Sarah E. Hitchcock-DeGregori

Deciphering the design of the tropomyosin molecule

The crystal structure at 2.0-Å resolution of an 81-residue N-terminal fragment of muscle α-tropomyosin reveals a parallel two-stranded α-helical coiled-coil structure with a remarkable core. The high alanine content of the molecule is clustered into short regions where the local 2-fold symmetry is broken by a small (≈1.2-Å) axial staggering of the helices. The joining of these regions with neighboring segments, where the helices are in axial register, gives rise to specific bends in the molecular axis. We observe such bends to be widely distributed in two-stranded α-helical coiled-coil proteins. This asymmetric design in a dimer of identical (or highly similar) sequences allows the tropomyosin molecule to adopt multiple bent conformations. The seven alanine clusters in the core of the complete molecule (which spans seven monomers of the actin helix) promote the semiflexible winding of the tropomyosin filament necessary for its regulatory role in muscle contraction.

Inhibition of the Arp2/3 complex-nucleated actin polymerization and branch formation by tropomyosin

The actin filament network immediately under the plasma membrane at the leading edge of rapidly moving cells consists of short, branched filaments, while those deeper in the cortex are much longer and are rarely branched. Nucleation by the Arp2/3 complex activated by membrane-bound factors (Rho-family GTPases and PIP(2)) is postulated to account for the formation of the branched network. Tropomyosin (TM) binds along the sides of filaments and protects them from severing proteins and pointed-end depolymerization in vitro. Here, we show that TM inhibits actin filament branching and nucleation by the Arp2/3 complex activated by WASp-WA. Tropomyosin increases the lag at the outset of polymerization, reduces the concentration of ends by 75%, and reduces the number of branches by approximately 50%. We conclude that TM bound to actin filaments inhibits their ability to act as secondary activators of nucleation by the Arp2/3 complex. This is the first example of inhibition of branching by an actin binding protein. We suggest that TM suppresses the nucleation of actin filament branches from actin filaments in the deep cortex of motile cells. Other abundant actin binding proteins may also locally regulate the branching nucleation by the Arp2/3 complex in cells.

Tropomyosin has discrete actin-binding sites with sevenfold and fourteenfold periodicities

Analysis of the periodic distribution of amino acids in tropomyosin has revealed the presence of seven or 14 quasi-equivalent actin-binding sites. We tested the hypothesis of periodic actin-binding sites by making deletions of chicken striated alpha-tropomyosin cDNA using oligonucleotide-directed mutagenesis. The deletions corresponded to one-half (amino acid residues 47 to 67), two-thirds (residues 47 to 74) and one actin-binding site (residues 47 to 88), on the basis of there being seven sites. The mutant cDNAs were expressed as fusion and non-fusion proteins in Escherichia coli and analyzed for actin binding and regulatory function. Fusion tropomyosin binds to actin with an affinity similar to that of muscle tropomyosin. Of the mutant fusion tropomyosins, only that with a full site deleted retained actin affinity and the ability to inhibit the actomyosin S1 ATPase, though it was less effective than wild-type. We conclude that an integral number of half-turns of the tropomyosin coiled-coil, and the consequential sevenfold periodicity, as well as the correct orientation of the ends with respect to each other, are important for actin binding. On the other hand, non-fusion tropomyosin binds well to actin only in the presence of troponin, and the binding is calcium-sensitive. Assay of non-fusion mutant tropomyosins showed that mutants with deletion of one-half and one actin binding site both had high affinity for actin, equal to or slightly less than wild-type. The ability of these two mutants to regulate the actomyosin or acto-S1 ATPase with troponin in the absence of calcium was indistinguishable from that of the wild-type. The normal regulatory function of the mutant with a 1/14 deletion (removal of a quarter turn or half a site) indicates that a 14-fold periodicity is adequate for regulation, consistent with the presence of two sets of seven alpha and seven beta quasi-equivalent actin-binding sites. An alternative explanation is that the alpha-sites are of primary importance and that proper alignment of the alpha-sites in every second tropomyosin, as when half a site is deleted, is sufficient for normal regulatory function. Deletion of a non-integral period (2/3 of a site) severely compromised actin-binding and regulatory function, presumably due to the inability of the mutant to align properly on the actin filament.

