FULL PAPER Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase Zhang,Ya°(张亚)Lin,F(林赋)Deng, Xiaodong°(邓晓东) 王任小)Ye, Deyong*(叶德泳) School of pharmacy, Fudan University, Shanghai 201203, China b State Key Lab of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, Ch Sphingomyelin synthase (SMS) produces sphingomyelin and diacylglycerol from ceramide and phosphatidyl- soline. It plays an important role in cell survival and apoptosis, inflammation, and lipid homeostasis, and therefore has been noticed in recent years as a novel potential drug target. In this study, we combined homology modeling, molecular docking, molecular dynamics simulation, and normal mode analysis to derive a three-dimensional struc- ture of human sphingomyelin synthase(hSMs )in complex with sphingomyelin. Our model provides a reasonable explanation on the catalytic mechanism of hSMSl. It can also explain the high selectivity of hSMsI towards phos- hocholine and sphingomyelin as well as some other known experimental results about hSMSl. Moreover, we also derived a complex model of D609, the only known small-molecule inhibitor of hSMSI so far. Our hSMSI model may serve as a reasonable structural basis for the discovery of more effective small-molecule inhibitors of hSMSI Keywords sphingomyelin synthase, molecular modeling, molecular dynamics Introduction round of catalysis Due to its pharmaceutical implications, SMS Sphingomyelin synthase(SMS) is the enzyme that been noticed as a potential drug target in recent ye functions at the last step in the synthesis of shingo- myelin. It recognizes ceramide and phosphatidylcholine Huitema et al. reported the sequence of SMs using (PC)as substrates to produce sphingomyelin(SM)and functional cloning strategy in yeast.But the diacylglycerol (DAG)(Figure 1). Its activity influences three-dimensional structure of SMS remains unresolved the levels of SM, PC, ceramide, and DAG directly in so far. Without such structural information, it remains as living body, and is closely related with cell survival and a challenge to understand the catalytic mechanism of apoptosis, inflammation, and atherosclerosis SMS and discover potent inhibitors of SMS accordingly SMS has two known subtypes, SMSI and SMS2, In fact, very few small-molecule compounds that can which are classified by their cellular localizations. regulate the biological function of SMS have been re- SMSI is found merely in the trans-Golgi apparatus, and ported in literature. To the best of our knowledge, the SMS2 is primarily found in the plasma membranes. only known SMS inhibitor so far is D609(Figure 2) As most lipid phosphate phosphatase family, SMs which was reported to have a weak inhibitory activity catalyzes the choline phosphotransferase reaction possi-(ICso=500 umol-L-') against SMS in vitro, but no ac- bly through a similar mechanism. First, a double-chain tivity in vivo due to its unstable chemical structure.13-15 choline phospholipid(PC or SM)enters and binds to a In this study, we combined homology modeling, mo- single site of the enzyme. Then, a nucleophilic attack on lecular docking, and molecular dynamics simulation to the lipid-phosphate ester bond is executed by His328 in derive a three-dimensional structural model of human the assistance by Asp332. After the formation of a cho- line phosphohistidine intermediate and the release of sphingomyelin synthase 1(hSMS1). Our model can DAG or ceramide, His285 acts as a nucleophile to at reasonably explain some known experimental results tack on the carbon attached to the primary hydroxy regarding hSMSl. It can be applied to future structure- group on ceramide or DAG. Finally, the product (SM or based discovery of novel small-molecule inhibitors of PC) is released from the active site to allow the nex hSMS I dyye@shmu.edu.cn,wangrx/@mailsiocaccn;Tel 021-51980117,0086-021-54925128,Fax:0086-021-51980125 d January 5, 2011; revised February 18, 2011; accepte China(Nos. 30973641, 20902013), a special research fund for the Doctoral Program of Education from the Chinese Ministry of Education 0090071ll and an open grant from the State Key Laboratory of Bio-organic and Natural Products Chemistry, Chinese Academy of Sciences WILEY Chin. . Chem. 2011, 29, 1567--1575 C2011 SIOC, CAS, Shanghai, WILEY-VCH Verlag gmbH Co KGaA, Weinheim ONLINE LIBRARY
