Fig 1.
Ribbon representation of the 3D structure of the Lmod2s1/αTM1a1-14Zip complex (PDB ID 6UT2).
N- and C-termini of Lmod2s1 and αTM1a1-14Zip are marked. The ribbon of Lmod2s1 is shown in cyan. The N-terminal Gly residue and the first 14 residues of Tpm1.1 in αTM1a1-14Zip are colored in black, and the C-terminal GCN4 sequence is colored in brown. The side chain of Leu25 in the link motif connecting two Lmod2 helices is shown in cyan and labeled. The residues of a transiently forming loop Lys12-Glu14 in Lmod2s1 are also labeled. 3D, three-dimensional; Lmod, leiomodin; PDB, Protein Data Bank.
Fig 2.
Mapping of 15N-Lmod2s1 resonance peak shifts caused by chemical/sequence alterations in the model Tpm peptide onto the Lmod2s1 sequence and the 3D structure of the Lmod2s1/αTM1a1-14Zip complex.
(A) Shown on a magenta background are 15N-Lmod2s1 residues with the most affected 15N-HSQC cross-peaks upon the replacement of the N-terminal Gly residue in αTM1a1-14Zip with an acetyl group. (B) Shown on an orange background are 15N-Lmod2s1 residues with the most affected 15N-HSQC cross-peaks upon the replacement of αTM1a1-14Zip (containing 14 N-terminal residues of Tpm) with αTM1a1-28Zip (containing 28 N-terminal residues of Tpm). (C) Mapping of the most affected regions of 15N-Lmod2s1 (shown in magenta) upon replacement of the N-terminal Gly residue in αTM1a1-14Zip with an acetyl group onto the 3D model of the complex. (D) Mapping of the most affected residues of 15N-Lmod2s1 (shown in orange) upon replacement of αTM1a1-14Zip with αTM1a1-28Zip onto the 3D model of the complex. 3D, three-dimensional; HSQC, heteronuclear single-quantum coherence; Lmod, leiomodin; Tpm, tropomyosin.
Fig 3.
Schematic comparison of the effects of αTM1a1-14Zip and αTM1a1-28Zip on the 15N-HSQC spectrum of 15N-labeled Lmod2s1 mapped onto the Lmod2s1 amino acid sequence.
Compound chemical shift changes (Δδ) upon Lmod2s1/αTM1a1-14Zip complex formation were calculated as Δδ = [(ΔδH)2 + (ΔδN/6.5)2]1/2 [79], where δH and δN are chemical shifts of 1H/15N nuclei of backbone amides. Peak attenuation upon the titration of Lmod2s1 with αTM1a1-28Zip (data for 7:1 Lmod2s1: αTM1a1-28Zip molar ratio were used) was calculated as a relative intensity of a 1H-15N cross-peak in comparison with the intensity of the corresponding peak in the absence of αTM1a1-28Zip. Each set of data was divided into four groups by calculating quartile values and displayed as”no color,” green, yellow, and red in the order of increasing spectral effects. Since cross-peaks from pairs of Lmod2s1 residues L10/L37 and I16/L21 overlap (see Supplementary Materials in [27]), the effect of αTM1a1-28Zip titration on their individual resonance peaks could not be determined. Hence, the residues L10, I16, L21, and L37 are labeled in the bottom sequence as gray. Lmod, leiomodin; HSQC, heteronuclear single-quantum coherence.
Fig 4.
GFP-Lmod2[L25G] is unable to elongate thin filaments compared with GFP-Lmod2 WT.
(A) Representative images of rat neonatal cardiomyocytes expressing GFP, GFP-Lmod2 WT, or GFP-Lmod2[L25G]. GFP-Lmod2[L25G] was unable to localize to the pointed ends, near the M-line (“M”) of sarcomeres, unlike GFP-Lmod2 WT. Cells were stained with phalloidin to mark F-actin and α-actinin to indicate the Z-disc (“Z”). Turquoise lines show a gap in F-actin staining across the M-line. Scale bar = 1 μm. (B) Thin filament lengths from rat neonatal cardiomyocytes transfected with GFP, GFP-Lmod2 WT, or GFP-Lmod2[L25G] (S1 Data). Statistical data are shown as mean ± SEM, n = 49–78 total measurements from 10–20 cells per culture, three cultures; *p = 0.0177, **p = 0.0013, one-way ANOVA. F-actin, filamentous actin; GFP, green fluorescent protein; Lmod, leiomodin; WT, wild type.
