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Isolated Polar Amino Acid Residues Modulate Lipid Binding in the Large Hydrophobic Cavity of CD1d Shinsuke Inuki, Toshihiko Aiba, Natsumi Hirata, Osamu Ichihara, Daisuke Yoshidome, Shunsuke Kita, Katsumi Maenaka, Koichi Fukase, and Yukari Fujimoto ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00674 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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ACS Chemical Biology

Isolated Polar Amino Acid Residues Modulate Lipid Binding in the Large Hydrophobic Cavity of CD1d Shinsuke Inuki,† Toshihiko Aiba,†, ‡ Natsumi Hirata,† Osamu Ichihara, § Daisuke Yoshidome, § Shunsuke Kita,|| Katsumi Maenaka,|| Koichi Fukase,‡ Yukari Fujimoto*,† †Graduate

School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama,

Kanagawa 223-8522, Japan ‡Department

of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho,

Toyonaka, Osaka 560-0043, Japan §Schrödinger

K. K., 17F Marunouchi Trust Tower North, 1-8-1 Marunouchi Chiyoda-ku, Tokyo 100-

0005, Japan ||Graduate

School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan

[email protected]

TOC:

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Abstract: The CD1d protein is a non-polymorphic MHC class I-like protein that controls the activation of natural killer T (NKT) cells through the presentation of self and foreign lipid ligands, glycolipids, or phospholipids, leading to the secretion of various cytokines. The CD1d contains a large hydrophobic lipid binding pocket: the A’ pocket of CD1d, which recognizes hydrophobic moieties of the ligands, such as long fatty acyl chains. Although lipidprotein interactions typically rely on hydrophobic interactions between lipid chains and the hydrophobic sites of proteins, we showed that the small polar regions located deep inside the hydrophobic A’ pocket, could be used for the modulation of the lipid binding. A series of the ligands, -galactosyl ceramide (-GalCer) derivatives containing polar groups in the acyl chain, was synthesized, and the structure-activity relationship studies demonstrated that simple modification from a methylene to an amide group in the long fatty acyl chain, when introduced at optimal positions, enhanced the CD1d recognition of the glycolipid ligands. Formation of hydrogen bonds between the amide group and the polar residues was supported by molecular dynamics (MD) simulations and WaterMap calculations. The computational studies suggest that localized hydrating water molecules may play an important role in the ligand recognition. Here, the results showed that confined polar residues in the large hydrophobic lipid binding pockets of the proteins could be potential targets to modulate the affinity for its ligands.

Introduction Lipidprotein interactions play fundamental roles in the regulation of a variety of biological processes, such as signaling or trafficking in cells.1 Fatty acids, ceramides, glycolipids, and lipid proteins can bind strongly or selectively to specific proteins to regulate diverse biological activities, and these interactions have recently received significant attention as therapeutic targets for a variety of disorders.2 Lipidprotein interactions typically rely on hydrophobic interactions between lipid alkyl chains and the hydrophobic sites of proteins; however, certain lipid-binding areas in proteins include small hydrophilic regions, which can form specific hydrogen bonds with a ligand, and in some cases, these regions are located deep inside the hydrophobic binding pockets.3 In recent years, several researchers have ACS Paragon Plus Environment

