Coiled Coil Architecture Analysis

Evaluation with the coiled coil analysis program TWISTER (Strelkov & Burkhard 2002) shows that the pitch varies locally along the filament and total coiled coil turns of 1.78 for conformer 1 and 1.82 for conformer 2 can be measured. The ~90° phase shift orientation also corresponds to the longer low molecular weight tropomyosin, exhibiting ~2.75 turns of the coiled-coil per molecule. This three quarter pitch relates to the non-integral number of repeats, so the actin interacting residues are consistent along the thin filament, despite overlap complex and superhelical winding (Hitchcock-DeGregori & Singh, 2010). Based on full length and fragment structures of Tpm1, the average pitch is estimated to be 140 – 148 Å (Brown et al., 2005; Whitby & Phillips, 2000). Average pitch length of respective Cdc8p conformers 1 and 2 are 131.4 Å and 128.5 Å. The overall range bandwidth of interhelical radius is similar to observed ranges of Tpm1 (Brown et al., 2005; Minakata et al., 2008; Strelkov & Burkhard, 2002). The coiled coil radius distributions are very similar for both conformers (Figure 30a), except the region around the kink (residue 50 to 80).

Here, conformer 1 fluctuates strongly in radius, whereas the conformer 2 transitions smoother into the low radius segment of the central heptad break (Figure 30a, dashed line). When comparing Cdc8p with all vertebrate Tpm isoforms, no repetition of classic alanine cluster can be found. Only one segment at the N-terminus (residues A11 to A25) shows a classic alanine cluster with a characteristically low interhelical radius and a subsequent 2-3° bend (Brown et al. 2001). Furthermore, three broken core segments with high radius can be estimated, which is caused by heptad breaking residues (shown for Tpm1, Minakata et al. 2008; Lehman et al., 2019) (Figure 30a). One complex broken core segment is located before the kink, where K39 is on d-position and S50on a-position. This results to a broadly connected region with wider interhelical radius. Two further broken core regions can be found after the kink, where N98 is located on a-position and R130 on d-position. In both of latter cases the broken core segments show high interhelical radii (Figure 30).

Aromatic residues F119 and Y144 are located on ‘a’ and ‘d’-positions, they are associated with low interhelical distances. One aromatic residue of the hydrophobic core (Y44) is located in the center of the broken core segment of K39 and S50. For all three aromatic residue pairs on hydrophobic core position, the π-electron systems are non-stacked and oriented to neighboring residues (Chapter 3.4.5). This allows the coiled coil to reach close together to the closest interhelical radii besides the kink. The local staggering angle (LSA) (Nitanai et al., 2007) and filament curvature (Strelkov & Burkhard, 2002) are similar for both conformers, except for the bandshift break (Figure 29). Different LSA and curvature patterns for the different conformers of the same protein were previously demonstrated (Minakata et al. 2008). The curvature profile for the kinked conformer 1 displays a prominent kink of 23.2° at the residues 71 to 73

Results

64 (Local bending angle calculated after Nitanai et al., 2007). The coiled coil curvature of the straight conformer 2 displays two single kinks in this region which depict the chicane-like character (Figure 28c +Figure 30, upper half). The local staggering angle profile for both conformers is similar (LSA calculation adapted from Nitanai et al., 2007) (Figure 29, lower half). Two exceptions are both flanking regions of the kink. Whereas the staggering of relaxed conformer 2 is mostly negative (90° is unstaggered, <90° is negative staggering)(Figure 29d), the tensional conformer 1 is positively staggered in that region (>90° is positive staggering) (Figure 29, lower half, blue boxes). The broken core residues as well as the aromates on ‘a’ and ‘d’-position are mostly located in unstaggered segments. Only K39 shows slight inversion of staggering between the two conformers. For both conformers N98 is located in a negative staggered segment. Local twisting strength is described by the local pitch length (Strelkov & Burkhard, 2002) and is for both conformers depicted in Figure 30b. Both graphs are very similar, except minor variation at the broken core patterns and the pre-kink segment. The local pitch length pattern of the only classic alanine cluster in Cdc8p drops below 100 Å, which indicates strong winding. This is a described pattern for such segments (Minakata et al., 2008). The broken core segment between K39 and S50 displays a broad peak with low winding. More precisely, S50 is located in the sub average region beyond the peak and K39 in a low winding peak. The connection of broken core segments with low winding is also described in literature (Brown et al., 2001; Lakkaraju & Hwang, 2009; Minakata et al., 2008). The two broken cores around N98 and R130 are both located in low winding peaks, which both differ slightly in height and width. The post kink aromatic residues on heptad core positions (F119 and Y144) lie in overwinding valleys (Figure 30b), exhibiting local pitch lengths between 100 and 115 Å. The bandshift heptad break shows for both conformers very strong overwinding with local pitch lengths of approximately 30 Å.

Results

65

Figure 29: Coiled coil architecture of both conformers – LSA and curvature. The left Y-axis and the lower graphs represent the local staggering angle LSA (scheme of LSA measurement in bottom left corner). Stagger threshold of 90° is marked with a dashed line. The colors are in accordance with an established color code. Blue boxes represent the stagger inversion segments. The right Y-axis and the upper graphs represent the coiled coil curvature (scheme of curvature measurement is in top right corner).

Average curvature of 1.42° is marked with a dashed line. Alanine stagger, kink region, broken core residues, and hydrophobic core located aromatic residues are marked

Results

66

Results

67

Figure 30: Coiled coil architecture of both conformers – Interhelical radius and local pitch length. a) Interhelical radius of the coiled coil of the two conformers along the molecule. Tight regions including the kink are additionally marked with the residue range. Broken core regions and important residues are marked. Conformer 1 is represented by the blue graph and conformer 2 by the magenta graph. Scheme of interhelical distance measurement is depicted bottom right. b) Blue and pink graphs represent the local pitches of conformer 1 and 2, respectively. Locations of aromatic heptad core residues are depicted in green. Broken core residues are shown in black. The overall average pitch length of both conformers is 130 Å and is marked as horizontal, dashed lines. Scheme of local pitch measurement is depicted bottom right.

Figure 31: Aromatic residues on heptad core positions. a) Left Y-axis and graph in black shows the initial distance of the Cα -pairs. Red Y-axis and graph represent the standard deviation of the Cα distance over 50 ns MD-simulation with NAMD. Heptad breaking residues are marked with filled red dots. Green filled dots mark the positions of aromatic residues in a/d-positions.

Green circles indicate stabilized segments with low fluctuation. Hard kink, alanine-cluster, and broken core segments are marked. b+c) According pairs of Y44 and Y144 (respectively,) with inter- and intrachain interactions (yellow dotted lines

Results

68