Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy

Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.


Abstract
Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes 26 controlling the homeostasis of ceramides. They are implicated in human pathophysiology, 27 including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We 28 report here the crystal structure of the human ACER type 3 (ACER3). Together with 29 computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a 30 seven transmembrane domain architecture and a catalytic Zn 2+ binding site in its core, similar 31 to adiponectin receptors. Interestingly, we uncover a Ca 2+ binding site physically and 32 functionally connected to the Zn 2+ providing a structural explanation for the known regulatory 33 role of Ca 2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 34 mutant found in leukodystrophic patients. expressed in most tissues 10-12 . Very little is known at the molecular level: ACERs are 80 localized intracellularly in the membrane of the endoplasmic reticulum-Golgi apparatus 81 network and their activity, mainly directed against ceramides with long unsaturated acyl 82 chains (C18:1, C20:1 and C24:1), was shown to be Ca 2+ -dependent 10,12-14 . 83 The critical role of ACERs in human physiology and, in particular ACER3, was recently 84 revealed by clinical data demonstrating that ACER3 deficiency leads to progressive 85 leukodystrophy in early childhood 15 . This study demonstrated that patients were homozygous 86 for a p.E33G ACER3 mutant and that this mutation impaired the ACER3 ceramidases activity 87 in patients' cells. When compared to healthy individuals, this loss of function resulted in 88 higher level of several ceramide species in the blood, in particular for the ACER3 preferred 89 substrates, C18:1 and C20:1 ceramides. It was proposed that these aberrant levels of 90 ceramides in the brain could result in an incorrect central myelination leading to the clinical 91 phenotype associated with the ACER3 mutant. However, in mice, while ACER3 knock-out 92 results in an aberrant accumulation of various ceramides, it does not affect myelination. 93 Instead, this deficiency induces the premature degeneration of Purkinje cells and cerebellar 94 ataxia 16 . In the periphery, in mice, the modulation of C18:1 ceramide levels by ACER3 95 regulates the immune response through the upregulation of cytokines while its deficiency 96 increases colon inflammation and its associated tumorigenesis 17 . Moreover, in vitro results 97 obtained in human cells revealed that ACER3 contributes to acute myeloid leukemia (AML) 98 pathogenesis 18 . Indeed, it was found that ACER3 expression negatively correlates with the 99 survival of AML patients, and that ACER3 is essential for the growth of AML cells as the sh-environment formed by the residues S160 TM5 , S178 TM6 and H231 TM7 in which we found 155 some electron density that we modeled as a water molecule (Fig. 2b). Such a 156 hydrophilic patch might play a role in ceramide binding. Directly below this area 157 appeared a large electron density in the calculated 2Fo--Fc map as well as in the 158 (unbiased) polder OMIT map 22 (see methods) (Extended data Fig. 3) that we 159 tentatively modeled as a monoolein. Indeed, monoolein by weight forms 54% of the 160 lipidic cubic phase (concentration in the mM range) and displays an acyl chain moiety 161 chemically identical to that of C18:1 ceramide (Extended data Fig. 3), the preferred 162 substrate of ACER3 14 . 163 At the bottom of the intramembrane pocket, we unambiguously identified a Zn 2+ ion 164 (Extended data Fig. 3 and methods). This Zn 2+ is coordinated by three His residues 165 (H81 TM2 , H217 TM7 and H221 TM7 ) and a water molecule forming hydrogen bonds with 166 S77 TM2 and D92 TM3 (Fig. 2d). The residues forming this Zn 2+ binding site are strictly 167 conserved among the ACER family, and across species from yeast to human, highlighting 168 the importance of this site in the biological function of these proteins (Extended data 169 Fig. 4). Given the well--characterized ceramidase activity of ACER3, these structural data 170 definitively establish ACERs as Zn 2+ --dependent intramembrane enzymes. M96 TM3 , F103 TM3 , V153 TM5 , L156 TM5 , V157 TM5 and F182 TM6 (Fig. 3b). Surprisingly, the 180 site of unsaturation with the sp 2 carbons is surrounded by a polar environment 181 constituted by the sulfhydryl group of C100 TM3 and three hydroxyl groups of S99 TM3 , 182 Y149 TM5 and S228 TM7 (Fig. 3c). It is not clear from the structure alone whether this 183 feature has a role in the C18:1 vs C18:0 ceramide substrate preference. The sphingosine 184 moiety remains partially accessible to the membrane leaflet and interacts with a set of 185 hydrophobic residues from TM5 and TM6 (Fig. 3b). The ceramide carbonyl group and 186 its primary alcohol directly interact with the Zn 2+ ion, resulting in an octahedral 187 coordination sphere (Fig. 3d). The ceramide is further stabilized by polar contacts 188 between its carbonyl and the amine group of W220 TM7 side chain, as well as its primary 189 alcohol and the carboxylate of D92 TM3 . Interestingly, the crystallographic water molecule 190 that bridges S77 TM2 , D92 TM3 and the Zn 2+ ion appears to be suitably placed for a 191 nucleophilic attack on the ceramide carbonyl (Fig. 3d), suggesting a general acid--base 192 catalytic mechanism in which D92 TM3 acts as a proton acceptor/donor (similar to e.g.,

