Structure and mechanism of human diacylglycerol acyltransferase 1

bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 1 Structure and mechanism of human diacylglycerol acyltransferase 1 2 Lie Wang1,*, Hongwu Qian2,*, Yin Nian1,*, Yimo Han2,*, Zhenning Ren1, Hanzhi Zhang1, Liya 3 Hu1, B. V. Venkataram Prasad1, Nieng Yan2,#, Ming Zhou1,# 4 5 1 6 of Medicine, Houston, TX 77030, USA. 7 2 8 * These authors contributed equally 9 # Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Correspondence to Ming Zhou ([email protected]) and Nieng Yan ([email protected]). 10 Summary 11 Human diacylglycerol O-acyltransferase-1 (hDGAT1) synthesizes triacylglycerides and is 12 required for dietary fat absorption and fat storage. The lack of 3-dimensional structure has 13 limited our understanding of substrate recognition and mechanism of catalysis, and hampers 14 rational targeting of hDGAT1 for therapeutic purposes. Here we present the structure of 15 hDGAT1 in complex with a substrate oleoyl Coenzyme A at 3.1 Å resolution. hDGAT1 forms a 16 homodimer and each protomer has nine transmembrane helices that carve out a hollow chamber 17 in the lipid bilayer. The chamber encloses highly conserved catalytic residues and has separate 18 entrances for the two substrates fatty acyl Coenzyme A and diacylglycerol. The N-terminus of 19 hDGAT1 makes extensive interactions with the neighboring protomer, and is required for 20 enzymatic activity. 21 22 Keywords: DGAT1; MBOAT; acyl-CoA; diacylglycerol; cryoEM 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 24 25 Introduction Diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) is an integral membrane protein 26 that synthesizes triacylglycerides (TG or triglycerides, Figure S1) from two substrates, 27 diacylglycerol (DAG or diglyceride) and fatty acyl Coenzyme A (acyl-CoA) (1). The primary 28 physiological function of DGAT1 in mammals is fat absorption and storage (2, 3). In humans, 29 DGAT1 is highly expressed in epithelial cells of the small intestine and DGAT1 activity is 30 essential for dietary fat absorption (4, 5). DGAT1 is also found in other organs or tissues such as 31 liver where it synthesizes fat for storage and in female mammary glands where it produces fat in 32 the milk (6). In plants, DGAT1 synthesizes seed storage lipids (seed oils) that are widely utilized 33 for food or biofuels (7, 8). In addition to the synthesis of TG, hDGAT1 is also implicated as a 34 host virulence factor for hepatitis C and rotaviruses (9-11). Dgat1-/- mice are viable and show 35 significantly reduced TG in all tissues, increased sensitivity to leptin and insulin, and resistance 36 to obesity when kept on a high-fat diet (12, 13). These results have generated considerable 37 interest in targeting hDGAT1 for treating hypertriglyceridemia and fatty liver disease, and for 38 controlling obesity, diabetes, coronary heart diseases (14, 15). 39 DGAT1 belongs to a large superfamily of membrane-bound O-acyl transferases 40 (MBOAT, http://pfam.xfam.org/family/MBOAT) that are found in all kingdoms of life. MBOAT 41 family includes enzymes such as acyl-CoA:cholesterol acyltransferase (ACAT) that attaches a 42 fatty acid to a cholesterol and is crucial for bile acid sysnthesis (16), and protein-serine O- 43 palmitoleoyltransferase (PORCUPINE or PORCN) that adds a palmitoleate to a conserved serine 44 residue to activate the WNT protein (17, 18). Members of the MBOAT family have a highly 45 conserved histidine residue required for the transferase activity and are predicted to have 8-11 46 transmembrane segments (16, 19-24). Crystal structure of a bacterial member of MBOAT, DltB, 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 47 was reported recently (25). DltB catalyzes the transfer of a D-alanine from an intracellular 48 protein DltC to a lipoteichoic acid located on the extracellular side of the membrane (Figure S1). 49 However, the structure of DltB is not a suitable model for hDGAT1 because of their low 50 sequence identity (~20%) and very different substrates. As we will see in Discussion, DltB and 51 DGAT1 likely evolved from a common ancestor but have diverged significantly to accommodate 52 differences in their substrates. 53 Because of the physiological and pharmacological importance of DGAT1 and its 54 significance as a model for other related MBOAT members, it is important to understand the 55 molecular details of how DGAT1 works in terms of substrate recognition and mechanism of 56 catalysis and inhibition. We expressed and purified human DGAT1, and solved the structure of 57 hDGAT1 in complex with a substrate oleoyl-CoA at an overall resolution of 3.1 Å by using 58 single-particle cryo-electron microscopy (cryoEM). 59 60 Functional characterization of purified hDGAT1 61 Full-length hDGAT1 was over-expressed and purified (Figure 1A, Methods). The purified 62 hDGAT1 elutes on a size-exclusion column with a main peak at an elution volume of ~11.7 ml, 63 which corresponds to a molecular weight of ~150 kDa (Figure S2A). Since each hDGAT1 64 protomer has a molecular weight of ~55 kDa and with the detergent micelle, the elution peak is 65 consistent with a dimeric hDGAT1. The dimer is also clearly visible on an SDS-PAGE, 66 indicating that it is stable enough to be partially resistant to denaturing conditions (Figure 1A). 67 We noticed that there is a minor peak at ~10.4 ml that likely corresponds to a tetramer (Figures 68 1A and S2A). A number of previous studies also showed that DGAT1 from plants and mammals 69 could form either dimer or tetramer (20, 26, 27), however, it is not clear whether both of the 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 70 oligomeric states exist in the native endoplasmic reticulum membranes and whether the 71 oligomeric state has an impact on the enzymatic functions. 72 During the purification process, a thick white layer of fat appeared after the 73 centrifugation step (Figure S2B), indicating that the heterologously expressed hDGAT1 is 74 functional in cells. To find out whether the purified hDGAT1 dimer remains functional, we 75 measured the activity of the purified hDGAT1 by following the production of Coenzyme A 76 (Figure S2C). Rapid production of Coenzyme A occurs in the presence of oleoyl-CoA, 1,2- 77 dioleoyl-sn-glycerol (1,2-DAG) and the purified hDGAT1 (Figures S2C-S2D). In contrast, 78 Coenzyme A production was not observed when either 1,2-DAG or hDGAT1 was omitted from 79 the reaction mixtures. Coenzyme A production was almost completely suppressed in the 80 presence of T863, a known hDGAT1 inhibitor (28) (Figure S2D). These results indicate that the 81 purified dimeric hDGAT1 preserves its enzymatic function. 