Supplementary information

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1 Supplementary information Superoxide dismutase 1 is positively selected in great apes to minimize protein misfolding Pouria Dasmeh 1, and Kasper P. Kepp* 2 1 Harvard University, Department of Chemistry and Chemical Biology, Cambridge, MA, USA 2 Technical University of Denmark, DTU Chemistry, DK-2800 Kongens Lyngby, Denmark. Current address: Department of Biochemistry and Cedergren Center for Bioinformatics and Genomics, Faculty of Medicine, University of Montreal, 2900 Edouard-Montpetit, Montreal, Quebec H3T 1J4, Canada. * Corresponding Author kpj@kemi.dtu.dk Phone: S1

2 Figure S1. Multiple sequence alignment (Clustal Omega) of SOD1 in species studied in this work. Charged amino acids are marked in red and blue, respectively. Consensus amino acids are shown at the bottom of the alignment. S2

3 S3

4 Figure S2. Complete multiple sequence alignment of SOD1 in rodents and primates. Identical and similar positions are shown in blue and pink, respectively (made using the software msa from R[1]). Consensus amino acids are shown at the bottom of the alignment. S4

5 Figure S3. Tree from DataMonkey used to conduct relaxation tests. S5

6 Table S1. Codons detected to be under positive selection using different methods as explained in the main text. Datamonkey PAML SLAC FEL REL FUBAR MEME M8_rel Branchsite test[2] Table S2. Branches detected to be under positive selection. Branch leading to Corrected p-value a ω+ b Pr [ω=ω+] c Pig Beaver Great apes Opossum a: p-values corrected for multiple sampling using the Holm-Bonferooni method[3]. b: dn/ds for positively selected sites, c: fraction of sequence evolving with ω+. Table S3. Logarithm of likelihood function for null and alternative model in which relaxation is ignored and assumed. Branch set a ω1 b ω2 ω3 log L c LR (p-value) d Reference 0.00 (65%) (23%) 3.65 (12%) Test 0.00 (65%) (23%) 3.65 (12%) Unclassified (80%) (17%) 54.1 (2.8%) Reference (79%) (14%) 6.66 (6.6%) 6.20 (p = 0.013) Test (79%) (14%) 2.85 (6.6%) Unclassified (80%) (17%) 75.3 (2.8%) a: Branches refer to test (i.e., primates), alternative (i.e., rodents) and unclassified (i.e., rest) branch sets. b: different ωs refer to different site classes assumed for residues with the fraction of residues written in parenthesis. c: logarithm of likelihood function. d: Twice the log-likelihood (i.e., selection criteria) and the p-value associated with it in parenthesis. S6

7 Computed ΔΔG (kcal/mol) Computed ΔΔG (kcal/mol) Popmusic 2.1 y = 1.48x R² = Experimental normalized ΔΔG (Byström et al.) I-Mutant 2.0 y = 1.25x R² = Experimental normalized ΔΔG (Byström et al.) Figure S4. Correlation between experimental G (normalized G(norm) from Ref. [4]) and computed G using I-Mutant 2.0 and Popmusic 2.1. S7

8 Table S4. Data used for benchmarking Popmusic 2.1 and I-Mutant 2.0 (Figure S4). The normalized free energy changes G(norm) are taken from Ref. [4] Mutant G(norm) I-Mutant G(norm) Popmusic A4V V7E G37R L38V G41D G41S H43R H46R H48Q D76V D76Y L84V G85R N86D N86K N86S D90A D90V G93A G93D G93R G93S G93V E100G E100K D101G D101N I104F S105L L106V I113T G114A D124V D125H S134N N139D N139K L144F L144S V148G S8

9 # mutations ΔΔG (kcal/mol) Figure S5. The number of mutations with a ΔΔG in a certain range (in kcal/mol) for all possible mutations in SOD1 computed with Popmusic; most introduced mutations are destabilizing, and the distribution agrees well with the general behavior of such distributions[5]. S9