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characterized and evaluated a buried hydrogen bond behavior in an effort to predict the druggability of the putative binding sites.4,5 Barril et al. demonstrated that formation of shielded hydrogen bonds between ligands and their receptor proteins increased the kinetic stability of the complexes.6 On the other hands, some groups reported that hydrogen bond strengths depended on the polarity of surrounding environments.7 Kelly and co-workers demonstrated that the strength of hydrogen bonds in hydrophobic environments can increase by up to 1.2 kcal/mol in comparison with that of hydrogen bonds in polar environments, and provide forces responsible for protein folding.8 However, these kind of confined hydrogen bonds in large hydrophobic lipid binding pockets, such as for recognition site of fatty acids, have not been investigated in detail. The MHC class I-like molecule CD1d is a nonpolymorphic antigen-presenting glycoprotein, the ligands of which include glycolipids.9 These ligands bind to the hydrophobic groove of CD1d and then activate natural killer (NK) T cells by means of T cell receptor (TCR) recognition of CD1d-ligand complex.10 NKT cell activation leads to the secretion of various cytokines, e.g., IFN-, TNF, IL-4, IL-5, IL-10 and, IL-13, which then initiate various immune responses and modulate the balance of immune system.11 IFN- and TNF support inflammatory responses and activate the cellular immune responses (the Th1 (T-helper 1) response).12 By contrast, IL-4, IL-5, IL-10, and IL-13 mainly modulate humoral immunity (the Th2 (T-helper 2) response). It was reported that the production levels and balance of the cytokines depended on the ligand structure,13 and furthermore, the binding affinity of glycolipids to CD1d was found to correlate well with cytokine production by NKT cells.14,15 In particular, enhancing the stability of the glycolipids/CD1d complex could potentially increase the IFN- production. The control

over

the

cytokine

levels

and

balance

is

important

for

the

development

of

effective immunotherapies, such as anti-cancer or anti-virus (Th1 response) or anti-parasite (Th2 response) immunity. The immune regulation via CD1d is also recently examined for controlling the immune tolerance to prevent graft rejections.16 For these reasons, researchers have developed a number of CD1d ligands based on natural or synthetic compounds.17 The glycolipid, -galactosyl ceramide with a saturated acyl chain (C26:0) (-GalCer, KRN7000, Figure 1), is one of the most potent 3 ACS Paragon Plus Environment


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ligands18 and is derived from a marine natural product. Several structure–activity relationship (SAR) studies based on -GalCer derivatives have been performed.13 Only a few compounds with more potent activities against NKT cells than -GalCer have been identified thus far.13 -GalCer consists of a sugar head, ceramide, and an acyl chain component. According to the crystal structures of murine CD1d complexed with -GalCer, the sugar head group is mainly recognized by TCR on the NKT cell.19,20 The crystal structure also showed that CD1d includes two large hydrophobic pockets: the A’ pocket and the F’ pocket, which recognize lipid hydrophobic moieties. The ceramide part, including the long alkyl chain, can bind to the F’ pocket. In contrast, the acyl chain part can be accommodated in the A’ pocket of CD1d, a large hydrophobic binding groove lined with hydrophobic

residues.

Previous

SAR

studies

have

suggested

that the strength of the

hydrophobic interactions in the A’ pocket significantly impact cytokine production.13 Truncation of the saturated acyl chain of -GalCer, such as C20:0 -GalCer, can actually reduce global cytokine production.21 In particular, the cytokine measurements on mouse splenocytes showed that IFN- levels for C20:0 -GalCer were markedly reduced in comparison with those for C26:0 -GalCer. The A’ pocket includes a few hydrophilic residues, Cys12, Gln14, Ser28, or His38, which are expected to form hydrogen bonds to ligands;19 however, the significance of these residues have not been evaluated in detail. We focused on these regions to investigate the importance of limited hydrophilic regions in a lipid binding site and to examine whether these sites are available for ligandprotein interactions. Some research groups introduced several functional groups into the fatty acid chain of -GalCer, and evaluated their activities.22-27 Among them, Kim and co-workers focused on the hydrophilic residues in mCD1d and designed truncated -hydroxy fatty acid-containing -GalCer derivatives, revealing that the compound retained an activity comparable to that of -GalCer.25 On the other hands, in the crystal structure of mCD1d with a -GalCer derivative, GalA-GSL, which contained a relatively short C14 acyl chain, the CD1d complex also included a palmitic acid (C16) in the A’ pocket, and the carboxylic group of the palmitic acid interacted with the Cys12 side chain.28 In the present study, we ACS Paragon Plus Environment