A systematic nomenclature for mammalian tropomyosin isoforms.

Tropomyosin, a ubiquitous protein in animals and fungi, is associated with the actin cytoskeleton and is involved with stabilising actin filaments and regulating the interaction of the filament with other actin binding proteins. The protein is best known for its role in regulating the interaction between actin and myosin in muscle contraction but in recent years its role as a major player in the organisation and dynamics of the cytoskeleton has been increasingly recognised. In mammals Tpm is expressed from four distinct genes and alternate splicing of each gene can produce a total of up to 40 different mRNA variants most of which are expressed as proteins. We are expecting a renaissance in the study of tropomyosins as the roles of these different isoforms are beginning to be deciphered. However, it is our belief that such a renaissance is being limited by confusion over the naming systems for the tropomyosin isoforms. These result in even experienced workers struggling to reconcile work done in different laboratories and at different times. We propose here a systematic nomenclature for tropomyosin based on the best current practice. We recommend the adoption of these names and a cross-reference to the table of alternate names and accession numbers for protein sequences is included here. The National Center for Biotechnology Information (NCBI) website has been amended to include the nomenclature for the human, mouse and rat genes.

Tropomyosin: Function Follows Structure

Tropomyosin is known as the archetypal coiled coil, being the first to be sequenced and modeled. Studies of the structure and dynamics of tropomyosin, accompanied by biochemical and biophysical analyses of tropomyosin, mutants and model peptides, have revealed the complexity and subtleties required for tropomyosin function. Interruptions in the canonical coiled coil allow for bends and regions of local instability that are required for tropomyosin to bind to the helical actin filament. This chapter highlights insights gained from recent structural studies as they relate to variations in tropomyosins coiled-coil structure that are essential for binding to actin and the relationship of periodic repeats to actin molecules in the filament.

Evolutionarily conserved surface residues constitute actin binding sites of tropomyosin

Tropomyosin (Tm) is a two-chained, α-helical coiled-coil protein that associates end-to-end to form a continuous strand along actin filaments and regulates the functions and stability of actin in eukaryotic muscle and nonmuscle cells. Mutations in Tm cause skeletal and cardiac myopathies. We applied a neoteric molecular evolution approach to gain insight into the fundamental unresolved question of what makes the Tm coiled coil an actin binding protein. We carried out a phylogenetic analysis of 70 coding sequences of Tm genes from 26 animal species, from cnidarians to chordates, and evaluated the substitution rates (ω) at individual codons to identify conserved sites. The most conserved residues at surface b, c, f heptad repeat positions were mutated in rat striated muscle αTm and expressed in Escherichia coli. Each mutant had 3–4 sites mutated to Ala within the first half or the second half of periods 2–6. Actin affinity and thermodynamic stability were determined in vitro. Mutations in the first half of periods 2, 4, and 5 resulted in the largest reduction in actin affinity (> 4-fold), indicating these mutations include residues in actin-binding sites. Mutations in the second half of the periods had a ≤ 2-fold effect on affinity indicating these residues may be involved in other conserved regulatory functions. The structural relevance of these results was assessed by constructing molecular models for the actin-Tm filament. Molecular evolution analysis is a general approach that may be used to identify potential binding sites of a protein for a conserved protein.

Alteration of Tropomyosin Function and Folding by a Nemaline Myopathy-Causing Mutation