82 We further characterized the purified hDGAT1 to establish basic parameters of its 83 enzymatic reaction. The initial rate of Coenzyme A production at different concentrations of 84 oleoyl-CoA was measured and it follows a Michaelis-Menten type relationship with KM and Vmax 85 of 14.6 ± 1.3 µM and 956.6 ± 36.1 nmol/mg/min, respectively (Figure 1). The KM and Vmax 86 values are comparable to those previously reported of hDGAT1 in microsomes (29, 30), and the 87 Vmax value is equivalent to a turnover rate of ~1/second for each hDGAT1 protomer. Enzymatic 88 activity was also measured at different concentrations of DG which has a KM and Vmax of 597.1 89 ± 94.5 µM and 3310 ± 279.1 nmol/mg/min (Figure 1C). We did not observe substrate inhibition 90 up to 100 µM of oleoyl-CoA, which is different from a previous study on DGAT1 from the plant 91 Brassica napus (31). hDGAT1 has almost no preference for oleoyl-CoA (KM= 14.6 ± 1.3 µM ; 92 Vmax= 956.6 ± 36.1 nmol/mg/min) versus stearoyl-CoA (KM= 8.6 ± 1.3 µM ; Vmax= 839.4 ± 49.9 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 93 nmol/mg/min), or palmitoleoyl-CoA (KM= 6.2 ± 0.9 µM ; Vmax = 838.6 ± 31.6 nmol/mg/min) 94 versus palmitoyl-CoA (KM = 6.4 ± 1.1 µM ; Vmax = 767.8 ± 34.0 nmol/mg/min) as acyl donors 95 but has a slower Vmax and higher KM for decanoyl-CoA (Figures S2G-S2I, Table S2). For acyl 96 acceptors, hDGAT1 has a clear preference for DAG over the two mono-acyl glycerols (Figure 97 S2F, Table S2). Divalent cations such as Mg2+ or Ca2+ has no significant effect on the enzymatic 98 reaction (Figure S2E, Table S2), consistent with previous results from both the mammalian and 99 plant DGAT1s (32-34). In addition to the enzymatic activity, we estimated binding affinity of 100 oleoyl-CoA to hDGAT1 in a scintillation proximity assay in which we used oleoyl-CoA to 101 compete the binding of 3H-acetyl-CoA, and the result shows that oleoyl-CoA binds to hDGAT1 102 with an IC50 of 18.2 ± 5.2 nM (Figure 1D). 103 104 Overall structure of hDGAT1 105 hDGAT1 structure was solved by single-particle cryoEM. Due to the modest size of hDGAT1 106 particles (~110 kDa), conditions for grid preparation and data collection were extensively 107 optimized to achieve desired contrast and particle density (Figure S3A, Methods). Data were 108 processed following the flow chart shown in Figure S3B and detailed in Methods. A density map 109 was reconstructed to an overall 3.1 Å resolution with C2 symmetry imposed using 408,945 110 particles (Figure S3B). Furthur refinement using 275,945 particles produced a map that has 111 almost identical resolution but better density for bound lipids and detergents (Figure S3B). 112 Resolution for helices close to the core of the dimer reaches 2.7 Å while regions close to the 113 peripheral of the dimer has lower resolution likely due to their relatively higher mobility (Figure 114 S3C). 115 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 116 The density map is of sufficient quality to allow de novo building of residues 64 to 224 and 239 117 to 481, which include all the transmembrane helices, one oleoyl-CoA, and 5 partially resolved 118 lipid/detergent molecules, and the structure was refined to proper geometry (Figure S4, Table 119 S1). The first 63 and the last 5 residues, and residues 225-238 which is part of a cytosolic loop, 120 were not resolved. Residues 112 to 120, which is part of a luminal loop, were partially resolved 121 and built as poly-alanines. 122 123 hDGAT1 dimer has a dimension of ~105 by 55 by 48 Å and is shaped like a canoe (Figures. 2A- 124 2D). Based on the positive-inside rule (35), the N-terminus of hDGAT1 resides at the cytosolic 125 side (Figure S5). This assignment is also consistent with the previous consensus based on 126 biochemical studies (20, 36, 37) and allows for unambiguous placement of the C-terminus to the 127 lumen side of the ER. Each hDGAT1 protomer has nine transmembrane helices, TM1-9, and 128 three long loops, an ER luminal (extracellular) loop EL1 between TM1 and 2, an intracellular 129 loop IL1 between TM4 and 5, and a second intracellular loop IL2 between TM6 and 7 (Figures 130 2E-F). Both IL1 and IL2 are structured and composed of highly conserved amino acid sequences 131 (Figure S6). 132 133 In each protomer, TM2-9 and the two intracellular loops IL1 and 2 form a distinctive structural 134 fold that we define as the MBOAT fold (Figures 2E-2F and and 3A-3D). TM1, which is not part 135 of the MBOAT fold, is isolated from the rest of the transmembrane helices and linked to the 136 MBOAT fold by the long ER luminal loop EL1 (residues 110 to 125). EL1 is partially structured 137 and extends ~35 Å along the luminal side of the protein (Figures 2E and 2I). 138 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 139 The Dimer interface 140 Although TM1 seems suspended in the membrane when a protomer is viewed in isolation, the 141 space between the TM1 and the rest of the protomer (the MBOAT fold) is filled by the TM1 142 from the neighboring protomer so that the two protomers form a domain-swapped homodimer 143 (Figures 2A-F). TM1 makes extensive hydrophobic interactions with both TM6 and TM9 from 144 the neighboring subunit (Figures 2G-I). Crossover of the TM1 helix brings the N-terminus of one 145 protomer close to the intracellular side of its neighbor. Residues 64 to 80 of the N-terminus 146 interact with both IL1 and IL2 of the neighboring subunit. Overall, the dimer interface has an 147 extensive buried surface area of 684.5 Å2. The two TM1s only make a single contact at Ile80 148 located close to the intracellular side of the membrane, and the space between them is filled with 149 2 lauryl maltose neopentyl glycol (LMNG) molecules and 4 partially resolved lipid molecules 150 (Figures S5A-H). 