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thus designed and synthesized novel glycolipid ligands containing polar functional groups in the fatty acid part, in an effort to effectively modulate binding, to investigate the potential utility of these molecules as novel CD1d ligands, and to characterize the contributions of their hydrogen bonding interactions toward the ligand activities using NKT cell activation assays and molecular dynamics (MD) simulations.29

Figure 1. Structure of -galactosyl ceramide with a saturated acyl chain (C26:0) (-GalCer, KRN7000). Our ligand design strategy is based on the modification from a methylene to an amide group in the long fatty acyl chain to allow the interaction between the polar residues and the ligands.

Results and Discussion

Figure 2. Hydrophilic and hydrophobic map of the A’ pocket in mCD1d (PDB: 3G08, at 1.6 Å resolution)19, generated by SiteMap. The color of the surface represents the nature of the pocket: the light blue mesh indicates hydrophobic regions and the red mesh indicates hydrophilic regions. The GalCer derivative structure is shown in the orange stick model.


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Ligand Design. We first sought to develop new ligands based on the -GalCer derivatives in an effort to further our understanding of the structural features. Several SAR studies and crystal structures of CD1d--GalCer are available to facilitate this study.13,19 Initially, we analyzed one of the mCD1d-GalCer derivative crystal structures (PDB: 3G08)19 using the computational tool SiteMap30 to obtain detailed information about the mCD1d A’ pocket (Figure 2). We found that this pocket featured two spatially limited hydrophilic regions that included hydrogen bonding donors and acceptors. One region consisted of a Ser28 residue, and the other region contained a Cys12 residue. In order to allow the hydrogen bonds to form between the polar residues and ligands, we designed four novel types of GalCer analogues that contain amide groups in their long acyl chains (Figure 3): (1) -GalCer type 1a-e: having one amide-containing acyl chain (C26:0); (2) C20:0 -GalCer type 2a-e: having one amide containing truncated acyl chain (C20:0);14,21 (3) Ar group-containing -GalCer type 3a-d: having one amide containing terminal Ar group attached to an acyl chain (these compounds were designed based on the compounds reported by Wong et al., which exhibited a stronger Th1 cytokine response than GalCer);23 (4) bis-amide-containing -GalCer type 4a,b: having two amide-containing acyl chains. We expected that the amide groups of the (1), (2), and (3) ligands and that of the type (4) ligand (in the middle of the acyl chain) could interact with the Ser28 residue, and the amide groups closer to the terminus of the acyl chain in the (4) type ligands could interact with the Cys12 residue. The amide group was selected as the polar moiety because it is readily introduced into the acyl chain, and it can potentially function as both a hydrogen bond donor and acceptor.


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Figure 3. Structures of the α-GalCer analogues.

Synthesis of the Ligands. The -GalCer analogues containing amide groups, presented in Figure 3, were prepared as indicated in Scheme 1. The synthesis of the key intermediate 5 proceeded efficiently from

D-galactose

and phytosphingosine according to the procedure published in the literature.31

Reduction of the azide group of 5 with PPh3 and H2O,31 followed by acylation with the Z-protected amino acid derivatives in the presence of WSC·HCl, gave the compounds 6a-e (m = 6-10). Removal of the Z and Bn groups of 6a-e with Pd(OH)2/C and subsequent acylation with various acids and DMTMM32 led to the desired -GalCer analogues in 20-50% yields. The low-moderate yields of acylation


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steps were partly due to the difficulties in the purification of the desired analogues. Next, we prepared the Ar group containing the -GalCer analogues 3a-d (Scheme 1). The key intermediate 5 was converted to the trifluoroacetyl protected derivatives 7a (m = 7) and 7b (m = 10) as described above. Cleavage of the trifluoroacetyl group under basic conditions, acylation with acid chlorides, and removal of the Bn groups gave the desired analogues 3a-d.

