and its potential cross-link partner affords covalently connected coiled-coil motifs upon furan activation. We describe for the first time the reaction of the activated furan warhead with cysteine and tyrosine, besides the previously reported lysine, thus enhancing the versatility of the furan cross-link methodology by the possibility to target different amino acids. The present in vitro validation of “furan-armed” α-helices provides further grounds for exploiting furan technology in the development of furan-modified ligands/proteins to target proteins in a covalent way through various amino acid side chains.

Novel insertion mutation (Arg1822_Glu1823dup) in MYH6 coiled-coil domain causing familial atrial septal defect

ObjectiveAtrial septal defect, secundum (ASD Ⅱ, OMIM: 603642) is the second common congenital heart defect (CHD) in China. However, the genetic etiology of familial ASD II remains elusive. Methods and resultsUsing whole-exome sequencing (WES) and Sanger sequencing, we identified a novel myosin heavy chain 6 (MYH6) gene insertion variation, NM_002471.3: c.5465_5470dup (Arg1822_Glu1823dup), in a large Chinese Han family with ASD II. The variant Arg1822_Glu1823dup co-segregated with the disease in this family with autosomal dominant inheritance. The insertion variant located in the coiled-coil domain of the MYH6 protein, which is highly conserved across homologous myosin proteins and species. In transfected myoblast C2C12 cell lines, the MYH6 Arg1822_Glu1823dup variant significantly impaired myofibril formation and increased apoptosis but did not significantly reduce cell viability. Furthermore, molecular simulations revealed that the Arg1822_Glu1823dup variant impaired the myosin α-helix, increasing the stability of the coiled-coil myosin dimer, suggesting that this variant has an effect on the coiled-coil domain self-aggregation. These findings indicate that Arg1822_Glu1823dup variant plays a crucial role in the pathogenesis of ASD II. ConclusionOur findings expand the spectrum of MYH6 variations associated with familial ASD II and may provide a molecular basis in genetic counseling and prenatal diagnosis for this Chinses family.

Structural and Functional Peculiarities of Cytoplasmic Tropomyosin Isoforms, the Products of TPM1 and TPM4 Genes

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.