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

Abstract

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.

Keywords: Circular dichroism; Dilated cardiomyopathy; Intrinsically disordered regions; Leiomodin; Nuclear magnetic resonance; Tropomodulin.

Figure 1

The schematic view of the binding sites of Tmod1 and Lmod2 molecules. TM1, TM2 tropomyosin-binding sites; A1, A2, A3 actin-binding sites; LRR leucine rich repeats. The inset shows the amino acid sequence of the tropomyosin-binding site of Lmod2 and the first tropomyosin-binding site of Tmod1. Identical residues are shown in red.

Figure 2

Sequences of the model Tpm1.1 peptides. The residues of αTM1a encoded by exon 1a of the Tpm1 gene are in red and are underlined. The number of residues of 1a is shown on left. A Gly residue (shown with g) was added to the N-terminus of each peptide to mimic acetylation. The C-terminal GCN4 sequence is shown in black. The positions a and d of the heptad repeat of coiled-coil molecules are listed under the corresponding residues.

Figure 3

2D ¹⁵N-HSQC spectra of ¹⁵N-labeled Lmod2s1 in the presence and absence of αTM1a_1-14Zip. The spectra were recorded on a Varian VNMRS 600 MHz spectrometer. The concentration of Lmod2s1 was 0.22 mM. The blue spectrum was recorded in the absence of αTM1a_1-14Zip. The green and red spectra were obtained in the presence of 0.12 mM and 0.24 mM αTM1a_1-14Zip, respectively.

Figure 4

Comparison of the Lmod2s1 NMR spectra in the presence of Tpm1.1 model peptides of varying lengths. The 2D ¹⁵N-HSQC spectra of ¹⁵N-labeled Lmod2s1 were recorded in the presence of a >20% stoichiometric excess of αTM1a_1-14Zip (red), αTM1a_1-21Zip (green), or αTM1a_1-28Zip (blue). Arrows show the cross-peaks of the Lmod2s1/αTM1a_1-14Zip complex that are markedly shifted with respect to those of the Lmod2s1/αTM1a_1-28Zip complex. The cross-peaks of residues 4–6 are not affected by binding and are shown in boxes.

Figure 5

Chemical shift index analysis of the unbound ¹⁵N/¹³C-Lmod2s1. The sequence of the peptide is displayed on top. Assigned atoms are displayed as green boxes. Chemical shifts consistent with α-helical or β-sheet structure are displayed as bars with red or blue circles, respectively. The position of two α-helices is shown schematically at the bottom of the figure.

Figure 6

Representative unfolding curves of peptide complexes and CD spectra of αTM1a_1-21Zip. Binding of 40 µM Lmod2s1 or Tmod1s1 to equimolar concentration of αTM1a_1-21Zip (a) wild type (WT) and (b) [K15N] was assayed using CD spectroscopy. The graphs illustrate the unfolding curves of the individual peptides (Lmod2s1, Tmod1s1, αTM1a_1-21Zip), the mixtures (Lmod2s1/αTM1a_1-21Zip, Tmod1s1/αTM1a_1-21Zip) and the sums of the individual unfolding curves (Lmod2s1+αTM1a_1-21Zip, Tmod1s1+αTM1a_1-21Zip). The lines show fitted curves to the experimental data. Spectra of αTM1a_1-21Zip and αTM1a_1-21Zip[K15N] at (c) 0°C and (d) 15°C.

Figure 7

Crystal structure of Tpm1.1 (PDB #1IC2) demonstrating distances between Asp14 and Lys15. Chimera software was used to calculate the distances.