Interplay between R513 methylation and S516 phosphorylation of the cardiac voltagegated sodium channel

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1 Supplemental Information Interplay between R513 methylation and S516 phosphorylation of the cardiac voltagegated sodium channel Amino Acids Pedro Beltran-Alvarez, 1, * Ferran Feixas, 2, Sílvia Osuna, 2, Rubí Díaz-Hernández, 1 Ramon Brugada, 1, * Sara Pagans 1, * 1 Cardiovascular Genetics Center, Institut d Investigació Biomèdica de Girona Dr. Josep Trueta, and Department of Medical Sciences, School of Medicine, University of Girona, ES Girona, Spain 2 Institut de Química Computacional i Catàlisi (IQCC) and Departament de Química, Universitat de Girona, Campus Montilivi, ES Girona, Spain * Contributed equally as corresponding authors. Sara Pagans: sara.pagans@udg.edu, Ramon Brugada: rbrugada@idibgi.org, Pedro Beltran-Alvarez: pbeltran@idibgi.org. Contributed equally. Supplemental Methods: Molecular Dynamics Online Resources S1 to S10 1

2 Supplemental Methods Molecular Dynamics All molecular dynamics (MD) simulations were done with the AMBER 12 package (Case et al. 2012) in an in-house graphics processing unit (GPU) cluster starting from the extended conformation of the peptide (Goetz et al. 2012, Salomon-Ferrer et al. 2013). Non-standard residue parameters for the methylated arginine found at position 7 of RmeS_short were obtained by using the antechamber module of Amber12 and the general AMBER force field (GAFF), (Wang et al. 2004). Partial charges were set to fit the electrostatic potential generated at the HF/6-31G(d) level by using the RESP model as implemented in Gaussian 09 (Frisch et al. 2009). For each system, the leap module was used to neutralize the peptide with counterions and to solvate the systems with a buffer region of 10 Å in all directions that resulted in a total of ~8,000 water molecules described by means of the TIP3P model (Jorgensen et al. 1983). The entire system was initially minimized in a two-step geometry optimization. First, a restrained minimization of solvent molecules and ions was performed using the whole peptide restrained by a harmonic potential with a force constant of 500 kcal mol -1 Å -2. Second, a minimization of the whole system was conducted with no restrains. The systems were progressively heated from 0 to 300 ᵒK during 120 ps and equilibrated in a NPT ensemble for 2 ns. Subsequently, a 50 ns MD simulation for each system was performed in the NVT ensemble with a Langevin thermostat using the PMEMD module of Amber 12 with the ff99sbildn force field (Hornak et al. 2006, Lindorff-Larsen et al. 2010). Long-range electrostatic interactions were described by means of the Particle Mesh Ewald summation method, while short-range non-bonded interactions were truncated using an 8 Å cutoff. Finally, to enhance conformational sampling of folded states, 100 ns of accelerated molecular dynamics simulations were computed using the dual boost approach (Hamelberg et al. 2004, Hamelberg et al. 2007). Supplemental References: Case DA, Darden TA, Cheatham III TE, Simmerling CL, Wang J, Duke RE, Luo R, Walker R, Zhang W, Merz KM, Roberts B, Hayik S, Roitberg A, Seabra F, Swails J, Götz AW, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wolf RM, Liu J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Cai Q, Ye X, Wang J, Hsieh M-J, Cui G, Roe DR, Mathews DH, Seetin MG, Salomon-Ferrer R, Sagui C, Babin V, Luchko T, Gusarov S, Kovalenko A, and Kollman PA (2012), AMBER 12, University of California, San Francisco. Goetz AW, Williamson MH, Xu D, Poole D, Le Grand S, Walker RC. (2012) Routine microsecond molecular dynamics simulations with AMBER - Part I: Generalized Born. J Chem Theory Comput 8, Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai, H, Vreven T, Montgomery JA Jr, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam NJ, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, 2

3 Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas Ö, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ. Gaussian, Inc., Wallingford CT. (2009). Hamelberg D, Mongan J, McCammon JA. (2004) Accelerated molecular dynamics: A promising and efficient simulation method for biomolecules. J Chem Phys, 120(24), Hamelberg D, de Oliveira CAF, McCammon JA. (2007) Sampling of slow diffusive conformational transitions with accelerated molecular dynamics. J Chem Phys, 127, Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C. (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65, Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79, Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO, & Shaw DE. (2010) Improved side-chain torsion potentials for the Amber ff99sb protein force field. Proteins 78, Salomon-Ferrer R, Goetz AW, Poole D; Le Grand S, Walker RC. (2013) Routine microsecond molecular dynamics simulations with AMBER - Part II: Particle Mesh Ewald. J Chem Theory Comput 9, Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. (2004) Development and testing of a general Amber foce field. J Comp Chem 25,