10 Table S5. Substitutions occurring in all branches of the studied phylogeny and associated changes in thermodynamic stability computed with I-Mutant 2.0 and Popmusic 2.1, and solvent accessibility of the substituted site s(sasa in %). Notice that the two methods have reverse sign conventions for G, so that stabilizing effects corresponds to positive for I-Mutant and negative for Popmusic. Details: "Calculation" describes whether the G value was obtained as the reverse substitution (with inversed sign of the G value) in the 2C9V structure, or as a composite substitution, requiring two substitutions to be added. For example, 1 T > A is "reverse" because A is the wild type residue in the 2C9V structure, so the mutation A to T was computed and the sign inversed. Similarly, if neither the start X or end Y residue was present in the structure but rather a third residue Z, the site substitution of interest was computed as G (XZ) + G (ZY) (referred to as "composite"). Summary of changes along branches. IMUTANT SASA POPMUSIC Calculation Branch 1: C9V 1 T > A # reverse S > N reverse K > E T > S composite K > T composite S > T reverse E > L reverse E > N composite S > A reverse N > E reverse K > T reverse SUM Branch 2: L > M composite E > V composite D > E composite E > Q A > P reverse P > E composite P > S reverse K > Q reverse Branch 3: SUM Branch 4: T > R composite E > Q SUM Branch 5: M > T reverse A > E S10

11 SUM Branch 6: Branch 7: Branch 8: T > M G > S reverse N > G G > E R > Q composite F > Y L > H K > A S > R E > Q SUM Branch 9: (Mouse) 12 N > V > L E > I > SUM Branch 10: (Rat) 12 N > T > V composite Q > E composite 57 E > I > S > T T > A SUM Branch 11: (Rabbit) D > G reverse N > G > S Q > E H > D composite A > N > T S > K composite Q > L reverse S11

12 54 57 E > I > K > S D > N N > D reverse S > L E > D reverse H > M I > V M > L reverse E > D Q > P SUM Branch 12: G > N composite V > L SUM Branch 13: (Squirrel) 12 N > A > K composite N > K composite N > A G > N P > K T > A L > Q K > T T > I G > D reverse E > Q composite E > P composite SUM Branch 14: (Beaver) N > P > S I > V N > G G > S > R composite Q > L reverse E > I > K > L S12

13 77 80 E > Q G > N composite N > D reverse S > N E > D A > T SUM Branch 15: S > K Q > E reverse K > T V > L E > A H > N SUM Branch 16: (Naked_Mole_rat) 12 N > Q > H K > Q A > T > A composite T > N Q > K composite 57 E > I > N > E composite S > F A > P composite SUM Branch 17: (Guinea_pig) 12 N > T > I reverse 25 A > G > A composite T > V Q > K composite 57 E > I > K > Q T > A composite E > P composite SUM S13

14 Branch 18: Branch 19: (Tree_Shrew) 2 M > L composite 12 N > G > E composite V > L composite S > T composite T > M composite 57 E > I > L > E K > S E > Q T > I N > D S > V E > A composite K > R SUM Branch 20: Branch 21: (Galago) 1 A > T N > P > A K > Q A > V > M composite S > K composite T > A Q > D composite 57 E > I > L > Q D > N V > E N > I composite V > M S > G SUM Branch 22: H > N A > E S14

15 25 26 G > S V > K S > W R > S Q > L K > R N > K composite E > D M > L SUM Branch 23: T > A K > E SUM Branch 24: (Marmoset) N > E > I > K > S composite D > V SUM Branch 25: (Capuchin) 12 N > E > I > Branch 26: G > S SUM Branch 27: K > Q I > F T > K S > G SUM Branch 28: (Baboon) N > N > S E > I > K > N composite S15

16 SUM Branch 29: (Macaque) 12 N > E > I > Branch 30: T > I G > D SUM Branch 31: (Gibbon) 12 N > W > Y S > R E > I > SUM Branch 32: M > T T > K Q > E Q > A K > D destab 114 S > C exp SUM Branch 33: (Orangutan) Q > K S > R K > E A > V E > I > D > S SUM Branch 34: S > G SUM Branch 35: (Gorilla) 12 N > E > S16