Scheme 1. Synthesis of the -GalCer analogues 1a-e, 2a-e, 3a-d, 4a, and 4b.

The Antigen Presenting Cell (APC)-Free Assay. Initially, we evaluated the binding potential of our designed/synthesized ligands to mCD1d proteins and the characterization of the functional NKT cell responses to the ligands using an established APC-free assay (Figure 4).21,33,34 This assay was conducted using the mCD1d fusion protein bound to the culture plate. The ligands were loaded onto the fusion proteins and presented to 2E10 NKT hybridoma cells that tend to produce IFN- and IL-4 upon the ligand stimulation.35 After 48 h incubation of the 2E10 hybridoma in the presence of the ligand-loaded mCD1d fusion proteins, we quantified the IL-2 and IFN- cytokine secretion levels produced by NKT cell activation (Figure 4), using an enzyme-linked immunosorbent assay (ELISA). We first investigated ACS Paragon Plus Environment

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the -GalCer derivatives, as shown in Figure 4a and 4e. Although the analogues 1a (m = 6) and 1b (m = 7) showed activities comparable to that of -GalCer, the derivatives 1c (m = 8), 1d (m = 9), and 1e (m = 10) exhibited much higher levels of IL-2 and IFN- production. The tendency of activities of the C20:0 -GalCer ligands were similar to those of the -GalCer derivatives. Treatment with 2c (m = 8), 2d (m = 9), or 2e (m = 10) increased cytokine production compared with the corresponding C20:0 -GalCer (Figure 4b and 4f).14,21 The amide position attached through a linker length of m = 810 increased cytokine production. Taken together, it should be emphasized that the introduction of only one amide group into the lipid moiety dramatically increased cytokine production. Although these compounds with an amide group at different positions presumably have similar physical properties, the amide positions in the acyl chain had significant effects on their activities, suggesting that site-specific interactions between these amide groups and the polar residues, such as Ser28 in the A’ pocket of CD1d, were critical to the ligand binding interactions. Further analysis revealed that the Ar group containing the -GalCer analogues 3a, 3b, and 3c (m = 7) displayed activities that were approximately equal to those of GalCer. Among these ligands, the fluorine-containing analog 3b produced slightly higher levels of cytokines (Figure 4c and 4g). By contrast, 3d (m = 10) induced a notable increase in IL-2 and IFN- production compared with the analogues, suggesting that, among the Ar group-containing -GalCer analogues, the amide positions could be important factors for the cytokine production as well. The introduction of another amide group to the 1e type ligand, as in 4a, 4b produced similar levels of cytokine induction to that of 1e (Figure 4d and 4h).


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Figure 4. Antigen presenting cell (APC)-free assay for lipid binding to mCD1d with the indicated ligands (100 nM). a) IL-2 secretion induced by the analogues 1a-e, b) analogues 2a-e, c) analogues 3a-d, d) analogues 4a, 4b, e) IFN- secretion induced by the analogues 1a-e, f) analogues 2a-e, g) analogues 3a-d, h) analogues 4a, 4b. -GalCer and C20:0 -GalCer were used as references. The graphs show mean ± standard error for triplicate values, and results shown were representative of two or three independent experiments.

Coculture Assay with RBL.CD1d, and 2E10 Hybridoma Cells. We next conducted coculture assays using mCD1d-expressing cells (RBL.CD1d) and 2E10 NKT hybridoma.34 The RBL.CD1d and 2E10 hybridoma were coincubated in the presence of the various concentrations of ligands. After 48 h of culturing, IFN- and IL-2 release was measured by ELISA. Lipid ligands (e.g., α-GalCer) can be loaded onto CD1d through endosomal trafficking; however, some researchers have reported that several lipidcontaining ligands are loaded directly onto CD1d molecules at the cell surface without trafficking to endosomal compartments.36 Thus, in addition to the binding potential of the ligands to CD1d, the level of cytokine production in the coculture assay could also be affected by the ligand loading process in the 10 ACS Paragon Plus Environment