FULL PAPER * E-mail: dyye@shmu.edu.cn.; wangrx@mail.sioc.ac.cn.; Tel.: 0086-021-51980117, 0086-021-54925128; Fax: 0086-021-51980125 Received January 5, 2011; revised February 18, 2011; accepted Apil 28, 2011. Project supported by the National Natural Science Foundation of China (Nos.30973641, 20902013), a special research fund for the Doctoral Program of Higher Education from the Chinese Ministry of Education (No. 20090071110054), and an open grant from the State Key Laboratory of Bio-organic and Natural Products Chemistry, Chinese Academy of Sciences. Chin. J. Chem. 2011, 29, 1567—1575 © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1567 Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase Zhang, Yaa (张亚) Lin, Fub (林赋) Deng, Xiaodonga (邓晓东) Wang, Renxiao*,b (王任小) Ye, Deyong*,a (叶德泳) a School of Pharmacy, Fudan University, Shanghai 201203, China b State Key Lab of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China Sphingomyelin synthase (SMS) produces sphingomyelin and diacylglycerol from ceramide and phosphatidylcholine. It plays an important role in cell survival and apoptosis, inflammation, and lipid homeostasis, and therefore has been noticed in recent years as a novel potential drug target. In this study, we combined homology modeling, molecular docking, molecular dynamics simulation, and normal mode analysis to derive a three-dimensional structure of human sphingomyelin synthase (hSMS1) in complex with sphingomyelin. Our model provides a reasonable explanation on the catalytic mechanism of hSMS1. It can also explain the high selectivity of hSMS1 towards phosphocholine and sphingomyelin as well as some other known experimental results about hSMS1. Moreover, we also derived a complex model of D609, the only known small-molecule inhibitor of hSMS1 so far. Our hSMS1 model may serve as a reasonable structural basis for the discovery of more effective small-molecule inhibitors of hSMS1. Keywords sphingomyelin synthase, molecular modeling, molecular dynamics Introduction Sphingomyelin synthase (SMS) is the enzyme that functions at the last step in the synthesis of sphingomyelin. It recognizes ceramide and phosphatidylcholine (PC) as substrates to produce sphingomyelin (SM) and diacylglycerol (DAG) (Figure 1).1 Its activity influences the levels of SM, PC, ceramide, and DAG directly in living body, and is closely related with cell survival and apoptosis, inflammation, and atherosclerosis.2-9 SMS has two known subtypes, SMS1 and SMS2, which are classified by their cellular localizations. SMS1 is found merely in the trans-Golgi apparatus, and SMS2 is primarily found in the plasma membranes.10,11 As most lipid phosphate phosphatase family, SMS catalyzes the choline phosphotransferase reaction possibly through a similar mechanism. First, a double-chain choline phospholipid (PC or SM) enters and binds to a single site of the enzyme. Then, a nucleophilic attack on the lipid-phosphate ester bond is executed by His328 in the assistance by Asp332. After the formation of a choline phosphohistidine intermediate and the release of DAG or ceramide, His285 acts as a nucleophile to attack on the carbon attached to the primary hydroxyl group on ceramide or DAG. Finally, the product (SM or PC) is released from the active site to allow the next round of catalysis.12 Due to its pharmaceutical implications, SMS has been noticed as a potential drug target in recent years. Huitema et al. reported the sequence of SMS using functional cloning strategy in yeast.10 But the three-dimensional structure of SMS remains unresolved so far. Without such structural information, it remains as a challenge to understand the catalytic mechanism of SMS and discover potent inhibitors of SMS accordingly. In fact, very few small-molecule compounds that can