Smooth muscle contractile cytoskeleton in health and disease

Figures

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  Fig. 1. Basics of smooth muscle myosin. A: Schematic diagram of the two heavy chains (red and blue), and how they are divided up into motor, lever and tail domains. The light chains bind to the lever. The myosin head is comprised of motor, lever and light chains. B: SMM molecules, in which the RLCs are dephosphorylated, form a shutdown compact molecule, in which the two myosin heads interact with each other, and the coiled coil wraps around the heads. Phosphorylation of the RLCs in the shutdown myosin molecule disrupts the shutdown state, allowing the resulting extended molecule to assemble into filaments

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  Fig. 2. Smooth muscle myosin (SMM) heavy chain isoforms. A: Shows the four different isoforms. SM1B and SM2B have a 7 amino acid insert in the motor domain, and SM1A and SM1B have a longer non-helical tailpiece (NHT) as shown. B: shows the sequence for SM1A C-terminal coiled-coil tail, organised as heptad repeats (see inset diagram). The a (red font) and d (black, bold font) residues form the hydrophobic seam. The sequences of the C-terminal non-helical tail piece for each myosin are shown below. Residues shown in purple (bold) have been found to be mutated in disease (from the Human Genome Database), mainly resulting in thoracic aortic aneurysms. C: shows a surface view for the structure of SMM (PDB:6Z47) heads region. Both free and blocked head contain Mg. ADP. Pi in the nucleotide binding site. The position of loop 1 (the sequence of which varies between the isoforms) is indicated. The positions of Ser19 residues in the two RLCs in indicated. D: shows the pseudoatomic model for the full-length myosin (Scarff et al. 2020). This was not uploaded to the PDB, however the equivalent full-length model of NM2A is available on the PDB (PDB:9SZR). The models showing how the coiled coil wraps…

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  Fig. 3. Mutations in SMM. A: shows the numbers of mutations for every 100 residues along the sequence, based on those reported in the HGMD database (accessed Nov. 2024). Over 70% of mutations lie in the coiled coil. The regions of coiled coil shown in A are identified in the full-length model in B, for Seg-2 (from Bend-1 to Bend-2) and Seg-3 (from Bend-2 to the end of the coiled coil) using the same colour scheme, with residue numbers as indicated. (List of missense mutations can be found in Table S1. Terminating mutations are excluded). Pseudoatomic model in B, not available on the PDB (see legend to Fig. 2)

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  Fig. 4. A: Sequence alignment between the two smooth muscle actin isoforms: smooth muscle ɣ-actin (ACTG2) and smooth muscle -actin (ACTA2). The 4 subdomains in actin are indicated by dashed boxes. Areas of interaction with myosin, tropomyosin are indicated (see Key). Residues reported in HGMD (Human Genome Mutation Database) reported to be missense mutations are indicated in red, and in bold red if 2 or more missense occur in the same residue. Terminating mutations are excluded. B: The structure of a single actin subunit from an actin filament structure (PDB: 8RU0; (Oosterheert et al. 2024) showing the 4 subdomains, specific loops involved in longitudinal contacts along a single strand of actin monomers in the filaments, and the pathogenic helix and sensor loop in relation to Mg2+ ATP in the central region of the molecule. C: shows two subunits from the two separate strands in the filament (PDB: 8RU0) to show the lateral interactions between the two strands. (List of missense mutations can be found in Table S2 & 3). D. A plot to show the frequency of mutations in ACTA2 and ACTG2: The number of missense mutations in groups of 30 residues (e.g. 0‑29, 30–59 and so on) were counted and then…

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  Fig. 5. Alignment of the two of the three smooth muscle isoforms of tropomyosin (TPM1.4 and TPM1.3). The positions of a and d residues in the heptad repeat are indicated. The positions of each exon coding region are indicated, with dotted lines for splicing that is not in frame, and solid for in frame. Exons shared with Tpm1.1, which codes for an isoform of tropomyosin found in striated (cardiac and skeletal) muscle are indicated in blue font, with exon 6b shared between TPM1.4 and 1.1 only, shown in green. Positions of missense mutations in the protein encoded by shared exons in the TPM1 gene (obtained from HGMD) are indicated by the blue coloured boxes, except for Exon 6b, where they are shown in green. Residues with more than 1 mutation are indicated by an asterisk. (The third SMC isoform, TPM2.1, is not shown). B. Exon usage by Tpm1.3 and 1.3 (smooth muscle) and Tpm1.1 (striated). C: Plot to show numbers of mutations per exon, for the shared exons between TPM1.1 and TPM1.4 and/or TPM1.3. (Mutations listed in Table S4)

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  Fig. 6. The sequence and ɑ-helical regions of H-caldesmon. A. Domain 1 contains a region of ɑ-helix that likely binds to the S-2 (the first part of the coiled coil: Subfragment-2), predicted to be in the region just below the interacting heads in shutdown SMM, as shown. Alphafold3 predicted structure for this region of caldesmon, together with structure of SMM (PDB:6Z47 (Scarff et al. 2020) (Fig. S2A, B). B: Sequence and the Alphafold3 predicted structure for domain 2 (Fig. S2C) predicts a long single ɑ-helix, only found in this long form of caldesmon. The presence of this long single ɑ-helix has been demonstrated experimentally. C: Sequence and Alphafold3 predicted structure (Fig. S2D) of domain 3, which is predicted to bind weakly to actin. Waggawagga (Simm et al. 2015) also predicts most of this region to be a second SAH) domain (Fig. S1). D: Sequence and Alphafold predicted structure of domain 4, which contains tropomyosin, calmodulin and strong actin binding sites, as shown (This prediction is the least likely to be correct, as predicted by Alphafold: Fig. S2E)

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  Fig. 7. Diagram to show where caldesmon and calponin may lie in the thin filament, and how caldesmon bridges across to the thick filament to interact with myosin. The C-terminal region, calponin and F-actin were modelled using Alphafold (Fig. S3). As Alphafold modelled tropomyosin poorly (as assessed by a visual inspection), tropomyosin in the Alphafold model was replaced by tropomyosin on F-actin from a cryoEM structure reported previously (PDB: 5NOG) (Risi et al. 2017) using ChimeraX (https://www.rbvi.ucsf.edu/chimerax). The N-terminal of caldesmon is associated with the S-2 region of myosin (Fig. 6A). The pink rectangle indicates the rod domain of smooth muscle myosin (coiled coil) that would lie within the myosin filament. The central SAH domain of calponin is shown as a blue rectangle. The C-terminal caldesmon is shown in its predicted region on the thin filament (as predicted by Alphafold modelling). Calponin is predicted (by Alphafold3) to run in the groove between the two actin strands in the filament. (Diagram not to scale)