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On the other hand, the described structure of the soluble NCDase has nothing in 235 common with ACER3 structure apart from the three histidines coordinating the Zn 2+ 236 (Extended Data Fig. 6). In particular, catalytic sites belong to two clearly distinct 237 scaffolds: intramembranous for ACER3 and membrane--associated for NCDase with a 238 water--soluble domain predominantly constituted of beta--sheets (Extended Data Fig. 6) 239 forming the so--called beta--triangle hydrolase scaffold 9 . These clearly distinct structures 240 are in agreement with the different cellular localization as well as with the functional 241 specialization of these enzymes 3 .

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A Ca 2+ binding site within the N--terminus 244 Perhaps the most exciting/interesting feature of the ACER3 structure is the presence of 245 a Ca 2+ binding site within the N--terminus domain (Fig. 5a). Ca 2+ was shown to modify 246 the enzymatic activity of ACER3 but the molecular basis for this effect was unknown.

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The Ca 2+ ion is coordinated by six oxygens from the D19 carboxylate (bidentate), the 248 W20 backbone carbonyl, the E22 backbone carbonyl, the N24 side chain carbonyl and 249 the E33 carboxylate (monodentate) (Fig. 5b) are strictly conserved among the ACER family (Fig. 5c) in agreement with the Ca 2+ --258 dependent ceramidase activity described for ACER1, ACER2 and ACER3 10,12--14 . 259 Moreover, this domain is also strictly conserved across species from yeast to human 260 (Fig. 5c, Extended Data Fig. 4). This strict conservation during evolution further 261 highlights the paramount regulatory role of Ca 2+ in the enzymatic function.

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Interestingly, based on the crystal structure and MD simulations results, two residues 263 appear to play critical roles in physically linking the Ca 2+ and the Zn 2+ sites, W20 and 264 E33. Indeed, in the structure, the two residues interact simultaneously with both metal 265 sites (Fig. 5d). First, E33 carboxylate interacts with the Ca 2+ ion and is in close proximity 266 to H81 TM2 side chain, while H81 TM2 coordinates the Zn 2+ (Fig. 5d). Interestingly, E33 267 carboxylate rearranges during MD simulations to form a hydrogen bond with H81 TM2 268 side chain (Fig. 5d,e). Second, W20 coordinates Ca 2+ through its backbone carbonyl, and 269 its indole ring forms a His--aromatic complex with H81 TM2 and H217 TM7 side chains towards the cytoplasm (Fig. 5f). We hypothesize that the functional effect of Ca 2+ on the 276 catalytic activity of ACER3 originates from this direct link, with changes to the Ca 2+ site 277 propagating to the Zn 2+ catalytic site.