regulate the biological function of SMS have been reported in literature. To the best of our knowledge, the only known SMS inhibitor so far is D609 (Figure 2), which was reported to have a weak inhibitory activity (IC50＝500 µmol•L－1 ) against SMS in vitro, but no activity in vivo due to its unstable chemical structure.13-15 In this study, we combined homology modeling, molecular docking, and molecular dynamics simulation to derive a three-dimensional structural model of human sphingomyelin synthase 1 (hSMS1). Our model can reasonably explain some known experimental results regarding hSMS1. It can be applied to future structurebased discovery of novel small-molecule inhibitors of hSMS1
FULL PAPER Ceramide HaNON Figure 1(A)hSMSI-catalyzed synthesis of sphingomyelin( SM) from phosphatidylcholine(PC).(B) Some other phosphatides related to ceramide, including phosphatidylethanolamine(PE), phosphatidic acid(Pa), phosphatidylserine(PS)and phosphatidylglycerol(PG) The transmembrane domain of hSMSI is composed of six transmembrane helices(TMs). We employed 15 SK different computational methods to predict the locations of these TMs on the hSMSI sequence. Most of them produced consistent predictions (Table 1). In order to select an appropriate template for modeling the TMs of Figure 2 Chemical structures of D609(ICs0=500 umolL-I hSMSl. we retrieved a total of 61 entries with six TMs from the membrane structural proteins database against SMS) and the corresponding dimer d609-dixanthogen (no inhibition activity against SMs). PdbTm(htTp: //pdbtm. enzim. hu). After careful evaluations, which will be explained in the later Results Computational methods and Discussion section, we selected the crystal structure of Escherichia coli GlpG(PDB entry: 2IC8)as the tem Homology modeling of the hSMSl/lipid complex plate(Table 2)35 The Modeler function(as imple- structure mented in the Discovery Studio software suite), was The amino acid sequence of hSMSI used in our employed to generate a total of 30 structural models study, which has 413 residues in total, was retrieved based on this template from PubMed (access ID=NP 671512). The hSMSI As for the extracellular or intracellular loop of has an N-terminal domain, a transmembrane domain, hSMSl, loop 3(residues 235--274)is relatively long and a C-terminal domain. 6 Jiang et al. demonstrated (Figure 3). Therefore, it should be modeled based on an recently that truncation of N-terminal and C-terminal of appropriate template. PDB entry IBWO(residues 144 core structure of hSMSI(M130-Q353)was modeled in 463%quence identity =34. 1%; sequence similarity hSMSI would not abort its activity. Thus, only the 181)(se as selected as the template for this purpose our study Locations of the extracellular or intracellular This structure was selected throughout the entire PDB loop and transmembrane domain on the sequence of database according to the sequence similarity computed hSMSI were predicted by using the PSI-PRED Server by the FASTa algorithm. , No qualified template wa he predicted extracellular or intracellular loops and found for other loops of hSMSI though. These loops transmembrane segments are given in Figure 3. This were constructed from scratch by using the modeler prediction is basically consistent with the results re module in the Discovery Studio software suite. A total ported by Huitema et al. in a previous study of 30 structural models also generated for each 1568www.cjc.wiley-vch.deO2011SioC,Cas,Shanghai&WileY-VchVerlagGmbh&Co.Kgaa,WeinheimChin.j.chem.2011,29,1567-1575
