The N2A region of titin has a unique structural configuration

Figures

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  Figure 1.. N2A location and Ig domain components of titin N2A with their unique loop features. (A) Left: Location of N2A in the sarcomere. Thin filaments shown in blue; thick filaments in yellow; Z-line gray rectangle. Titin’s tandem domains are orange and the PEVK is gray. Right: Domain composition of the N2A region. (B) Alignment of human titin sequences of N2A Ig domains (I80-I83) and representative domains from constitutive (I9-I11) and skeletal (I67-I69) tandems. BC loops are highlighted in blue, FG β-hairpin in orange, and CD loops in shades of green. (C) Ig domain components of the N2A region. I81, I82, and I83 domains are as elucidated crystallographically in this work; I80 was modeled using I-TASSER (Yang and Zhang, 2015). Ig domains are shown in cartoon representation, with BC, CD, and FG loop features colored as in B. C, C terminus; N, N terminus.

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  Figure 2.. Domain interfaces create a unique tandem architecture. (A) Residues involved in interdomain interactions are shown as sticks colored by atom type (N, mid-blue; O, red; C, as regions shown in Fig. 1, where the FG loop is orange, the BC loop is royal blue, and all other regions are white). Hydrogen bonds are shown as dashed lines. (B) Comparison of chain architecture in titin Ig tandems from N2A (I81-I83), constitutive (I9-I11; PDB accession no. 5JDD), and skeletal (I67-I69; PDB accession no. 2RIK) I-band regions. Structures are shown as cartoon ribbons with the surface shown as gray underlay. BC, CD, and FG features are colored as in A. To facilitate visual comparison, structures are displayed so that the middle Ig domain adopts a same orientation (C) View along the longitudinal molecular axis showing the relative orientation of domains in each tandem. Left as a cartoon ribbon and right as a schematic where the domains are a circle and the CD loops are represented as lines. BC, CD, and FG features are colored as in A. C, C terminus; N, N terminus.

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  Figure 3.. Electrostatic surface potential of I81-I83. Electrostatic potential energy at the I81-I83 protein surface reflecting charge distribution. The surface is colored by a gradient of potential energy values ranging from −15 kcal/mol.e (red) to +15 kcal/mol.e (blue; e is the charge of one electron).

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  Figure 4.. Transfection of fluorescently tagged human titin constructs into C2C12 cells. (A and B) Cells were transfected with constructs encoding titin GFP-I80-UN2A-I81 or mCherry-I81-I83. Undifferentiated cells (2 d, A; or 7 d, B) were transfected and counterstained with Alexa-647–phalloidin to visualize F-actin (red) and/or obscurin IQ-64 epitope antibody (Blondelle et al., 2019) to visualize obscurin (blue). Obscurin was used as a sarcomeric marker protein that localizes in a cross-striated pattern in mature, differentiated C2C12 myoblasts. Scale bar, 20 µm.

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  Figure 5.. F-actin cosedimentation of titin N2A components. (A and B) F-actin cosedimentation of UN2A-I81 (A) and I81-I83 (B) in the presence and absence of calcium. No cosedimentation is observed in either the presence or absence of calcium. Lanes are labeled as follows: P, pelleted fraction; S, soluble fraction; M, molecular weight marker.

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  Figure 6.. N2A and its interaction partners. Titin N2A is shown in forest green. The PEVK region is shown as a gray spring. Calcium is shown associated with PEVK. CAPN3/p94 (purple) associates with the I80-UN2A and I82-PEVK regions (Hayashi et al., 2008). MARPs (yellow) bind to UN2A-I81 (Zhou et al., 2016; Miller et al., 2003; Lun et al., 2014) and mediate the association of myopalladin to the node. SMYD2 localizes to UN2A and recruits HSP90 to N2A (Donlin et al., 2012). PKA (orange) phosphorylates S9578 in UN2A (Adams et al., 2019; Lanzicher et al., 2020).