151 Previous studies on a plant DGAT1 have identified part of the N-terminus as intrinsically 152 disordered protein, and showed that deletion of the N-terminus before TM1 led to a loss of the 153 enzymatic activity (31, 38, 39). In the hDGAT1 structure, the first 63 residues are not resolved 154 and likely is disordered while residues 64-80 are well-resolved but do not have clear secondary 155 structures. These residues are resolved likely due to their extensive interactions to the 156 intracellular surface of the MBOAT fold (Figure S7A-E). We asked whether the interactions 157 between the N-terminus and the MBOAT fold core may affect the enzymatic activity. Deletion 158 of residues 2-64 (ΔN65) slightly reduced Vmax (563.9 ± 32.5 nmol/mg/min ) but has almost no 159 effect on KM (13.9 ± 2.6 µM , Figure 2J). In contrast, deletion of the entire N-terminus to the first 160 residue of TM1 (residues 2-84, ΔN84) abolishes the enzymatic activity (Figure 2J). The N- 161 terminus is not required for dimer formation because ΔN84 remains a dimer after purification 7 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 162 (Figure S7F). To narrow down the region of functional significance, we made three additional 163 shorter deletion mutations, ΔN70, ΔN75 and ΔN80, and measured their enzymatic activities. All 164 three have significantly lower enzymatic activity than the wild type, and it appears that larger the 165 deletion, lower the enzymatic activity (Figures 2J and S7G, Table S2). These results are 166 consistent with the recognized role of the N-terminus, however, further structural and functional 167 studies are required to determine the precise impact of the N-terminus on structure and function 168 of hDGAT1. 169 170 The reaction chamber and oleoyl CoA binding site 171 The MBOAT fold in hDGAT1 carves out a large hollow chamber in the hydrophobic core of the 172 membrane (Figure 3A-D). The almost universally conserved histidine 415 in the MBOAT family 173 of enzymies are found inside of the reaction chamber and on TM7 (Figures 2F and S8). TM2-9 174 segregates into three groups that form three sidewalls of the chamber: TM2, 3 and 4 pack into a 175 bundle that forms the first sidewall; TM5 and 6 are both very long with almost 40 amino acids 176 each, and the two helices coil into a unit that tilts roughly 56 degree to the membrane norm to 177 form the second sidewall; TM7, 8 and 9 form a panel and the third sidewall (Figure 3A-D). The 178 cytosolic ends of TM7 and 8 is ~19 Å apart, creating a side entrance to the reaction chamber 179 (Figures 3B and 3E). IL1 and IL2 are located at roughly the cytosolic surface of the membrane 180 and form the floor of the chamber. IL1 (residues 222 to 261) is composed of a helix flanked by 181 two long strands, while IL2 (residues 352 to 396) has a long amphipathic helix (AH, residues 182 380 to 394) preceded by a short helix and a loop. 183 The structure of hDGAT1 was solved in the presence of 1mM oleoyl-CoA. A large non- 184 protein density is found at the cytosolic side of the reaction chamber close to IL2 and it extends 8 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 185 deep into the reaction chamber (Figures 3E-3F). An oleoyl-CoA can be modeled into this density, 186 with the adenosine 3’,5’-diphosphate at the cytosolic entrance, the 4-phosphate panthothenic acid, 187 β-alanine and β-mercapto-ethylamine extend progressively into the reaction chamber, and the 188 acyl chain residing in a hydrophobic pocket inside of the reaction chamber (Figure 3G). Tyr390, 189 and Lys400 line the entrance of the acyl-CoA binding site, Gln375, Trp377, Asn378, His382 and 190 Ser411 line the tunnel leading to the active site, and Trp334, Phe337, Phe338, Phe342, Trp364, 191 Phe373, Trp374, and Trp377 line the hydrophobic pocket for the acyl chain (Figures 3H and 192 S8A-S8G). The activated thioesther is located to the vicinity of His415, poised for an attack from 193 the activated hydroxyl of DAG. The position of the thioester is stabilizd by interaction between 194 the carbonyl oxygen of the fatty acid and the side chain of Gln465 on TM9 (Figures 3H and 195 S8G). A conserved Pro466 creates a kink on TM9 that brings Gln465 closer to the acyl-CoA 196 (Figure 3H). 197 IL2 has a crucial role in acyl-CoA binding. Its V-shaped helix-turn-helix motif forms the 198 entrance for the acyl-CoA, and a number of residues on the two helices make direct contact to 199 the acyl-CoA (Figure 3I). The loop preceding the helices contains the FYXDWWN motif, which 200 was identified in both DGAT1 and ACAT as important for enzymatic activites (16, 40, 41) and is 201 highly conserved (Figure S6). Trp364, the first trptophan in the motif, forms part of the 202 hydrophobic pocket for the acyl chain, and although the rest of the motif does not have direct 203 contact with the acyl-CoA, the FYXDWWN motif packs tightly against the helix-turn-helix 204 motif (Figure 3I), and thus mutations in the former could affect the enzymatic activity. 205 Interestingly, the FYXDWWN motif also makes extensive contact with the N-terminus from the 206 neighboring protomer (Figure 3I), and perturbations to these interactions caused by N-terminal 9 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 207 deletions could affect the enzymatic activity although the N-terminus does not make direct 208 contact with the bound oleoyl-CoA. 209 To assess the functional impact of residues in the active site and ones that line the acyl- 210 CoA binding site, we mutated these residues, one at a time, and measured their enzymatic 211 activity. His415Ala abolishes the enzymatic activity, consistent with its role in catalysis. Point 212 mutations to residues that line the entrance of the acyl-CoA binding site reduces the enzymetic 213 activity by 30 to 70%, while mutations to the rest of the binding pocket, Trp377, Asn378, His382, 214 Ser411, have a larger impact with a loss of more than 80% activity (Figure 3J). Since His415 and 215 Ser411 are part of the highly conserved SxxxHEY motif that was shown to be crucial for 216 enzymatic activity in a related MBOAT enzyme, ACAT (16), we mutated the glutamate and 217 found that Glu416Leu abolishes the enzymatic activities (Figures 3B, 3C and 3E). Although 218 results form these initial mutational studies are largely confirmatory, the structure provides a 219 framework for further studies that will lead to more precise understanding of substrate 220 recognition and the mechanism of catalysis. 221 222 Gateway for DAG and TG 223 The reaction chamber has a very large opening to the hydrophobic core of the membrane, and the 224 opening is framed by TM4 on one side and TM6 on the other side, and by part of the IL1 225 (residues 234-245) on the cytosolic side (Figures 3B-D). Residues line the two sides of the 226 entrance are mostly hydrophobic, Val192, Leu196, Met199, and ILE203 on TM4, and Phe337, 227 Leu341, Leu346, and Val349 on TM6 (Figure 4A). A tubular density is found near the entrance 228 and extends into the reaction chamber (Figures 4B and 4C). Although an acyl chain could be 229 modeled into the density, we cannot identify the ligand. We speculate that this large opening 10 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 230 would allow entrance of DAG to the reaction chamber from either leaflet of the lipid bilayer, and 231 exit of the product TG (Figure 5). Consistent with this hypothesis, mutating Leu346 to a bulkier 232 side chain Trp produces an enzyme that has no enzymatic activity but retains binding to acetyl 233 CoA (Figure S8I, Table S2). 