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cells. The IL-2 production levels of most of the analogues were equal to or greater than that of -GalCer (Supporting Information, Figure S1). The IFN- levels are plotted in Figure 5. All ligands exhibited cytokine induction in a dose-dependent manner. As shown in Figure 5a, the cytokine production depended on the amide position in the acyl chain of the -GalCer derivatives, in good agreement with the APC cell-free assay results (Figure 4a); 1c-e (m = 8-10) displayed higher activities among the ligand analogues. By contrast, C20:0 -GalCer type (2a-e) exhibited no difference in cytokine induction, but all ligands (2a-e) induced a significant increase in IFN- production compared with C20:0 -GalCer (Figure 5b). The Ar group-containing -GalCer ligands 3a-c displayed activities comparable to that of -GalCer, while 3d produced slightly higher levels of cytokines (Figure 5c). The bis-amide-containing -GalCer 4a, 4b ligands demonstrated better cytokine secretion than -GalCer and 1e, indicating that the amide groups closer to the acyl chain terminus could also serve an important role in ligandprotein interactions or the ligand loading process (Figure 5d).

Figure 5. Coculture assay results obtained using the RBL.CD1d and 2E10 hybridoma. a) IFN- secretion in the presence of analogues 1a-e, b) analogues 2a-e, c) analogues 3a-d, d) analogues 4a, 4b.


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-GalCer, and C20:0 -GalCer were used as references. The graphs show mean ± standard error for triplicate values, and results shown were representative of two or three independent experiments.

IFN- and IL-4 Secretion by Mouse Splenocytes. Next, to investigate whether the designed ligands can stimulate the cytokine release from iNKT cells, we measured the levels of IFN- and IL-4 induction in mouse splenocytes. Primary NKT cells produce various cytokines including IFN- (the Th1 response) and IL-4 (the Th2 response), when stimulated with ligands. The IFN- and IL-4 levels were measured in the supernatant of mouse splenocytes cultured after 48 h treatment with -GalCer or the designed ligands 1e, 2e, 3d, or 4b (Figure 6). The effects of these analogues on IFN-γ and IL-4 induction levels were determined by ELISA. The amide-containing ligand 1e induced higher levels of IFN- and IL-4 cytokine secretion compared with -GalCer and C20:0 -GalCer. The amide-containing ligand 2e and bis-amide containing -GalCer 4b provided better IL-4 cytokine secretion. These results indicated that the amide-containing -GalCer analogue 1e, were potent CD1d ligands inducing both IFN- and IL-4 production, while 2e, 3d and 4b had the higher selectivity towards IL-4 production, in comparison with the reported derivatives.13

Figure 6. IFN- and IL-4 secretion by mouse splenocytes after stimulation by 1 nM -GalCer, C20:0 GalCer, 1e, 2e, 3d, or 4b. a) IFN- secretion in the presence of analogues, b) IL-4 secretion in the presence of analogues, -GalCer, and C20:0 -GalCer were used as references. The graphs show mean


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± standard error for triplicate values, and results shown were representative of three independent experiments.

Figure 7. WaterMap analysis and MD simulations. a-c) Hydration sites and dewetted regions identified near Ser28 in a) apo mCD1d, b) mCD1d complexed with an -GalCer derivative with a saturated acyl chain (C24:0), or c) with the amide-containing ligand (C24:0 equivalent to 1d). The hydration sites are drawn as spheres, color-coded by free energy relative to that of bulk water. The regions shown in the mesh indicate the dewetted region. Helices 1, 2, the hydration sites, and the dewetted regions in other area were omitted for clarity. For the apo structure, the ligand is shown in the figure only as a positional reference and was not included in the calculations. d) Number of time-averaged H-bonds present between the amide group of 1a-e and the polar residues in the pocket A’. The vertical axis indicate the total numbers of direct and water-bridged H-bonds.


