Journal of Muscle Research and Cell Motility

Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation

Wenjun Zheng*, Sarah E. Hitchcock-DeGregori§, and Bipasha Barua§

*Department of Physics, University at Buffalo, Buffalo, New York 14260§Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey 08854

E-mail: wjzheng@buffalo.edu

Supporting InformationTable S1 Summary of 40 -Tpm -Tpm mutations in periods P1-P7

period mutations from-Tpm to -Tpm P1P2P3P4P5

P6P7

L19I(e), A31Q(c), S36C(a), D41E(f), L43Q(a), V44Q(b), S45A(c) L57V(a), D58E(b), A63S(g), L64V(a), D66E(c), L73Q(c)

S132N(f), Q135M(b), I143L(c), I146M(f), A158S(d)I172L(d), S174G(f), D175E(g), A179S(d), L185V(c), S186A(d), G188S(f), A191G(b), E192D(c), T199I(c)E212D(b), Q216T(f), V227L(c), S229E(e), D230E(f)T247A(b), S252T(g), L260V(a), L265M(f), H276N(c), M281I(a), I284L(d)

Table S2 Comparison of minimal Tpm-actin distances between MD simulation and other structural models of Tpm-actin complex

minimal Tpm-actin CA-CA distance (Å)all residue pairs charged residue pairs non-charged residue pairs

wild-type MD*

Li model(Li et al. 2011)4A7F(Behrmann et al. 2012)4A7H(Behrmann et al. 2012)4A7L(Behrmann et al. 2012)3J8A (von der Ecken et al. 2015) 3J4K (Sousa et al. 2013)

5.62±0.41 7.28±0.57 6.22±0.39

7.82 8.48 9.85

7.81 8.26 7.81

7.28 7.28 7.40

7.56 8.36 8.76

8.31

7.09

*averaged over five actin subunits (AC07, AC09, AC11, AC13 and AC15, see Fig 1a) grep calc_mindis2ac out_tmac_1 | awk -f awkfile_avg_std

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Table S3 Radial distance of Tpm from the F-actin axis

radial distance* (Å)average minimum maximum

wild-type MD

Li model(Li et al. 2011)

41.84±0.68 32.85±0.82 53.87±3.60

44.47 36.89 51.81

*assessed by the average, minimum, or maximum of distances between all Tpm residues and the F-actin axis

Table S4 Fitting of Tpm-actin binding data to reparameterize and

mutant Kd ratio between mutant and WT target Gmutant-GWT

(kcal/mol)fitted Gmutant-GWT with G=EvdW+Eelec * (=0.08, =0.12) (kcal/mol)

L185R D137L

D175N

E180G

E54K

0.62 (Gupte et al. 2015)no effect (Matyushenko et al. 2015; Sumida et al. 2008)2.83 (Kremneva et al. 2004)1.26 (Golitsina et al. 1997)2.6 (Janco et al. 2012)3.78 (Kremneva et al. 2004) 2.62 (Golitsina et al. 1997) 2.1 (Janco et al. 2012)3.52 (Mirza et al. 2007)

-0.3 0.0 0.5

0.6

0.8

-0.1 0.0 0.3

0.6

0.9dG fitting of alpha and beta (dg_tmac.c) -> a.out

* Further adding flexibility parameters (such as or persistent length) into G does not improve the fitting, which supports our choice to ignore entropy in the calculation.

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Figure S1. Results of local flexibility analysis of the E62Q mutant in comparison with the wild-type Tpm

using the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for the wild type and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the wild type and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA1, ALA5, ALA6, and E218).

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Figure S2. Results of local flexibility analysis of the A63V mutant in comparison with the wild-type Tpm

using the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for the wild type and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the wild type and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA1, ALA3, ALA4, ALA6, and E218).

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Figure S3. Results of local flexibility analysis of the K70T mutant in comparison with the wild-type Tpm

using the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for the wild type and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the wild type and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA1, ALA3, ALA5, and E218).

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Figure S4. Results of local flexibility analysis of the V95A mutant in comparison with the wild-type Tpm

using the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for the wild type and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the wild type and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA2, ALA4, and ALA5).

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Figure S5. Results of local flexibility analysis of the D137L mutant in comparison with the wild-type

Tpm using the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for the wild type and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the wild type and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA1, ALA2, D137, ALA5, and E218).

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Figure S6. Results of local flexibility analysis of the E54R mutant in comparison with the WT Tpm using

the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue position of Tpm are shown as points in the upper and lower half of each panel, respectively (colored blue for the WT and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters ALA1-ALA6 (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the WT and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near D137, E218, and ALA5 in P5).

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Figure S7. Results of local flexibility analysis of the E180G mutant in comparison with the wild-type

Tpm using the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for the wild type and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the wild type and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near D137, ALA5, and E218).

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Figure S8. Results of local flexibility analysis of the L185R mutant in comparison with the wild-type

Tpm using the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for the wild type and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the wild type and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA1, ALA5, and E218).

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Figure S9. Results of local flexibility analysis of the E192K mutant in comparison with the wild-type

Tpm using the following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for the wild type and red for the mutant). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between the wild type and the mutant Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA1, ALA2, ALA3, ALA4, and E218).

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Figure S10. Results of local flexibility analysis of the -Tpm in comparison with the -Tpm using the

following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for -Tpm and red for -Tpm). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between -Tpm and -Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA1, ALA3, ALA4, ALA5, and E218).

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Figure S11. Results of local flexibility analysis of the -Tpm in comparison with the -Tpm using the

following parameters: (a) , (b) , (c) , (d) AS, (e) diameter. The average (AVE) and standard deviation (SD) of the above parameters as a function of a/d residue positions of Tpm are shown as points (colored blue for -Tpm and red for -Tpm). The data points for AVE (SD) are shown in the upper (lower) half of each panel. The green vertical lines mark the center positions of six Ala clusters (A22, A78, A120, A155, A183, and A239). The black dashes give the SD differences between -Tpm and -Tpm. Regions with pronounced SD differences are marked by arrows (e.g., near ALA1-5, and D137).

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