234 235 Discussion 236 hDGAT1 structure defines a conserved MBOAT structural fold, which forms a large chamber in 237 the hydrophobic core of the membrane so that the acyl transfer reaction is isolated inside of the 238 chamber. The structure shows that an acyl CoA is recognized by residues at the cytosolic side of 239 the reaction chamber and a hydrophobic pocket inside of the chamber. Since the hydrophobic 240 acyl chain of an acyl CoA likely is buried in the membrane, we speculate that a slit between 241 TM7 and 8 allows entry of the acyl chain into the chamber (Figure 5A). TM8 is less well 242 resolved than the neighboring helices (Figure S4), suggesting that it is more mobile in the 243 membrane and thus could move to accommodate entry of the acyl chain. DAG likely enters the 244 reaction chamber through the large opening between TM4 and TM6 (Figure 5A). The glycerol 245 backbone of a DAG can reach the catalytic center His415 by interacting with hydrophilic 246 residues in the vicinity of His415 (Figure 5B), while the two hydrophobic aliphatic acyl chains of 247 DAG could remain partially accommodated by the hydrophobic core of the membrane. We 248 speculate that the conserved His415 facilitates the acyl transfer reaction by activating the free 249 hydroxyl on DAG, and the presence of Glu416 could enhance the activation. The activated 250 hydroxyl oxygen then attacks the thioester on the fatty acyl-CoA to form a new ester bond 251 (Figure 5C). The product, TG, could retraces the entrance pathway of DAG back into the 252 membrane while CoA dissipates into the cytosol (Figure 5D). 11 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 253 254 We noticed that the bacterial DltB protein also has a core of 8 helices that fold into a similar 255 architecture (Figure S9D-F), although it has a total of 11 transmembrane helices (25). In DltB, 256 the intracellular loops are placed more towards the center of the membrane perhaps to 257 accommodate the two substrates coming from either side of the membrane. As a result, the 258 MBOAT fold in DltB does not carve out a reaction chamber in the membrane. The large opening 259 between TM4 and TM6 is covered by two extra helices that are not part of the MBOAT fold 260 (Figure S9A-F ). It is interesting to note that the binding site for acyl-CoA in hDGAT mirrors the 261 substrate binding site in DltB. DltC, with a covalently linked D-alanyl Ppant group, is a substrate 262 to DltB and equivalent to a fatty acyl-CoA to hDGAT1. In the DltB-DltC complex, DltC is 263 positioned at the equivalent location of acyl-CoA to the MBOAT fold. Overall, DltB is shaped 264 like an hourglass that allows the two substrates to approach the reaction center from either sides 265 of the membrane, and the transfer of an acyl group across the membrane (Figure S9G-J). These 266 observations highlight the versatility of the MBOAT fold that can be adapted for different 267 functions. 268 269 Acknowledgments 270 This work was supported by grants from NIH (DK122784 and HL086392 to MZ), Cancer 271 Prevention and Research Institute of Texas (R1223 to MZ), the Robert Welch Foundation 272 (Q1279 to BVVP), Ara Parseghian Medical Research Foundation (to N.Y. and Y.H.), and the 273 New Jersey Council for Cancer Research (to H.Q.). N.Y. is supported by the Shirley M. 274 Tilghman endowed professorship from Princeton University. We thank Paul Shao for technical 275 support during EM image acquisition. We acknowledge the use of Princeton’s Imaging and 12 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 276 Analysis Center, which is partially supported by the Princeton Center for Complex Materials, 277 and the National Science Foundation (NSF)-MRSEC program (DMR-1420541). 278 279 Author Contributions 280 M.Z., L.W., Y.N. and Z.R. conceived the project. L.W., Y.N., H.Q., Z.R., Y.H., H.Z. 281 conducted experiments. L.W., Y.N., H.Q., Z.R., Y.H., N.Y., and M.Z. analyzed data. L.H. and 282 B.V.V.P. advised on model building and refinement. L.W., Z.R. and M.Z. wrote the initial 283 draft and all authors participated in revising the manuscript. 284 285 Competing interests 286 The authors declare no competing financial interests. 287 288 Corresponding authors 289 Correspondence to Ming Zhou ([email protected]) and Nieng Yan ([email protected]). 290 291 13 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 292 Figures and Figure Legends 293 294 Figure 1. Purification and functional characterization of hDGAT1. A, Size-exclusion 295 chromatography profile of purified hDGAT1. Inset: SDS–PAGE of the purified hDGAT1. B-C. 296 Initial rate of reaction versus oleoyl-CoA (B) or DAG (C) concentration. Data in B were fit with 297 a Michaelis-Menten equation, and data in C were fit with an allosteric sigmoidal equation 298 (Methods). D. Competitive binding of oleoyl-CoA measured against its competition against 0.25 299 µM of 3H-acetyl-CoA. Data were fit with a single-site competitive binding isotherm (Methods). 300 In B, C and D, Each symbol is the average of three repeats. Error bars are standard errors of the 301 mean (s.e.m.) 302 14 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 303 304 Figure 2. Structure of hDGAT1. A–D Structure of hDGAT1 dimer is shown in cartoon and 305 surface representations as viewed from within the plane of the membrane (A, C), or the 306 intracellular side of the membrane (B, D). Approximate position of the ER membrane is marked 307 as grey shade. E. Cartoon representation of an hDGAT1 protomer in two orientations. F. 308 Topology of hDGAT1. The position of His415 is marked as a yellow star. G-I, dimerization 309 interface of hDGAT1 viewed in three orientations. One protomer is shown as grey cartoon but 310 with its TM1 and the N-terminus in surface. The other protomer is shown as rainbow colored 311 cartoon and marked with an outline. J. Enzymatic activity of N-terminal truncations of hDGAT1. 312 Initial rate of reaction versus oleoyl-CoA concentration. Each symbol represents the average of 15 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 313 three repeats. Error bars are s.e.m.. Solid lines are fit of the data points with a Michaelis-Menten 314 equation. 