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WaterMap Analysis. Considering the reports on positive correlation between the cytokine production and the binding affinities of glycolipids to CD1d,14,15 the improved activities of the amide-containing ligands suggested that the amide interacted with the polar residues, such as Ser28 and Gln14, in the hydrophilic region of the pocket A’ described above. Because these polar residues are positioned deep inside the hydrophobic pocket and confined within a small region, perturbation of the microenvironment, including the network of hydrating water molecules, by ligand binding may result in the formation of hydrogen bonds shielded by hydrophobic surroundings6 and/or the formation of localized high-energy water molecules.37,38 Since these perturbations were expected to significantly affect the binding free energy of the ligand, it was important to understand the changes in the microenvironment caused by the amide group of the ligand 1a-e placed in this region. To this end, the hydration state of the hydrophilic region was analyzed using WaterMap.37,38 WaterMap is a computational tool that uses molecular dynamics simulations and a statistical thermodynamic analysis of the resulting MD trajectories to assess the thermodynamic properties of localized water clusters (“hydration sites”) hydrating a ligand binding pocket.39-41 We performed WaterMap analyses of the apo mCD1d, the mCD1d in complex with an -GalCer derivative featuring a saturated acyl chain (C24:0), and the corresponding amide-containing ligand (C24 equivalent to 1d). The latter amide derivative had an amide group at the optimal position in the acyl chain (Figure 3, 4a and 4e). The high-resolution X-ray crystal structure of mCD1d (PDB: 3G08) was used as a template for the preparation of binding models. The hydration sites identified by WaterMap are shown in Figure 7a-c, with the free energies relative to bulk water color-coded. (See also Table S1, Supporting information) An analysis of the apo mCD1d structure predicted that the region in the pocket A’ where the saturated acyl chain of the -GalCer derivative binds was filled with multiple high-energy hydration sites. Displacement of these hydration sites should provide a large free energy of ligand binding (Figure 7a). In the X-ray crystal structure (PDB: 3G08), two conserved water molecules were involved in a tight H-bond network that included the amino acid residues Arg74, Trp40, and Ser28. The WaterMap MD simulation accurately reproduced the water molecules A and B. In addition to the ACS Paragon Plus Environment

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hydration site A and B, hydration site C was predicted to constitute a hydrogen bond network in this region. These water molecules are all entropically destabilized due to the strong positional and orientational constraints enforced by the H-bond network. (Table S1, Supporting information) The region shown in the mesh indicates the dewetted region42,43 within which very little water density was observed during the WaterMap analysis. The simulation of the (C24:0) ligand-bound structure (Figure 7b) revealed that most of the water molecules in the pocket were displaced by the ligand, and the space around Ser28 was tightly concealed by the acyl chain of the ligand. As a result, only water molecules A and B remained, and the presence of water C was energetically improbable. The absence of water C resulted in the increased volume of the dewetted region (Figure 7b), which is considered to carry a substantial energetic penalty. This result implied that although displacement of the unstable water molecules provided large binding energy to the -GalCer derivative, the effect was partially compromised by deterioration of the hydration stabilization of the polar residue. The binding model of the amide-containing ligand suggested that the amide group was placed at an ideal position to interact with Ser28 (Figure 7c). The analysis predicted that the introduction of the amide group not only provided hydrogen bond interactions with Ser28, it also improved the hydration state of the surrounding region compared with that of the (C24:0) ligand-bound structure. The improved polarity of the microenvironment warranted the presence of water C. It was predicted that the water molecules A, B, C, plus the ligand amide group and the surrounding amino acid residues, formed a wellordered hydrogen bond network resulted in the reduced size of the penalizing dewetted region. The hydration state of the region, thus, resembled that of the fully hydrated apo state. The WaterMap analysis suggested that the dramatic change in the H-bond/solvation network in this hydrophilic region contributed to the increase in the ligand activity.