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Remarkably, this discovery provides molecular insights into the E33G ACER3 mutation 279 carried by patients suffering leukodystrophy, which results in the loss of ACER3 280 ceramidase activity 15 . We used MD simulations to investigate the effect of this mutation 281 on the stability of the ACER3 structure relative to the CER18:1--bound wild type model 282 (Fig.  6). The effect observed on the flexibility of the Zn 2+ itself is rather mild due to the 283 integrity of its coordinating residues and the presence of the stabilizing C18:1 ceramide 284 molecule (Extended data Fig. 8). Of note, unlike the wild--type ACER3, the purified E33G 285 ACER3 mutant was highly unstable during purification and yielded mostly aggregated  In this study, we solved the crystal structure of ACER3 revealing a seven-transmembrane 299 domain architecture harboring a catalytic Zn 2+ binding site nearly identical to the one we 300 recently uncovered in ADIPORs 21 . Considering the fact that ACER3 and AdipoRs share a 301 similar fold, a common Zn 2+ catalytic site and similar functional enzymatic capability, it is 302 highly likely that ADIPORs are indeed genuine ceramidases. These discoveries are expanding 303 the family of ceramidases to, at least, seven members in humans. Given the established 304 functional link between fungal progesterone adipoQ receptor (PAQR) and ceramidase activity 305 26 , it is then tempting to speculate that all the members of the PAQR family 27 (11 proteins in 306 humans including ADIPOR1 and ADIPOR2) share this functional characteristic with ACER3. 307 Moreover, our data provide a structural explanation of the previously demonstrated regulatory 308 role of Ca 2+ on ACER3 enzymatic activity 12 . As it is known that high pH can increase the 309 binding of Ca 2+ , this might also constitute the molecular basis for the alkaline pH optima of 310 ACER ceramidase activity in vitro 12 . 311 Although ADIPORs seem to be ligand-dependent ceramidases (activated by adiponectin), the 312 intracellularly residing ACER3 might be regulated through changes in cytoplasmic Ca 2+ 313 concentration and/or local pH. In addition, the ACER3 structure and computational studies 314 highlight the role of E33 in the formation of the Ca 2+ binding site and provide insights into the 315 loss of ceramidase activity of the E33G-ACER3 mutant found in leukodystrophic patients. 316 This knowledge will enable the development of pharmacological chaperones specifically 317 designed to restore the enzymatic function of ACER3 mutant. This may constitute a potential 318 treatment option for individuals diagnosed with ACER3 mutation when leukodystrophy is 319 suspected (at the onset) and before severe clinical conditions manifest. In addition, our study 320 opens the way to the structure-based discovery of small molecules able to control the 321 ceramidase activity of ACER3. Ultimately, such modulators could have beneficial effects 322 against the pathogenesis of diseases involving C18:1 ceramide and ACER3 dysregulation 323 including colon cancer and AML. for serial synchrotron crystallographic data selection and merging procedure was developed at 403 the beamline (unpublished, code availability statement: the software will be made available 404 upon request). In order to identify correctly processed datasets, individual mini dataset was 405 first checked for indexing consistency, against user-provided reference unit-cell parameters. 406 Then, 194 datasets, identified as correctly indexed with a = 60.8 Å, b = 68.8 Å, and c = 257.0 407 Å in C2221 space--group, were scaled together using XSCALE 33 program in XDS package. 408 After a first round of scaling, many datasets, which have very low ISa values (described in 34 ), 409 were rejected against a cut-off value of 3.0. Thus, 77 datasets were selected out of 194 410 datasets, and scaled using XSCALE for second round. This yielded a scaled but unmerged 411 dataset with 100% completeness at 2.7Å resolution. The resolution cutoff was decided These 198 statistically equivalent datasets were scaled together using XSCALE program, 416 yielding a scaled but unmerged dataset with 100% completeness and high multiplicity at 3 Å 417 resolution. Later, these scaled but unmerged HKL files (output from XSCALE) were 418 converted into mtz files for structure determination. 