315 16 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 316 317 Figure 3. The reaction chamber and oleoyl-CoA binding. A-D. The reaction chamber (grey 318 surface) is shown in four orientations with the helices shown as cartoon. E-F. Density map of 319 oleoyl-CoA contoured at 7 σ in one hDGAT1 protomer shown as cartoon (E) or surface (F). In 320 (G), an oleoyl-CoA is modeled into the density and shown as spheres with carbon atoms colored 321 in yellow. The conserved SXXHEY motif was labeled as magenta spheres. H. Residues that 322 interact with oleoyl-CoA are shown in sticks with carbon atoms colored magenta. I. Interaction 323 between the FYXDWWN motif (magenta) with the N-terminus of the neighboring protomer 324 (cyan). J. Normalized enzymatic activity and oleoyl-CoA binding of hDGAT1 wild type and 325 mutants. (100 μM of oleoyl-CoA and 200 μM of 1,2-dioleoyl-sn-glycerol). Each bar represents 326 the average of three repeats. Error bars are s.e.m.. 327 17 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 328 329 Figure 4. Proposed gateway for DAG entry. A-B. An un-modeled tubular density extending 330 from the opening between TM4 and TM5 into the reaction chamber is viewed in two orientations. 331 C. Residues that line the TM4-5 opening are labeled as magenta sticks. 332 18 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 333 334 Figure 5. Proposed catalytic mechanism of hDGAT1. A. and B. A hDGAT1 monomer is 335 shown as a trapezoid in light blue and the reaction chamber in the shape of an inverted flask 336 colored in grey. TM7-9, acyl-CoA and DAG are shown schematically. The catalytic His415 is 337 marked in red on TM7. The CoA moiety of an acyl-CoA binds to hDGAT1 at the cytosolic 338 entrance of the tunnel and the rest of the acyl-CoA slides into the reaction chamber through a slit 339 between TM7 and TM8. The glycerol backbone of a DAG enters the chamber from a side 340 entrance and becomes almost horizontal with the two acyl-chains partially hosted in the 341 hydrophobic core of the membrane. C. After the reaction, CoASH exits the chamber through the 342 tunnel and the product TG could diffuse to either leaflet of the membrane. D. Proposed catalytic 343 mechanism. Both the 3-hydroxyl of DAG and the thioester of acyl-CoA are positioned near the 344 catalytic H415. E416 helps H415 activate the 3-hydroxyl on DAG for a nucleophilic attack on 345 the thioester of an Acyl-CoA. 346 347 19 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 348 Methods 349 Cloning, expression, and purification of human DGAT1 350 Human DGAT1 gene (accession number NP_036211) was codon-optimized and cloned into a 351 pFastBac dual vector(42) for production of baculovirus by the Bac-to-Bac method (Invitrogen). 352 High Five Cells (Thermofisher) at a density of ~3×106 cells/ml were infected with baculovirus 353 and grown at 27 °C for 48–56 h before harvesting. Cell membranes were prepared following a 354 previous protocol (42) and frozen in liquid nitrogen. 355 356 Purified membranes were thawed and homogenized in 20 mM HEPES, pH 7.5, 150 mM NaCl 357 and 2mM β-mercaptoethanol, and then solubilized with 1% (w/v) Lauryl Maltose Neopentyl 358 Glycol (LMNG, Anatrace) at 4 °C for 2 h. After centrifugation (55,000g, 45min, 4 °C), hDGAT1 359 was purified from the supernatant using a cobalt-based affinity resin (Talon, Clontech) and the 360 His-tag was cleaved by TEV protease. Oleoyl-CoA (20 μM) was added to reduce aggregation, 361 and hDGAT1 was then concentrated to 5 mg/ml (Amicon 100 kDa cutoff, Millipore) and loaded 362 onto a size-exclusion column (SRT-3C SEC-300, Sepax Technologies, Inc.) equilibrated with 20 363 mM HEPES, pH7.5, 150 mM NaCl, 0.01% glyco-diosgenin (GDN, Anatrace) for cryo-EM grid 364 preparation. For enzymatic assays, GDN was replaced with 1 mM (w/v) n-dodecyl-β-D- 365 maltoside (DDM, Anatrace). 366 367 hDGAT1 mutants were generated using the QuikChange method and the entire cDNA was 368 sequenced to verify the mutation. Mutants were expressed and purified following the same 369 protocol as wild type. 370 20 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 371 Cryo-EM sample preparation and data collection 372 The cryo grids were prepared using Thermo Fisher Vitrobot Mark IV. The Quantifoil R1.2/1.3 373 Cu grids were glow-discharged with air for 40 sec at medium level in a Plasma Cleaner (Harrick 374 Plasma, PDC-32G-2). Purified hDGAT1 was mixed with 1 mM of oleoyl-CoA and concentrated 375 to approximately 20 mg/ml. Aliquots of 3.5 µl purified hDGAT1 were applied to glow- 376 discharged grids. After being blotted with filter paper (Ted Pella, Inc.) for 3.5 s, the grids were 377 plunged into liquid ethane cooled with liquid nitrogen. A total of 2706 micrograph stacks were 378 collected with SerialEM(43) on a Titan Krios at 300 kV equipped with a K2 Summit direct 379 electron detector (Gatan), a Quantum energy filter (Gatan) and a Cs corrector (Thermo Fisher), at 380 a nominal magnification of 105,000 × and defocus values from -2.0 µm to -1.2 µm. Each stack 381 was exposed in the super-resolution mode for 5.6 s with an exposing time of 0.175 s per frame, 382 resulting in 32 frames per stack. The total dose rate was about 50 e-/Å2 for each stack. The stacks 383 were motion corrected with MotionCor2 (44) and binned 2 fold, resulting in a pixel size of 1.114 384 Å/pixel. In the meantime, dose weighting was performed (45). The defocus values were 385 estimated with Gctf (46). 386 387 Cryo-EM data processing 388 A total of 2,749,110 particles were automatically picked with RELION 2.1 (47-49). After 2D 389 classification, a total of 1,000,063 particles were selected and subject to a guided multi-reference 390 classification procedure. The references, one good and three bad, were generated with limited 391 particles in advance (Figure S3). Particles selected from multi-references 3D classification were 392 subjected to a global angular search 3D classification with one class and 40 iterations. The 393 outputs of the 31th-40th iterations were subjected to local angular search 3D classification with 21 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 394 four classes separately. Particles from the good classes of the local angular search 3D 395 classification were combined, yielding a total of 408945 particles. After handedness correction 396 and C2 symmetry application, 3D auto-refinement with an adapted mask yielded a 397 reconstruction with an overall resolution of 3.1 Å. Further 3D classification yielded a class of 398 275,945 particles and after 3D auto-refinement, yielded a map of 3.1 Å with improved density of 399 TM2, TM3, TM8 and lipids. 400 All 2D classification, 3D classification, and 3D auto-refinement were performed with RELION 401 3.0 and Cryosparc (50). Resolutions were estimated with the gold-standard Fourier shell 402 correlation 0.143 criterion (51) with high-resolution noise substitution (52). 403 404 Model building and refinement 405 For de novo model building of hDGAT1, a ploy-Alanine model was first built into the 3.1Å 406 density map manually in COOT (53). Structure refinements were carried out by PHENIX in real 407 space with secondary structure and geometry restraints (54). The EMRinger Score was 408 calculated as described (55). 