4 Online Resource S1. Example of phosphorylation time-course. RS_short (expected mass: Da) was incubated with PKA. Reaction rates of production of monophosphorylated RS_short (expected mass: Da) were calculated by dividing the area of phosphorylated RS_short by the area of RS_short, at different time points (inset). Online Resource S2. A. Side-by-side comparison of raw MALDI-TOF mass spectra of different concentrations of RS_short, and RmeS_short after 20, 40 and 60 min incubation with PKA. To allow comparison among different concentrations of the same peptide, and among different peptides, the same 3 time points are shown in all cases. For kinetic analyses, reaction rates were calculated using 5 7 time points within an appropriate time scale. For instance, reactions rates at high RS_short and RS_short_G514C concentrations were calculated using time points within the first 1 h of experiment. On the other hand, we used time points of up to 4 h to calculate reaction rates of RmeS_short. Expected masses are as follows: RS_short: Da; phosphorylated RS_short: Da; RmeS_short: Da; phosphorylated RS_short: Da. B. Raw MALDI-TOF mass spectra of different concentrations of RS_short_G514C after 20, 40 and 60 min incubation with PKA. Expected masses are as follows: RS_short_G514C: Da; phosphorylated RS_short: Da. Online Resource S3. LC-MS/MS analysis of phosphorylated RS_short. Only diagnostic fragment ions, which allow discrimination between phospho-s510 and phospho-s516, are labeled, as follows: green, precursor ions; red, b + ions; blue, y + ions. Online Resource S4. Molecular dynamics structures of RS_short (green) and RmeS_short (blue). The most representative conformation of each peptide sampled by a molecular dynamics trajectory is shown. R513 and methylated R513 are highlighted in sticks. The alignment of all Cα of both structures gave a root-mean square deviation (RMSD) of 0.4 Å. Online Resource S5. MALDI-TOF performance of peptides was not affected by introduction of methyl groups of the mutation G514C. A. MALDI-TOF mass spectrum of a 1 : 1 : 1 mixture, RS_short : RmeS_short : RS_short_G514C. Expected masses are as follows: RS_short: Da; RmeS_short: Da; RS_short_G514C: Da. B. MALDI-TOF mass spectrum of a 1 : 1 mixture, RmeS_short : Rme2S_short. Expected mass of Rme2S_short is Online Resource S6. A. MALDI-TOF mass spectra of RS_short, and RS_short incubated with PRMT3 for 3 h. Expected masses are as follows: RS_short: Da; monomethylated RS_short: Da; dimethylated RS_short: Da. B. MALDI-TOF mass spectra of RS_long, and RS_long incubated with PRMT3 for 2 h. Expected masses are as follows: RS_long: Da; monomethylated RS_long: Da; dimethylated RS_long: Da. 4

5 Online Resource S7. R513 is methylated by PRMT3. A. Expected fragments and m / z values after trypsin digestion of methylated RS_long. B. MALDI-TOF mass spectra of trypsin digests of RS_long (black), and of RS_long incubated with PRMT3 for 5 h (red). The tryptic fragment NHLSLTR (R513 bold and underlined) was observed in both cases (expected: Da). Previous incubation of RS_long with PRMT3 rendered new peaks at and Da, likely corresponding to mono- and di-methylated NHLSLTRGLSR. These peaks were only observed in methylated RS_long, most likely due to decreased trypsin performance in the presence of arginine methylation at R513. C. Same MALDI-TOF spectra in the mass range Da. Previous incubation of RS_long with PRMT3 (red) rendered new peaks at and Da, likely corresponding to mono- and di-methylated NHLSLTR. Online Resource S8. Methylated and phosphorylated peptides found after data mining our previous results (Beltran-Alvarez et al. 2011). Online Resource S9. Michaelis-Menten plot and apparent kinetic parameters of RS_short_G514C phosphorylation by PKA. Online Resource S10. Sequence alignment of region of interest of brain and cardiac isoforms of the voltage-gated sodium channel, highlighting R513 and S516 (bold and underlined, Na V 1.5 numbering) 5

6 Online Resource S1. 6

7 Online Resource 2. A. RS_short, 36.3 µm. Time points: 20, 40, 60 min. RmeS_short, 36.3 µm. Time points: 20, 40, 60 min. 7

8 RS_short, 12.1 µm. Time points: 20, 40, 60 min. RmeS_short, 12.1 µm. Time points: 20, 40, 60 min. 8

9 RS_short, 7.3 µm. Time points: 20, 40, 60 min. RmeS_short, 7.3 µm. Time points: 20, 40, 60 min. 9

10 RS_short, 4.8 µm. Time points: 20, 40, 60 min. RmeS_short, 4.8 µm. Time points: 20, 40, 60 min. 10

11 Peptide: RS_short, 3.6 µm. Time points: 20, 40, 60 min. Peptide: RmeS_short, 3.6 µm. Time points: 20, 40, 60 min. 11

12 B. RS_short_G514C, 36.3 µm. Time points: 20, 40, 60 min. RS_short_G514C, 12.1 µm. Time points: 20, 40, 60 min. 12

13 RS_short_G514C, 7.3 µm. Time points: 20, 40, 60 min. RS_short_G514C, 4.8 µm. Time points: 20, 40, 60 min. 13

14 RS_short_G514C, 3.6 µm. Time points: 20, 40, 60 min. RS_short_G514C, 1.8 µm. Time points: 20, 40, 60 min. 14

15 RS_short_G514C, 0.9 µm. Time points: 20, 40, 60 min. 15

16 Online Resource S3. 16

17 Online Resource S4. Online Resource S5. 17

18 Online Resource S6. 18

19 Online Resource S7. 19

20 Online Resource S8. Online Resource S9. Online Resource S10. 20