Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase CHEMISTRY ++++十十十十十+十十++++++++++十十十十十十十十十+++十十++++十十十十十十十十++++++++++十十十十十十十十十+++++++++十十十+十十十十十+++++++++++++十 MKEVVYWSPKKVADWLLENAMPEYCEPLEHFTGQDLINLTQEDEKKPPLCRVSSDNGORLLDMIETLKMEHHLEAHKNGHANGHLNIGVDIPTPDGSESI 110 130 140 150 170 180 200 +++++++十+++十+++++++++++++十++十工工工工工工 KIKPNGMPNGYRKEMIKIPMPELERSQYPMEWGKTFLAFLYALSCEVLTTVMISVVHERVPPKEVQPPLPDTFFDHFNRVQWAFSICEINGMILVGLWL 210 220 260 +++++++工工工工工X 00000XXXX工工工工工++++ OLLLKYKSIISRRFECIVGILYLYRCITMYVTILPVPGMHENCSPKLEGDWEAQLRRIMKLIAGGGLSITGSHNMCGDYLYSGHTVMLTLTYLEIKEYS 310 340 370 390 +++++++工工工工工工XXXo0000o--00000X 工工工++++++++++++十十++++++++++++十+++++++++++++++++++ PRRLWWYHWICWLLSVVGIECILLAHDHYTVDVVVAYYITTRLEWWYHTMANQQVLKEASQMNLLARVWWYRPFQYEEKNVQGIVPRSYHWPFPWPVVHL Figure 3 Transmembrane topology of hSMSl predicted by MEmsAt3. Here, the characters with broad lines, with thin lines and with- out line stand for transmembrane domains, loop regions, and terminal domains respectively (+ intracellular domains; the extracellular loop; O: Outside helix cap; X: Central transmembrane helix segment; 1: Inside helix cap) Table 1 Predicted locations of the trans-membrane helices on hSMS I Method N TMI TM3 TM4 TM5 TM6 AEMSTAT36M130-S154(25)Nl78W202(25)1210-1234(25)N275F295(21)1308-D332(25)V335-Q354(20) PSIPRED06E131H157(27)W821204(23)S209-1229(21)H285-E298(14)L304A325(22)V31-A351(21) Ssp26W32-H157(26)W82-K206(25)S2091228(20)T286F295(10)w305-A325(21)Y329353(25) Beta Pred26W132-R159(28)F184-k206(23)111-1234(24)T286-Y299(14)W306-A325(20)Y329-Q353(25) ConPred I235F136-V156(21)S185L205(21)F2151235(21) D279-￥299(21)Y307-D327(21) PROF- 6G133V156(24)R179K206(28)K2081233(26)T286K297(12)W306-L324(19)T330-M350(21) 6M130H157(28)Q181-L204(24)S209-1229(21)Y280-Y299(20)W305-A325(21)V331Q353(23) DPM 6M130-V160(31)Q181-k206(26)1210-1234(25)T286-F298(13)R302-H326(25)T330-Q353(24 DSC27 6M30-H157(28)W182-L205(24)1210-1233(24)V287-K297(11)R302-1323(22)D332Q353(22) GOR128 6M30-E158(29)R179L205(27)S209-P238(30)M276-k297021)I310-Y329(20)V331-Q353(23) GOR329 6M130V156(27)86-Y207(22)1210-1233(24)T286R302(17)L304A325(22)V331-Q353(23) 6W132-H157(26)1186-L205(20)S209-234(26)Y280-K297(18)R303-A325(23)V331-Q353(23) PHD 6E131-R159(29)Nl78L205(28)S209L235(27)C277-K297(21R303-L324(22)T330-Q353(24) Predator26Tl3V156(22)F184L205(2)S209T234(26)T286K297(12)W305-A325(21)T33353(24) 6T35-R159(25)W182-L205(24)S209-1234(26)M276k297(22)L304-324(21) ted number of Tms. Numbers in brackets are the lengths of tm Table 2 Comparison of the length of transmembrane helices on which the three key residues(H285, H328, and D332) hSMSI and GlpG(PDB entry: 21C8) could not form a reasonable arrangement in the catalytic Protein TM number TMI TM2 TM3 TM4 TM5 TM6 ock Iso excluded. Among the remaining hSMS I 6 252525212520 models, the one with the lowest probability density function(PDF)energy computed by Modeler was cho- GlpC 271720182118 sen for further refinement. In order to relax the steric repulsions between side chains, side chain refinement loop. All of the resulting models were then visually with restraints on backbone was performed. Each loop checked to exclude those having serious internal col was refined by high-level optimizations by using Mod- sions. Moreover, as for the third extracellular loop(loop eler. Side-chain refinement was performed 5, residues 327-339), two highly conserved residues cause the conformation of backbone could have bee H328 and D332 on this loop formed hydrogen bond, changed during the optimization performed at the pre- which could be used as an additional criterion to select vious step appropriate model for loop 5 Then. all of the whole structural models of hSMSI Molecular dynamics simulation of the hSMSI/SM were visually inspected to exclude those containing complex structure crossing loops or serious internal steric collisions Based on the final representing model of hSMSI Models on which the catalytic pocket were too small to SM was manually docked into the catalytic pocket in accommodate PC or SM were