419 420 Structure determination and refinement 421 The structure of ACER3-BRIL was determined by molecular replacement. Initial phases were 422 obtained using the BRIL molecule extracted from PDB 4RWD (chain B) as a search model in 423 PHASER 36 . After density improvement using the CCP4 program parrot 37 , the electron density 424 corresponding to ACER3 was still poor and it was only possible to build the C-terminal half 425 of helix 7 (h7 1/2 ) which is attached to the BRIL. Subsequently, an ab-initio model of ACER3 426 obtained from the I-TASSER webserver 28 was cut into fragments of two transmembrane 427 helices, which were used as additional search models in PHASER while keeping the BRIL-428 ACER3 h7 1/2 fragment as a fixed solution. This procedure allowed us to successfully place 429 two, and then four transmembrane helices. This in turn provided enough phase information to 430 enable manual rebuilding of ACER3 in COOT 38 , as well as autobuilding in BUCCANEER 39 . 431 Subsequently, the structure was refined using AUTOBUSTER 40 . At late stages of refinement, 432 translation libration screw-motion (TLS) parameters generated within AUTOBUSTER were 433 used. MolProbity was used to assess the quality of the structure 41 and indicated that 98% of 434 residues were within favored Ramachandran regions. The data collection and refinement 435 statistics are summarized in Extended Data Table 1 subjected to energy minimization, equilibration and production simulation using the 477 GROMACS input scripts generated by CHARMM-GUI 47 . Briefly, the system was energy 478 minimized using 5000 steps of steepest descent, followed by 375 ps of equilibration. NVT 479 (constant particle number, volume, and temperature) and NPT (constant particle number, 480 pressure, and temperature) equilibrations were followed by NPT production runs for both 481 systems. The van der Waals interactions were smoothly switched off at 10-12 Å by a force-482 switching function 48 , whereas the long-range electrostatic interactions were calculated using 483 the particle mesh Ewald method 49 . The temperature and pressure were held at 310.15 K and 1 484 bar, respectively. The assembled systems were equilibrated by the well-established protocol 485 in Membrane Builder, in which various restraints were applied to the protein, lipids and water 486 molecules, and the restraint forces were gradually reduced during this process. During positive ion mode. The source parameters used were as follows: source temperature was set at 520 300 °C, nebulizer gas (nitrogen) flow rate was 10 L/min, sheath gas temperature was 300 °C, 521 sheath gas (nitrogen) flow rate was 12 L/min and the spray voltage was adjusted to +4000 V. 522 The collision energy optimums for sphingosine (d17:1) and sphingosine (d18:1) were 5 eV.     TM4   ECL3   TM7   TM3  TM5   TM6  TM2   TM1   R203 ICL3   K204 ICL3   K205 ICL3   V206 ICL3   P207 ICL3   P208 ICL3   ECL1   ECL2   C196 TM6   C21 Nter   TM6  TM1  TM7 N-terminus   Cytoplasm   TM7  TM6  TM5   TM3   TM2   TM1   180°T   M7   TM6   TM5   TM3  TM2   In all panels, the Zn 2+ and Ca 2+ as well as the water molecule are represented as spheres (coloured orange, green and red, respectively).     TM4   ECL3   TM7  TM3  TM5   TM6  TM2   TM1   ECL1  ECL2   ICL1  ICL2   ICL3   BRIL   TEV  (a) Based on the interactions with docked ceramide C18:1 and the Zn 2+ catalytic site architecture in the structure, we propose a general acid-base catalysis mechanism for the hydrolysis of the amide bond by ACER3. In this mechanism, the zinc ion activates a water molecule for nucleophilic attack of the amide carbon in which D92 TM3 acts as a proton acceptor/donor (1 and 3). W220 side chain polarizes the amide carbonyl and stabilizes the oxyanion formed in the tetrahedral transition state