409 410 DGAT1 Activity assay 411 hDGAT1 activity was monitored using a fluorescence-based coupled-enzyme assay (56) in a 412 quartz cuvette at 37°C. The cuvette was read in a FluoroMax-4 spectrofluorometer (HORIBA) 413 with 340 nm excitation and 465 nm emission at 15 s internals. All assays were done in a buffer 414 with 20 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM β-mercaptoethanol, 0.5 mM DDM, 1% 415 TritonX-100. Final concentrations of NAD+, thiamine pyrophosphate and α-ketoglutarate were 416 0.25 mM, 0.2 mM and 2 mM respectively. The α-ketoglutarate dehydrogenase (αKDH) was 22 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 417 prepared from beef heart using a published protocol (57). Sufficient amount of αKDH was used 418 to ensure that the hDGAT1 reaction is the rate limiting step. The hDGAT1 concentration in the 419 assay was 40 nM. The oleoyl-CoA concentration was 2.5-100 µM in assays for KM and Vmax 420 determination, and 100 µM in all other tests. The concentrations of 1,2-dioleoyl-sn-glycerol and 421 other monoacylglycerols were 200 µM in the selectivity assay. The initial rates in various DG 422 concentrations were not well fit with the traditional Michaelis Menten equation, but could be fit 423 with an allosteric sigmoidal equation: Y=Vmax*Xh/(Km+Xh), in which X is DAG 424 concentrations, and h is the Hill coefficient. 425 426 Scintillation proximity assay 427 Binding of Oleoyl-CoA to hDGAT1 was estimated using a Scintillation Proximity Assay (SPA). 428 Purified hDGAT1 (with his tag) was absorbed onto Copper HIS-Tag PVT beads (Perkin Elmer, 429 RPNQ0095) and incubated with [3H]-Acetyl-CoA (ARC, ART0213A) for 30 min at ~22°C in 430 the binding buffer (20 mM Hepes pH 7.5, 150 mM NaCl, and 0.02% GDN). Each 100 µL 431 reaction mixture contains 600 ng hDGAT1, 0.25 µM [3H]-Acetyl-CoA and 2.5 mg/ml Copper 432 HIS-Tag PVT beads. Background binding was estimated in the presence of both 800 mM 433 imidazole or in the absence of protein. For dose response curves, 0.005 to 200 µM of cold 434 oleoyl-CoA was used to compete for the binding of 0.25 µM 3H-acetyl-CoA. The binding assay 435 was performed in a 96-well plate (Perkin Elmer) and the scintillation read out by a MicroBeta 436 2450 Microplate Counter (Perkin Elmer). Data were plotted in Graphpad 8.0 software and fit 437 with the following equation to obtain IC50: Y=Bottom+(Top-Bottom)/(1+10^(X-LogIC50)) 438 23 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 439 Supplemental Information 440 441 442 443 Figure S1. Comparison DltB and DGAT1. Both hDGAT1 and DltB have an acyl-group donor 444 and an acceptor. In the acyl-group donor row, the red dashed lines indicate the bonds that are 445 broken during acyl-transfer reactions. In the acyl-group acceptor row, the hydroxyl groups are 446 highlighted in red. In hDGAT1, acyl-CoA comes from the intracellular side while DAG comes 447 from inside of the membrane. In DltB, the Ppant-DltC is intracellular while the LTA is 448 extracellular. 449 24 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 450 451 Figure S2. Biochemical and functional characterization of hDGAT1. A. size exclusion 452 profile of hDGAT1. Elution volume of membrane proteins of known molecular weight, bcMalT 453 (100 kDa, green) (58), mouse SCD1 (41 kDa, blue) (42) and opossum UT-A (200 kDa) (59) are 454 marked by arrows. B. A white layer of fat appeared after membrane solubilization and 25 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 455 centrifugation. C. hDGAT1 reaction is coupled to that of α-ketoglutarate dehydrogenase (αKDH) 456 to monitor production of Coenzyme A in real time. D. Fluorescence of NADH plotted versus 457 time. hDGAT1 (40 nM) is mixed with 100 μM oleoyl-CoA in the presence of αKDH, NAD+ 458 (0.25 mM), α-ketoglutarate (2 mM), and thiamine pyrophosphate (0.2 mM). E. Normalized 459 activity of hDGAT1 in the presence of EDTA, 1mM of Ca2+ or Mg2+. Each reaction has 100 μM 460 of oleoyl-CoA and 200 μM of 1,2-dioleoyl-sn-glycerol. F. Normalized activity of hDGAT1 in 461 the presence of 200 μM of 1,2-dioleoyl-sn-glycerol, 1-oleoyl-sn-glycerol, or 2-oleoyl-sn-glycerol. 462 Each reaction has 100 μM of oleoyl-CoA. G-I. Enzymatic activity of hDGAT1 with different 463 acyl-CoAs. Solid line represents fit of the data points with a Michaelis-Menten equation. In E-H, 464 each symbol represents the average of three repeats. Error bars are s.e.m. 465 466 26 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 467 27 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 468 Figure S3. Cryo-EM Analysis of the hDGA1. A. Representative micrograph of hDGAT1 (left), 469 its Fourier transform (middle) and representative 2d class averages (right). Representative 470 particles are labeled in red circles. B. A flowchart for the cryo-EM data processing and structure 471 determination of the hDGAT1. Final maps of hDGAT1 and the gold-standard Fourier shell 472 correlation curves for the overall maps are shown. C. Local resolution maps calculated using 473 RELION 2.0. 474 28 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 475 29 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 476 Figure S4. EM Maps for hDGAT1. A. The overall map of hDGAT1 and its atomic model. B. 477 EM density for each TM helix, each intracellular loop and the N-terminus. 478 30 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 479 480 31 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 481 Figure S5. Dimerization interface and lipids. A-H. hDGAT1 dimer is shown in two 482 orientations as cartoon (A and F) and surface (B and G) representation. Lipids and detergent 483 molecules buried in the dimer interface or attached to the surface of hDGAT1 are shown as 484 sticks. C-E and H. Detailed view of each detergent/lipid molecule and its corresponding density. 485 I. hDGAT1 dimer is shown in three orientations as electrostatic surface representation. The 486 electrostatic potential is calculated using the APBS plugin (60). 487 488 32 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 489 33 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 490 491 Figure S6. DGAT1 sequence alignment. DGAT1 sequences of human (Uniprot accession 492 number O75907), mouse (Q9Z2A7), frog (XENLA, A0A1L8G0L4), fish (DANIO, Q6P3J0), 493 thale cress (ARABID, Q9SLD2) and human ACAT (P35610) are aligned using the Clustal 494 Omega server (61). Secondary structural elements of hDGAT1 are labeled above the alignment. 