FULL PAPER Zhang et al. 1568 www.cjc.wiley-vch.de © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chin. J. Chem. 2011, 29, 1567—1575 HO O O H O O HO H HN O HO PC SM Ceramide O O P O O H O O O O N O P O H HN O HO O O N Diacylglycerol + + SMS + - + - A O O P O O H O O O H3N O O P HO O H O O O O O P O O H O O O O H3N O O O O P O O H O O O OH HO H PE PA PS PG + + - O - - - - O O B Figure 1 (A) hSMS1-catalyzed synthesis of sphingomyelin (SM) from phosphatidylcholine (PC). (B) Some other phosphatides related to ceramide, including phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine (PS) and phosphatidylglycerol (PG). O S S K O S SS O S D609 D609 dixanthogen + - Figure 2 Chemical structures of D609 (IC50＝500 µmol•L－1 against SMS) and the corresponding dimer D609-dixanthogen (no inhibition activity against SMS). Computational methods Homology modeling of the hSMS1/lipid complex structure The amino acid sequence of hSMS1 used in our study, which has 413 residues in total, was retrieved from PubMed (access ID＝NP_671512). The hSMS1 has an N-terminal domain, a transmembrane domain, and a C-terminal domain.16 Jiang et al. demonstrated recently that truncation of N-terminal and C-terminal of hSMS1 would not abort its activity.17 Thus, only the core structure of hSMS1 (M130-Q353) was modeled in our study. Locations of the extracellular or intracellular loop and transmembrane domain on the sequence of hSMS1 were predicted by using the PSI-PRED Server.18 The predicted extracellular or intracellular loops and transmembrane segments are given in Figure 3. This prediction is basically consistent with the results reported by Huitema et al. in a previous study.10 The transmembrane domain of hSMS1 is composed of six transmembrane helices (TMs). We employed 15 different computational methods to predict the locations of these TMs on the hSMS1 sequence.19-33 Most of them produced consistent predictions (Table 1). In order to select an appropriate template for modeling the TMs of hSMS1, we retrieved a total of 61 entries with six TMs from the membrane structural proteins database PDBTM (http://pdbtm.enzim.hu).34 After careful evaluations, which will be explained in the later Results and Discussion section, we selected the crystal structure of Escherichia coli GlpG (PDB entry: 2IC8) as the template (Table 2).35 The Modeler function (as implemented in the Discovery Studio software suite)36,37 was employed to generate a total of 30 structural models based on this template. As for the extracellular or intracellular loop of hSMS1, loop 3 (residues 235—274) is relatively long (Figure 3). Therefore, it should be modeled based on an appropriate template. PDB entry 1BW0 (residues 144— 181) (sequence identity＝34.1%; sequence similarity＝ 46.3%) was selected as the template for this purpose. This structure was selected throughout the entire PDB database according to the sequence similarity computed by the FASTA algorithm.38,39 No qualified template was found for other loops of hSMS1 though. These loops were constructed from scratch by using the Modeler module in the Discovery Studio software suite. A total of 30 structural models were also generated for each
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 1569
Molecular 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 without 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 collisions. 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 chosen 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 Modeler. Side-chain refinement was performed again because the conformation of backbone could have been changed during the optimization performed at the previous 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
FULL PAPER Zhang et al. binding mode of SM was then optimized within the 1.0 MPa. After these preparative steps, a production of structural restraints of hSMSl(Figure 4) Minimization 10 ns long was conducted under the same temperature was conducted using the adopted basis New. and pressure. During this process, the temperature was ton-Raphson algorithm with the CHARMm force field controlled with the Hoover method whereas the pres- implemented in the discovery studio software sure was controlled by coupling to a pressure bath using extended system algorithm. The mass of the pressure piston was 1000 amu. Langevin piston collision fre et to 25.0 ps The dielectric constants of 332 protein and water molecules were set to 1.0 and 80.0 respectively. Distance cutoff in generating the list of pairs was 14. 