excluded. Models on favor of the SN2 nucleophilic substitution reaction. The Chin J. Chem. 2011, 29, 1567--1575 C2011 SIOC, CAS, Shanghai, WILEY-VCH Verlag gmbH Co KGaA, Weinheim wwcjc. wiley-vch. de 1569Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase Chin. J. Chem. 2011, 29, 1567—1575 © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cjc.wiley-vch.de 1569 Figure 3 Transmembrane topology of hSMS1 predicted by MEMSAT3. Here, the characters with broad lines, with thin lines and with￾out line stand for transmembrane domains, loop regions, and terminal domains respectively (＋: intracellular domains; -: the extracellular loop; O : Outside helix cap; X : Central transmembrane helix segment; I: Inside helix cap). Table 1 Predicted locations of the trans-membrane helices on hSMS1 Method Na TM1 TM2 TM3 TM4 TM5 TM6 MEMSTAT319 6 M130-S154 (25)b N178-W202 (25) I210-T234 (25) N275-F295 (21) I308-D332 (25) V335-Q354 (20) PSIPRED20 6 E131-H157 (27) W182-L204 (23) S209-T229 (21) H285-E298 (14) L304-A325 (22) V331-A351 (21) APSSP21 6 W132-H157 (26) W182-K206 (25) S209-I228 (20) T286-F295 (10) W305-A325 (21) Y329-Q353 (25) BetaTPred222 6 W132-R159 (28) F184-K206 (23) I211-T234 (24) T286-Y299 (14) W306-A325 (20) Y329-Q353 (25) ConPred II23 5 F136-V156 (21) S185-L205 (21) F215-L235 (21) D279-Y299 (21) Y307-D327 (21) PROF24 6 G133-V156 (24) R179-K206 (28) K208-T233 (26) T286-K297 (12) W306-L324 (19) T330-M350 (21) SSpro25 6 M130-H157 (28) Q181-L204 (24) S209-T229 (21) Y280-Y299 (20) W305-A325 (21) V331-Q353 (23) DPM26 6 M130-V160 (31) Q181-K206 (26) I210-T234 (25) T286-E298 (13) R302-H326 (25) T330-Q353 (24) DSC27 6 M130-H157 (28) W182-L205 (24) I210-T233 (24) V287-K297 (11) R302-L323 (22) D332-Q353 (22) GOR128 6 M130-E158 (29) R179-L205 (27) S209-P238 (30) M276-K297 (21) I310-Y329 (20) V331-Q353 (23) GOR329 6 M130-V156 (27) I186-Y207 (22) I210-T233 (24) T286-R302 (17) L304-A325 (22) V331-Q353 (23) MLRC30 6 W132-H157 (26) I186-L205 (20) S209-T234 (26) Y280-K297 (18) R303-A325 (23) V331-Q353 (23) PHD31 6 E131-R159 (29) N178-L205 (28) S209-L235 (27) C277-K297 (21 R303-L324 (22) T330-Q353 (24) Predator32 6 T135-V156 (22) F184-L205 (22) S209-T234 (26) T286-K297 (12) W305-A325 (21) T330-Q353 (24) SOPM33 6 T135-R159 (25) W182-L205 (24) S209-T234 (26) M276-K297 (22) L304-324 (21) T330-Q353 (24) a Predicted number of TMs. b Numbers in brackets are the lengths of TMs. Table 2 Comparison of the length of transmembrane helices on hSMS1 and GlpG (PDB entry: 2IC8) Protein TM number TM1 TM2 TM3 TM4 TM5 TM6 hSMS1 6 25 25 25 21 25 20 GlpG 6 27 17 20 18 21 18 loop. All of the resulting models were then visually checked to exclude those having serious internal colli￾sions. Moreover, as for the third extracellular loop (loop 5, residues 327—339), two highly conserved residues H328 and D332 on this loop formed hydrogen bond, which could be used as an additional criterion to select appropriate model for loop 5. Then, all of the whole structural models of hSMS1 were visually inspected to exclude those containing crossing loops or serious internal steric collisions. Models on which the catalytic pocket were too small to accommodate PC or SM were excluded. Models on which the three key residues (H285, H328, and D332) could not form a reasonable arrangement in the catalytic pocket were also excluded. Among the remaining models, the one with the lowest probability density function (PDF) energy computed by Modeler was cho￾sen for further refinement. In order to relax the steric repulsions between side chains, side chain refinement with restraints on backbone was performed. Each loop was refined by high-level optimizations by using Mod￾eler. Side-chain refinement was performed again be￾cause the conformation of backbone could have been changed during the optimization performed at the pre￾vious step. Molecular dynamics simulation of the hSMS1/SM complex structure Based on the final representing model of hSMS1, SM was manually docked into the catalytic pocket in favor of the SN2 nucleophilic substitution reaction. The