Mutations in the human TPM3 gene encoding gamma-tropomyosin (alpha-tropomyosin-slow) expressed in slow skeletal muscle fibers cause nemaline myopathy. Nemaline myopathy is a rare, clinically heterogeneous congenital skeletal muscle disease with associated muscle weakness, characterized by the presence of nemaline rods in muscle fibers. In one missense mutation the codon corresponding to Met-8, a highly conserved residue, is changed to Arg. Here, a rat fast alpha-tropomyosin cDNA with the Met8Arg mutation was expressed in Escherichia coli to investigate the effect of the mutation on in vitro function. The Met8Arg mutation reduces tropomyosin affinity for regulated actin 30- to 100-fold. Ca(2+)-sensitive regulatory function is retained, although activation of the actomyosin S1 ATPase in the presence of Ca(2+) is reduced. The poor activation may reflect weakened actin affinity or reduced effectiveness in switching the thin filament to the open, force-producing state. The presence of the Met8Arg mutation severely, but locally, destabilizes the tropomyosin coiled coil in a model peptide, and would be expected to impair end-to-end association between TMs on the thin filament. In muscle, the mutation may alter thin filament assembly consequent to lower actin affinity and altered binding of the N-terminus to tropomodulin at the pointed end of the filament. The mutation may also reduce force generation during activation.

A Periodic Pattern of Evolutionarily Conserved Basic and Acidic Residues Constitutes the Binding Interface of Actin-Tropomyosin

Background: The interface of actin with tropomyosin, the universal regulator of the actin filament, is unknown. Results: Mutagenesis of actin and tropomyosin revealed a pattern of residues required for complex formation in the closed state. Conclusion: The results support models of the actin-tropomyosin filament in the absence of myosin and troponin. Significance: A validated actin-tropomyosin model is required to understand regulation and disease mechanisms. Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the “closed state” where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp25-Glu334-Lys326-Lys328) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.

The Sequence of the Alternatively Spliced Sixth Exon of α-Tropomyosin Is Critical for Cooperative Actin Binding but Not for Interaction with Troponin

Tropomyosins, a family of highly conserved coiled-coil actin binding proteins, can differ as a consequence of alternative expression of several exons (Lees-Miller, J., and Helfman, D. (1991) BioEssays 13, 429–437). Exon 6, which encodes residues 189–213 in long, 284-residue tropomyosins, has two alternative forms, exon 6a or 6b, both highly conserved throughout evolution. In α-tropomyosin, exon 6a or 6b is not specific to any one of the nine isoforms. Exon 6b encodes part of a putative Ca2+-sensitive troponin binding site in striated muscle tropomyosins, suggesting that the exon 6-encoded region may be specialized for certain tropomyosin functions. A series of recombinant, unacetylated tropomyosin exon 6 deletion and substitution mutants and chimeras was expressed in Escherichia coli to determine the requirements of exon 6 for tropomyosin function. Functional properties of the tropomyosins were defined by actin affinity measured by cosedimentation, troponin T affinity using a newly developed biosensor assay, and regulation of the actomyosin MgATPase. The region of tropomyosin encoded by exon 6 affects actin affinity but not thin filament assembly, troponin T binding, or regulation with troponin. The tropomyosins with exon 6a or 6b function normally whether a striated muscle exon 9a or smooth/non-muscle exon 9d is present. However, the effect of deleting 21 amino acids encoded by exon 6 or replacing it with a GCN4 leucine zipper sequence depends on the COOH-terminal sequence.

Expression and biological activity of Baculovirus generated wild-type human slow α tropomyosin and the Met9Arg mutant responsible for a dominant form of nemaline myopathy

We have previously reported a Met9Arg mutation in the human skeletal muscle alpha tropomyosin gene (TPM3) associated with autosomal dominant nemaline myopathy [Nat. Genet. 9 (1995) 75]. We describe here the generation of wild-type (Wt-tpm3) and Met9Arg (M9R-tpm3) mutant human skeletal muscle slow alpha tropomyosin using the Baculovirus expression vector system (BEVS). This system produces correct posttranslationally modified recombinant tropomyosin proteins in insect cells. We show that the interactions of Wt-tpm3 with actin and tropomyosin are comparable to those of fast alpha tropomyosin isolated from chicken striated muscle. However, the recombinant M9R-tpm3 is at least 100 times less effective at binding actin than Wt-tpm3. This paper represents the first study of this mutation directly on the human isoform of tropomyosin that is involved in nemaline myopathy. It also represents the first time that human tpm3 has been produced using BEVS. This system can now be used to accurately demonstrate the effect of this (and other disease-associated tropomyosin mutations) on the interactions of tpm3 with the other protein components of the muscle thin filament, including those responsible for differing forms of nemaline myopathy.