495 Residues are colored based on their conservation using the ESPript server (62). Residues at the 496 acyl-CoA binding site are labeled with green triangles and those at the active site with red stars. 497 34 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 498 499 Figure S7. Interaction of the N-terminus with the neighboring protomer. A. hDGAT1 dimer 500 (cartoon) is viewed in two orientations. Detailed interactions between a hDGAT1 protomer and 501 the N-terminus from the neighboring protomer are shown in C-E. Residues involved in the 502 interactions are shown as sticks. F. Size-exclusion chromatography of N-terminal truncations of 503 hDGAT1. G. Competitive binding of various concentrations of cold oleoyl-CoA against 0.25 µM 504 of 3H-acetyl-CoA on different hDGAT1 N-terminal truncation mutations. Each symbol is the 505 average of three repeats, and error bars are s.e.m.. 35 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 506 507 Figure S8. Oleoyl-CoA binding binding site. A-C. Oleoyl-CoA (spheres) bound to hDGAT1 508 protomer (cartoon) viewed in three orientations. Detailed interaction between hDGAT1 and the 509 CoA moiety and the acyl-chain are shown in D-G. Residues involved in coordinating oleoyl- 510 CoA are shown as sticks with carbon atoms colored in magenta. H. Planar view of the interaction 511 between oleoyl-CoA and hDGAT1 generated by LigPlus (63, 64). I. Competitive binding for the 512 residues involved in oleoyl-CoA binding. Each symbol represents the average of three repeats, 513 and error bars are s.e.m.. Data were fit to a single site binding isotherm. 36 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 514 515 Figure S9. The MBOAT fold in hDGAT1 and DltB. Structures of hDGAT1 (A-C) and DltB 516 (D-F) are shown as cartoon in three different orientations. The equivalent helices in the MBOAT 517 fold are colored the same. Helices that are distinct in each protein, TM1 in hDGAT1 and H1-H3 518 in DtlB, are colored in grey. G and I. Cartoon representations of hDGAT1 and DltB structure. 519 AH2 in hDGAT1 and H13 in DltB adopts very different conformations and are highlighted in 37 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 520 blue. H and J. Cut-away surface illustrations of hDGAT1 and DltB showing their cytosolic 521 tunnels. The position of the conserved histidine residue is marked as a yellow star. In DltB, 522 active site is located to the thin layer separating the intra- and extracellular sides. 523 38 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 524 Table S1 | Summary of Cryo-EM data collection, processing and refinement 525 526 39 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 527 Table S2 | Summary of enzymological parameters of hDGAT1 and mutants 528 529 530 40 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 531 References 532 533 534 1. Cases S, et al. (1998) Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proceedings of the National Academy of Sciences 95(22):13018. 535 536 2. Bhatt-Wessel B, Jordan TW, Miller JH, & Peng L (2018) Role of DGAT enzymes in triacylglycerol metabolism. Archives of Biochemistry and Biophysics 655:1-11. 537 538 539 3. Liu Q, Siloto RMP, Lehner R, Stone SJ, & Weselake RJ (2012) Acyl-CoA:diacylglycerol acyltransferase: Molecular biology, biochemistry and biotechnology. Progress in Lipid Research 51(4):350-377. 540 541 4. Kayden HJ, Senior JR, & Mattson FH (1967) The monoglyceride pathway of fat absorption in man. The Journal of clinical investigation 46(11):1695-1703. 542 543 544 545 5. Mansbach CM & Gorelick F (2007) Development and Physiological Regulation of Intestinal Lipid Absorption. II. Dietary lipid absorption, complex lipid synthesis, and the intracellular packaging and secretion of chylomicrons. American Journal of Physiology-Gastrointestinal and Liver Physiology 293(4):G645-G650. 546 547 6. Kühn C, et al. (2004) Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle. Genetics 167(4):1873-1881. 548 549 7. Dyer JM, Stymne S, Green AG, & Carlsson AS (2008) High-value oils from plants. The Plant Journal 54(4):640-655. 550 551 8. Durrett TP, Benning C, & Ohlrogge J (2008) Plant triacylglycerols as feedstocks for the production of biofuels. The Plant Journal 54(4):593-607. 552 553 9. Vieyres G & Pietschmann T (2019) HCV Pit Stop at the Lipid Droplet: Refuel Lipids and Put on a Lipoprotein Coat before Exit. Cells 8(3):233. 554 555 10. Kim Y, et al. (2012) Novel triacsin C analogs as potential antivirals against rotavirus infections. European journal of medicinal chemistry 50:311-318. 556 557 11. Crawford SE & Desselberger U (2016) Lipid droplets form complexes with viroplasms and are crucial for rotavirus replication. Current opinion in virology 19:11-15. 558 559 12. Smith SJ, et al. (2000) Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat. Nature Genetics 25(1):87-90. 560 561 13. Chen HC, et al. (2002) Increased insulin and leptin sensitivity in mice lacking acyl CoA:diacylglycerol acyltransferase 1. The Journal of clinical investigation 109(8):1049-1055. 562 563 564 14. Zhao G, et al. (2008) Validation of Diacyl Glycerolacyltransferase I as a Novel Target for the Treatment of Obesity and Dyslipidemia Using a Potent and Selective Small Molecule Inhibitor. Journal of Medicinal Chemistry 51(3):380-383. 565 566 15. DeVita RJ & Pinto S (2013) Current Status of the Research and Development of Diacylglycerol O-Acyltransferase 1 (DGAT1) Inhibitors. Journal of Medicinal Chemistry 56(24):9820-9825. 41 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 567 568 16. Das A, Davis MA, & Rudel LL (2008) Identification of putative active site residues of ACAT enzymes. Journal of lipid research 49(8):1770-1781. 569 17. Nusse R (2005) Wnt signaling in disease and in development. Cell Research 15(1):28-32. 570 571 18. Nusse R, van Ooyen A, Cox D, Fung YKT, & Varmus H (1984) Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature 307(5947):131-136. 572 573 19. Hofmann K (2000) A superfamily of membrane-bound O-acyltransferases with implications for wnt signaling. Trends in biochemical sciences. 25(3):111-112. 574 575 576 577 20. McFie PJ, Stone SL, Banman SL, & Stone SJ (2010) Topological Orientation of AcylCoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation. Journal of Biological Chemistry 285(48):37377-37387. 578 579 580 21. Rios-Esteves J, Haugen B, & Resh MD (2014) Identification of key residues and regions important for porcupine-mediated Wnt acylation. The Journal of biological chemistry 289(24):17009-17019. 581 582 583 22. Yang J, Brown MS, Liang G, Grishin NV, & Goldstein JL (2008) Identification of the Acyltransferase that Octanoylates Ghrelin, an Appetite-Stimulating Peptide Hormone. Cell 132(3):387-396. 