Fig 5.
GFP-Lmod2[L25G] does not significantly displace Tmod1 from thin filament pointed ends.
(A) Representative images showing Tmod1 assembly in rat neonatal cardiomyocytes expressing either GFP, GFP-Lmod2 WT, or GFP-Lmod2[L25G]. Cells were stained for Tmod1 and α-actinin (red) to mark Z. Scale bar = 1 μm. (B) Percentage of cells having consistent Tmod1 assembly at thin filament pointed ends (S2 Data). Mean ± SEM, n = 124–158, total number of cells measured, four cultures (*p = 0.0427, **p = 0.0011, one-way ANOVA). GFP, green fluorescent protein; Lmod, leiomodin; M, M-line; Tmod, tropomodulin; WT, wild type; Z, Z-disc.
Fig 6.
Comparison of the head-to-tail Tpm overlap junction (PDB ID 2G9J, left) with the Lmod2s1/αTM1a1-14Zip complex (right).
The sequence alignment of the structurally homologous parts of the C-terminal helix of Lmod2s1 and C-terminus of Tpm1.1 is shown at the bottom of the figure. Similar residues are marked in green on the sequences and their side chains are displayed on the 3D structures. 3D, three-dimensional; Lmod, leiomodin; PDB, Protein Data Bank; Tpm, tropomyosin.
Fig 7.
A model of Lmod2 assembly at the pointed end.
The view on the left shows the Lmod2 assembly at the pointed end of the thin filament including the entire tropomyosin molecule bound to F-actin (S3 Data). The view on the right shows the magnified boxed part of the assembly. Letters A and B label two pointed-end actin molecules (colored in tan and gray, respectively, to facilitate interpretation of the figure). Tropomyosin is shown in cyan, actin (with the exception of the pointed-end actin protomer B in the right panel) in tan, and TpmBS1 of Lmod2 in green. The Lmod2 ABS1h fragment shared with Tmod1 (residues Pro60-Leu86 in Lmod2) is shown in blue (modeled from the complex of homologous Tmod1 ABS1 with actin, PDB ID 4PKG, [19]). The fragment includes the conservative amphiphatic helix (residues Arg66-Lys79 in Lmod2). Within the linker connecting TpmBS1 and ABS1h (residues Pro42-Thr59), residues Asn45-Arg51, which were found critical for Lmod2 ABS1 binding to actin [23], are shown in red-orange. The remaining linker residues are shown in orange. ABS2 (LRR) is shown in magenta (modeled from the complex of Lmod2 LRR with actin [PDB ID 5WFN, [42, 43]]). The linker connecting ABS1h and ABS2 (residues Gly87-Asn195) is in the back and shown in brown. ABS, actin-binding site; F-actin, filamentous actin; Lmod, leiomodin; LRR, leucine-rich repeat; PDB, Protein Data Bank; TpmBS1, tropomyosin-binding site.
Fig 8.
A model for the leaky cap created by Lmod2 binding at the pointed end of the thin filament.
Top figures show side views, and bottom figures display views from the top. (A) The initial position of Lmod2 assembled at the pointed end and corresponding to the structure shown in Fig 7. ABS1 is in a “closed gate” conformation with the blue helix of ABS1 obstructing the attachment of an actin molecules to actin A. (B) Step 1, an actin molecule 1 (shown in purple) attaches to actin B. Actin molecule 1 does not interfere with the Lmod2 binding, and the ABS1 of Lmod2 remains in the “closed gate” conformation. (C) An actin molecule 2 (shown in turquoise) attaches to actin A. Interstrand interactions between actin molecules 1 and 2 facilitate displacement of Lmod2 ABS1 helix, and ABS1 adopts an “open gate” conformation (the blue helix moves away from the point of intrachain actin-actin interaction interface). This allows further actin polymerization until new tropomyosin N-termini are available for Lmod or Tmod attachment. ABS, actin-binding site; Lmod, leiomodin; Tmod, tropomodulin.