17 58 I > S > F SUM Branch 36: (Human) 12 N > E > I > Branch 37: (Bat) 1 A > T M > T reverse 3 K > R N > H > R composite K > E A > N > T V > K reverse S > F E > K Q > E reverse 57 E > I > S > R K > T K > Q G > E composite K > E D > N N > E composite I > L E > K V > Q E > A composite I > V A > R K > R E > D T > K SUM Branch 38: Branch 39: A > E S17

18 11 11 D > Q Q > E E > V V > L S > A E > D reverse A > R K > Q Q > K SUM Branch 40: E > Q SUM Branch 41: G > D K > Q reverse SUM Branch 42: (Dog) 12 N > A > N > S G > E N > X E > I > N > I composite H > Y SUM Branch 43: (Fox) 1 1 E > T composite 2 2 M > T reverse Q > D reverse N > P > K E > Q reverse V > E reverse A > G > A composite P > L E > D composite E > I > S18

19 68 71 S > G K > T Q > E reverse V > M N > H composite S > H S > A L > M composite R > P composite D > E composite E > D T > K Q > K reverse SUM Branch 44: (Cat) 12 N > E > I > I > M SUM Branch 45: (Giant_panda) 12 N > A > K composite G > E composite N > G E > I > N > T composite I > L SUM Branch 46: G > E composite N > G E > D composite S > D A > P SUM Branch 47: (Horse) 2 2 M > L composite N > Q > H T > V composite S19

20 23 24 K > Q A > Q composite V > L S > K composite T > F composite T > E composite E > K Q > E reverse E > I > S > T P > A G > D reverse K > E D > N V > K N > D reverse I > M E > K E > K composite P > Q composite Q > P SUM Branch 48: M > T reverse P > T S > T N > T K > R SUM Branch 49: (Pig) N > H > Y composite Q > L A > G G > K G > V > L T > K reverse T > A E > I > L > E E > Q S20

21 D > Y S > A E > D A > T SUM Branch 50: Q > A G > D T > S reverse G > D reverse D > N T > I composite E > V S > P V > L H > Y Q > P SUM Branch 51: (Goat) 12 N > A > E > T > K E > I > T > K R > C SUM Branch 52: Branch 53: (Red_deer) 12 N > H > R composite 25 A > E > D > H composite 57 E > I > I > K composite P > S reverse Y > H reverse G > R S > N S21

22 P > Q SUM Branch 54: Branch 55: (Bovine) 12 N > A > E > E > I > P > K composite SUM Branch 56: (Sheep) 12 N > H > R composite 25 A > E > T > K E > I > T > K S > G SUM Branch 57: (Opossum) 1 A > V N > H > F composite A > Q composite G > V composite N > G G > E V > L T > S reverse T > K reverse T > A E > I > L > H K > T G > N composite N > T composite E > K V > H S22

23 S > E H > M E > A T > E SUM Branch 58: (Chicken) 8 8 L > M N > G > A Q > E T > V composite E > Q K > Q A > E > D Q > N reverse E > I > S > G K > Q E > A E > D G > D > G S > E S > T S > C reverse E > A K > R N > D K > L SUM S23

24 References [1] U. Bodenhofer, E. Bonatesta, C. Horejš-Kainrath, S. Hochreiter, msa: an R package for multiple sequence alignment, Bioinformatics. (2015) btv494. [2] J. Zhang, R. Nielsen, Z. Yang, Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level, Mol. Biol. Evol. 22 (2005) [3] S. Holm, A simple sequentially rejective multiple test procedure, Scand. J. Stat. 6 (1979) [4] R. Byström, P.M. Andersen, G. Gröbner, M. Oliveberg, SOD1 mutations targeting surface hydrogen bonds promote amyotrophic lateral sclerosis without reducing apo-state stability., J. Biol. Chem. 285 (2010) doi: /jbc.m [5] N. Tokuriki, F. Stricher, J. Schymkowitz, L. Serrano, D.S. Tawfik, The stability effects of protein mutations appear to be universally distributed., J. Mol. Biol. 369 (2007) doi: /j.jmb S24