0 A Switching function was used between 10. A and 12.0 A to treat non-bonding interactions RMSD values relative to the initial structure were moni- tored as an indication of equilibrium along the MD trajectory. The last snapshot on the resulting md tra- jectory was retrieved, minimized without restraint in couple with the implicit membrane model. This final 8 efined model of the hSMSI/SM complex is shown in Figure 5. In order to evaluate this structural model, the PROCHECK program(version 3. 5.4 was applied to check its stereochemical quality. The Ramachandran plot of this hSMS I structure produced by PROCHECK Figure 4 The binding mode of sphingomyelin to hSMS1 Three lipid complex structure nesis study on the hSMSl/ Computational muta residues, i.e. H285, H328 and D332, in the catalytic site are criti- cal for the catalytic mechanism of hSMSI We then performed computational mutagenesis on the hSMSl complex structure to evaluate the impor In order to refine the structural model of the tance of both the functional groups on the lipid substrate hSMSI/SM complex, it was further subjected to a and several amino acid residues on hSMSISuch results long-time molecular dynamics (MD)simulation will provide additional proofs on the catalytic mecha- which an implicit GB/SA model of bilayer membrane nism of hSMS1. Two types of mutations were pe was applied. Major parameters for setting the implicit formed accordingly: on the substrate side, SM was mu membrane GB/SA model include: Grid spacing for tated in turn into PC, PE, PA, PS, and PG(Figure 1) lookup table (DGP)=1.5 A: Half membrane switching whereas on the hSMsl side, several important amino length(MSW)=2.0 A; Half smoothing length(SW) 3 A; Non-polar surface tension coefficient turn into alanine. Both types of mutations were done (SGAMMA)=0. 418 kJ/(mol-A2). Number of angular using the Discovery Studio software by deleting or ntegration points 50 was used for volume integration adding some atoms while keeping original atoms as GB/SA calculation. To keep the catalytic site stable for much as possible. After mutation, two rounds of mini- SN2 nucleophilic substitution reaction, three pairs of mization were applied to the hSMSIAipid complex harmonic distance restraint were applied with harmonic structure with the same force field parameters described force constant 418(kJ/molA): atom pairs 1-2, 4-5. above. Finally, binding energies between the substrate and 6-7(Figure 4). Firstly, hSMS 1/SM was positioned molecule and hSMS I were calculated using in the center ot the implicit membrahe model. The pla- hSMSi/ipid complex (Table 3 and Table 4) Iting tered at ==0 with a membrane thickness of 30 A. To- Normal mode analysis and protein motion analy pology and coordinate files were generated with the of hSMS Discovery Studio software. Minimization was per formed using the program CHARMm(release c33b2) We conducted protein domain motion analysis for Atotalof5000stepsofsteepestdescentminimizationtheDyndomon-lineserver(http://fizz.cmp.uea.ac.uk/ and 5000 steps of conjugate gradient minimization were dyndomn 4 The initial and the last configuration of the performed subsequently As the first step of MD simulation, the entire system hSMS I/SM complex in MD simulation were submitted was heated froma to in100 ps. Then, the as inputs. The Dyn Dom server returned the information whole system was equilibrated for 400 ps under a con- of the " hinge axes"(Figure 7). In order to confirm the stant temperature of 310 K and a constant pressure of mes of protein domain motion ana 1570www.cjc.wiley-vch.deO2011SioC,Cas,Shanghai&WileY-VchVerlagGmbh&Co.Kgaa,WeinheimChin.j.chem.2011,29,1567-1575