584 585 23. Buglino JA & Resh MD (2010) Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog. PloS one 5(6):e11195-e11195. 586 587 588 24. Lin S, Lu X, Chang CCY, & Chang T-Y (2003) Human acyl-coenzyme A:cholesterol acyltransferase expressed in chinese hamster ovary cells: membrane topology and active site location. Molecular biology of the cell 14(6):2447-2460. 589 590 25. Ma D, et al. (2018) Crystal structure of a membrane-bound O-acyltransferase. Nature 562(7726):286-290. 591 592 26. Cheng D, et al. (2001) Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein. The Biochemical journal 359(Pt 3):707-714. 593 594 595 27. Zhang J, et al. (2014) Monoacylglycerol Acyltransferase-2 Is a Tetrameric Enzyme That Selectively Heterodimerizes with Diacylglycerol Acyltransferase-1. Journal of Biological Chemistry 289(15):10909-10918. 596 597 598 28. Cao J, et al. (2011) Targeting Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small molecule inhibitors for the treatment of metabolic diseases. The Journal of biological chemistry 286(48):41838-41851. 599 600 601 29. Yen C-LE, Monetti M, Burri BJ, & Farese RV (2005) The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters. Journal of Lipid Research 46(7):1502-1511. 42 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 602 603 604 30. Orland MD, et al. (2005) Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1737(1):76-82. 605 606 31. Caldo KMP, et al. (2017) Diacylglycerol Acyltransferase 1 Is Regulated by Its N-Terminal Domain in Response to Allosteric Effectors. Plant physiology 175(2):667-680. 607 608 609 32. Little D, Weselake R, Pomeroy K, Furukawa-Stoffer T, & Bagu J (1994) Solubilization and characterization of diacylglycerol acyltransferase from microspore-derived cultures of oilseed rape. The Biochemical journal 304 ( Pt 3)(Pt 3):951-958. 610 611 33. Lung S-C & Weselake RJ (2006) Diacylglycerol acyltransferase: A key mediator of plant triacylglycerol synthesis. Lipids 41(12):1073-1088. 612 613 34. Cases S, et al. (2001) Cloning of DGAT2, a Second Mammalian Diacylglycerol Acyltransferase, and Related Family Members. Journal of Biological Chemistry 276(42):38870-38876. 614 615 35. von Heijne G & Gavel Y (1988) Topogenic signals in integral membrane proteins. European Journal of Biochemistry 174(4):671-678. 616 617 618 36. Shockey JM, et al. (2006) Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum. The Plant Cell 18(9):2294. 619 620 621 37. Wurie HR, Buckett L, & Zammit VA (2011) Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells. Journal of Biological Chemistry 286(42):36238-36247. 622 623 624 38. Weselake RJ, et al. (2006) Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape. BMC biochemistry 7:24-24. 625 626 39. Panigrahi R, et al. (2018) Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus. Sci Rep 8(1):16665-16665. 627 628 629 40. Guo Z, Cromley D, Billheimer JT, & Sturley SL (2001) Identification of potential substratebinding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences. Journal of Lipid Research 42(8):1282-1291. 630 631 632 41. Oelkers P, Behari A, Cromley D, Billheimer JT, & Sturley SL (1998) Characterization of Two Human Genes Encoding Acyl Coenzyme A:Cholesterol Acyltransferase-related Enzymes. Journal of Biological Chemistry 273(41):26765-26771. 633 634 42. Bai Y, et al. (2015) X-ray structure of a mammalian stearoyl-CoA desaturase. Nature 524(7564):252-256. 635 636 43. Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152(1):36-51. 637 638 44. Zheng SQ, et al. (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14(4):331-332. 43 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 639 640 45. Grant T & Grigorieff N (2015) Measuring the optimal exposure for single particle cryo-EM using a 2.6 A reconstruction of rotavirus VP6. Elife 4:e06980. 641 46. Zhang K (2016) Gctf: Real-time CTF determination and correction. J Struct Biol 193(1):1-12. 642 643 47. Scheres SH (2015) Semi-automated selection of cryo-EM particles in RELION-1.3. J Struct Biol 189(2):114-122. 644 645 48. Scheres SH (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180(3):519-530. 646 647 49. Kimanius D, Forsberg BO, Scheres SH, & Lindahl E (2016) Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Elife 5. 648 649 50. Punjani A, Rubinstein JL, Fleet DJ, & Brubaker MA (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature Methods 14(3):290-296. 650 651 51. Rosenthal PB & Henderson R (2003) Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 333(4):721-745. 652 653 654 52. Chen S, et al. (2013) High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135:24-35. 655 656 53. Emsley P, Lohkamp B, Scott WG, & Cowtan K (2010) Features and development of Coot. Acta crystallographica. Section D, Biological crystallography 66(Pt 4):486-501. 657 658 659 54. Adams PD, et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography 66(Pt 2):213221. 660 661 55. Barad BA, et al. (2015) EMRinger: side chain-directed model and map validation for 3D cryoelectron microscopy. Nature methods 12(10):943-946. 662 663 56. Rana MS, et al. (2018) Fatty acyl recognition and transfer by an integral membrane Sacyltransferase. Science (New York, N.Y.) 359(6372):eaao6326. 664 665 57. Stanley CJ & Perham RN (1980) Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method. The Biochemical journal 191(1):147-154. 666 667 668 58. Ren Z, et al. (2018) Structure of an EIIC sugar transporter trapped in an inward-facing conformation. Proceedings of the National Academy of Sciences of the United States of America 115(23):5962-5967. 669 670 671 59. Levin EJ, et al. (2012) Structure and permeation mechanism of a mammalian urea transporter. Proceedings of the National Academy of Sciences of the United States of America 109(28):1119411199. 672 673 674 60. Baker NA, Sept D, Joseph S, Holst MJ, & McCammon JA (2001) Electrostatics of nanosystems: Application to microtubules and the ribosome. Proceedings of the National Academy of Sciences 98(18):10037. 44 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.896332. this version posted January 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 675 676 61. Sievers F, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular systems biology 7:539-539. 677 678 62. Robert X & Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research 42(W1):W320-W324. 679 680 63. Wallace AC, Laskowski RA, & Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Engineering, Design and Selection 8(2):127-134. 681 682 64. Laskowski RA & Swindells MB (2011) LigPlot+: Multiple Ligand–Protein Interaction Diagrams for Drug Discovery. Journal of Chemical Information and Modeling 51(10):2778-2786. 683 684 45