Structural diversity in the atomic resolution 3D fingerprint of the titin M-band segment

Figures

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  Fig 1. Sequence and structural diversity in titin M-band Ig domains. A) M-band domain organization in representative titin sequences from Homo sapiens (Q8WZ42), mouse (A2ASS6), horse (F6VG02), Atlantic salmon (A0A1S3PNI2), and zebrafish (A5X6X5). The preceding titin kinase domain has been included as a point of reference. The residue numbers indicated above correlate to the human titin sequence. Those domains for which high-resolution structures have been determined are colored in blue (X-ray) and cyan (NMR). M-band domains M7, M8, and M9 are missing in the sequences from Atlantic salmon and zebrafish. The length of the specified M-band domains and connecting loop regions are proportional to the human titin sequence. Calculated overall sequence identities, using the human titin sequence as reference, are indicated in % and are highlighted by a color spectrum: > 85%, red; >70%, orange; >50%, yellow; <50%, no color. B) Structure-based alignment of the M1, M3, M4, M5, M7, and M10 human titin sequences. Aligned residues are in upper case; residues with an uncertain alignment are in lower case. The positions of the secondary structural elements taken from the M1 structure are shown on top of…

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  Fig 2. High-resolution structures of M-band domains A) M1, B) M3, C) M4, D) M5, E) M7 and F) M10. Cartoon representations in two different orientations; the second view is rotated by approximately 90° around a vertical axis. The two opposite β-sheets ABDE and A’CC’FG are colored in magenta and cyan, respectively. The conserved 310 helix within the EF loop at the C-tip is colored in orange. Cysteines and the exposed specificity residue (residue position 14 in M1, cf. Fig 1B) are shown in sticks in atom-specific colors. Cysteines are highlighted in yellow (with sulfhydryl groups oriented towards hydrophobic core), orange (with potential for disulfide bridge formation), and red (with sulfhydryl group surface exposed). Zoom-ins of the N-tip (upper inlet) and C-tip (lower inlet) areas highlight those residues that are involved in specific side-chain mediated hydrogen bonds. For reasons of clarity, only hydrogen bonds formed by side chain/side chain interactions are shown. Residues that are conserved among different M-band sequences are labeled in red.

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  Fig 3. Conserved structural elements in M-band domains. A) Conservation of main chain hydrogen pattern: 6/6 structures, red; 5/6 structures, salmon; 4/6 structures, orange; 3/6 structures, yellow. The numbers of conserved hydrogen bonds are listed for the two β-sheets ABDE and A’CC’FG, and the C-tip area. B) Display of residue positions that are conserved among different M-band sequences (cf. Fig 1B) in salmon, and residues that are conserved for each M-band domain among different species but variable in different M-band domains in green. From the left to right: semi-transparent surface presentation (left), stick representation (center), and stick representation rotated by 90° around a vertical axis (right). The color scheme is consistent with Fig 2.

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  Fig 4. Mechanisms of M-band domain assembly. A) Structures of apo-M10 and M10 in complex with obscurin [20]. In the apo-M10 structure, β-strands with unused valences of hydrogen bond-mediated β-sheet formation are shown in strong colors and are labeled. The remaining β-strands are shown in paler colors. In the structure of the M10-obscurin complex, only β-strand B, interacting with β-strand G of obscurin (green), is shown in strong color (magenta). Other parts of the obscurin molecule are shown in pale green. B) Structure of the M4-SS-M4’ assembly. Colors are as in Fig 2, expect that the loops of the second M4’ molecule are shown in light pink. The zoomed-in inset shows structural details of the M4-M4’ disulfide bridge and the Zn2+ ion (blue sphere), which is symmetrically bound in a tetrahedral arrangement by two aspartates (D68, D68’) and two histidines (H70, H70’) from M4 and M4’. C) Superimposed and normalized gel filtration elution curves of M1, M3, M4, and M7 under reducing (green) and non-reducing (violet) conditions. For the M4 domain, in addition, the elution curve under non-reducing conditions in the presence of Zn2+ is shown (pale violet). D) Non-reducing (left) and reducing…