FULL PAPER Zhang et al. 1570 www.cjc.wiley-vch.de © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chin. J. Chem. 2011, 29, 1567—1575 binding mode of SM was then optimized within the structural restraints of hSMS1 (Figure 4). Minimization was conducted using the adopted basis Newton-Raphson algorithm40 with the CHARMm force field implemented in the Discovery Studio software. P O O O O N N H H N N H N O O H285 H328 D332 1 2 3 4 5 6 7 8 Figure 4 The binding mode of sphingomyelin to hSMS1. Three residues, i.e. H285, H328 and D332, in the catalytic site are critical for the catalytic mechanism of hSMS1. In order to refine the structural model of the hSMS1/SM complex, it was further subjected to a long-time molecular dynamics (MD) simulation in which an implicit GB/SA model of bilayer membrane41 was applied. Major parameters for setting the implicit membrane GB/SA model include: Grid spacing for lookup table (DGP)＝1.5 Å; Half membrane switching length (MSW)＝2.0 Å; Half smoothing length (SW)＝ 0.3 Å; Non-polar surface tension coefficient (SGAMMA)＝0.418 kJ/(mol•Å2 ). Number of angular integration points 50 was used for volume integration in GB/SA calculation. To keep the catalytic site stable for SN2 nucleophilic substitution reaction, three pairs of harmonic distance restraint were applied with harmonic force constant 418 (kJ/mol•Å2 ): atom pairs 1—2, 4—5, and 6—7 (Figure 4). Firstly, hSMS1/SM was positioned in the center of the implicit membrane model. The planar membrane is perpendicular to the z axis and centered at z＝0 with a membrane thickness of 30 Å. Topology and coordinate files were generated with the Discovery Studio software. Minimization was performed using the program CHARMm (release c33b2).42 A total of 5000 steps of steepest descent minimization and 5000 steps of conjugate gradient minimization were performed subsequently. As the first step of MD simulation, the entire system was heated from 0 K to 310 K in 100 ps. Then, the whole system was equilibrated for 400 ps under a constant temperature of 310 K and a constant pressure of 1.0 MPa. After these preparative steps, a production of 10 ns long was conducted under the same temperature and pressure. During this process, the temperature was controlled with the Hoover method43 whereas the pressure was controlled by coupling to a pressure bath using extended system algorithm.44 The mass of the pressure piston was 1000 amu. Langevin piston collision frequency was set to 25.0 ps－1 . The dielectric constants of protein and water molecules were set to 1.0 and 80.0, respectively. Distance cutoff in generating the list of pairs was 14.0 Å. Switching function was used between 10.0 Å and 12.0 Å to treat non-bonding interactions. RMSD values relative to the initial structure were monitored as an indication of equilibrium along the MD trajectory. The last snapshot on the resulting MD trajectory was retrieved, minimized without restraint in couple with the implicit membrane model. This final refined model of the hSMS1/SM complex is shown in Figure 5. In order to evaluate this structural model, the PROCHECK program (version 3.5.4)45 was applied to check its stereochemical quality. The Ramachandran plot of this hSMS1 structure produced by PROCHECK is shown in Figure 6. Computational mutagenesis study on the hSMS1/ lipid complex structure We then performed computational mutagenesis on the hSMS1 complex structure to evaluate the importance of both the functional groups on the lipid substrate and several amino acid residues on hSMS1. Such results will provide additional proofs on the catalytic mechanism of hSMS1. Two types of mutations were performed accordingly: on the substrate side, SM was mutated in turn into PC, PE, PA, PS, and PG (Figure 1); whereas on the hSMS1 side, several important amino acid residues near the binding pocket were mutated in turn into alanine. Both types of mutations were done using the Discovery Studio software by deleting or adding some atoms while keeping original atoms as much as possible. After mutation, two rounds of minimization were applied to the hSMS1/lipid complex structure with the same force field parameters described above. Finally, binding energies between the substrate molecule and hSMS1 were calculated using an implicit GB/SA membrane model for each resulting hSMS1/lipid complex (Table 3 and Table 4). Normal mode analysis and protein motion analysis of hSMS1 We conducted protein domain motion analysis for hSMS1 to study its major conformational motions with the DynDom on-line server (http://fizz.cmp.uea.ac.uk/ dyndom/).46 The initial and the last configuration of the hSMS1/SM complex in MD simulation were submitted as inputs. The DynDom server returned the information of the "hinge axes" (Figure 7). In order to confirm the outcomes of protein domain motion analysis, normal
Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase CHEMISTRY D3325 A28 R3 Figure 5 The hSMs 1/SM complex structure as the last snapshot of 10 ns molecular dynamics simulation. (A) hSMSI is rendered in ribbons. Several key residues around SM are shown in stick models. Dashed lines stand for hydrogen bonds. (B)hSMSI is rendered in the solvent accessible surface. SM is rendered in stick model. The arrow indicates where the choline moiety on SM is buried inside Table 3 Computed binding energies between hSMSl and some lipid substrates Complex Binding energy/(k.mol) ASMSI/PE 62.80 hSMS1/PA hSMSI/PS 63.76 hSMSI/PG 32.80 The binding energy between hSMSI and SM is taken as the energy rererence. Table 4 Computed binding energies between hSMSl mutations and SM 180-135-90-5 hhSMS I mutation Binding energy/ (kJmol) Phi° Figure 6 Ramachandran plot of the structural model of hSMSI D332A after molecular dynamics refinement. A: core alpha; a: allowed H285A 26.27 alpha; -a: general alpha; B: core beta; b: allowed beta, -b: gen-H328A 73.47 eral beta; L: core left-handed alpha; I: allowed left-handed alpha, R342A -l: general left-handed alpha, p: allowed epsilon,-p: genera Y338A epsilon. Glycines are shown as triangles. Here, 87.5% of residues 21.08 are in the most favored regions(A, B, L), 10.5% of residues are in the additionally allowed regions(a, b, 1, p), 1.0% of residues H274A 18.95 are in the generally allowed regions (a, b, -1, -p), and only L281A 1.0% of residues are in the disallowed regions F173A 17.61 mode analysis(NMA) was conducted to analyze the F3A+1A+74A+1281A5414 ntrinsic motions of the hSMSl structure. The eINemo The binding energy between SMSI and SM is set as the refer on-lineserver(http://igs-server.cnrs-mrs.fr/elnemo/ence Chin J. Chem. 2011, 29, 1567--1575 C2011 SIOC, CAS, Shanghai, WILEY-VCH Verlag gmbH Co KGaA, Weinheim wweje. wiley-vch.de 15
Molecular 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 1571 Figure 5 The hSMS1/SM complex structure as the last snapshot of 10 ns molecular dynamics simulation. (A) hSMS1 is rendered in ribbons. Several key residues around SM are shown in stick models. Dashed lines stand for hydrogen bonds. (B) hSMS1 is rendered in the solvent accessible surface. SM is rendered in stick model. The arrow indicates where the choline moiety on SM is buried inside. Figure 6 Ramachandran plot of the structural model of hSMS1 after molecular dynamics refinement. A: core alpha; a: allowed alpha; ~a: general alpha; B: core beta; b: allowed beta; ~b: general beta; L: core left-handed alpha; l: allowed left-handed alpha; ~l: general left-handed alpha; p: allowed epsilon; ~p: general epsilon. Glycines are shown as triangles. Here, 87.5% of residues are in the most favored regions (A, B, L), 10.5% of residues are in the additionally allowed regions (a, b, l, p), 1.0% of residues are in the generally allowed regions (~a, ~b, ~l, ~p), and only 1.0% of residues are in the disallowed regions. mode analysis (NMA) was conducted to analyze the intrinsic motions of the hSMS1 structure. The elNémo on-line server (http://igs-server.cnrs-mrs.fr/elnemo/ Table 3 Computed binding energies between hSMS1 and some lipid substrates Complex Binding energy/(kJ•mol－1 ) hSMS1/SM 0.00a hSMS1/PC －27.90 hSMS1/PE 62.80 hSMS1/PA 123.80 hSMS1/PS 63.76 hSMS1/PG 32.80 a The binding energy between hSMS1 and SM is taken as the energy reference. Table 4 Computed binding energies between hSMS1 mutations and SM hhSMS1 mutation Binding energy/(kJ•mol－1 ) Wild type 0.00a D332A －0.25 H285A 26.27 H328A 73.47 R342A 81.79 Y338A 21.08 F177A 7.57 H274A 18.95 L281A 14.60 F173A 17.61 F173A＋F177A＋H274A＋L281A 54.14 a The binding energy between SMS1 and SM is set as the reference