MD Simulations. The -GalCer derivatives 1a-e, having one amide group in the acyl chain displayed improved activities compared with -GalCer in terms of their ability to induce IL-2 and IFN- secretion ACS Paragon Plus Environment

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(Figure 4a and 4e). Although the activities varied significantly, they did not depend critically on the exact position of the amide substructure in the alkyl chain. Some tolerance for the amide position was allowed to achieve the optimal activity. These results could not be explained in terms of a rigid docking model in which the H-bonds between the ligand amide group and the polar amino acid residues in the region were either present or absent. In fact, the docking scores calculated for -GalCer and 1a-e using fixed binding poses showed a poor correlation with the activities (data not shown). Furthermore, the observation of b-factors for the ligand and the surrounding amino acid residues in the X-ray crystal structure of mCD1d (PDB: 3HE6,20 Supporting Information, Figure S2) clearly indicated that the acyl chain of -GalCer was not very ordered, and significant movement in the pocket during binding was expected. For these reasons, we performed 20 ns MD simulations of the mCD1d complexes with the amide ligand 1a-e, and the interactions between the ligand amide group and the polar amino acid residues in the pocket A’ were analyzed. Figure 7d shows the time-averaged number of H-bonds formed between the amide group and the polar residues in the pocket A’, including the water-bridged H-bonds. The amide groups of 1a and 1b could not form H-bonds with Ser28, and they occasionally interacted with Gln14 and Tyr73, which were positioned closer to the entrance to the pocket (Supporting Information, Figure S3). Although 1c interacted with Ser28 through 0.24 H-bonds on average, its amide position was far from optimal, and most of the H-bond interactions were with Gln14. 1d and 1e provided very similar interaction profiles, and the two compounds interacted extensively with both Ser28 and Gln14. The interactions were predominantly direct H-bonds to Ser28 and water-bridged Hbonds to Gln14. The time-averaged number of H-bonds formed by 1a-e agreed well with the activities of the ligands shown in Figure 4a and 4e, suggesting that the formation of H-bonds between the ligands and polar residues could contribute to the cytokine production levels.

Conclusions We found that the introduction of amide groups into the long fatty acyl chain of the CD1d ligand, GalCer, increased cytokine production through recognition by the lipid binding protein mCD1d. In 16 ACS Paragon Plus Environment

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particular, the compounds containing m = 9 or 10 methylene linkers (e.g. 1d or 1e) were more potent than -GalCer and its derivatives (e.g. 1a-c), indicating that the positions of the amides in the acyl chain were important for activity and could form site-specific interactions with the mCD1d protein. MD simulations and WaterMap analysis suggested that the enhanced activities resulted from the interactions between the amide groups and the polar residues, such as Gln14 or Ser28, in the A’ pocket, due to the formation of a hydrogen bond network. The hydrogen bonds located deep inside of hydrophobic pocket were shielded by the surrounding apolar residues so that their strengths could be increased.6,8 Additionally, the introduction of amide groups might cause conformational changes of the acyl chains, affecting their affinities for CD1d.21 Further investigations of the roles of these amide groups are now underway. The second amide groups closer to the terminus of the acyl chain in 4a and 4b did not affect the activities in the APC-free assay (Figure 4d and 4h), but enhanced the cytokine production in the coculture assay (Figure 5d), suggesting that the second amide groups might have positive effects on the ligand loading process in the cells. Taken together, we demonstrated that confined polar residues in the large hydrophobic area of the lipid binding pockets of mCD1d could be targeted as “hot spots” that influenced the affinity between the protein and its ligands. These effects provide guidelines for ligand design in the context of lipid binding proteins.

Methods Lipid Ligands. -GalCer and its derivative (C20:0--GalCer) were prepared following literature procedure.18 The amide-containing ligands were synthesized as described in Supporting information. All lipids were dissolved in DMSO and kept aliquoted at ‒30 °C.

Cell Culture. RBL.CD1d cells and 2E10 NKT hybridoma cells were cultured in RPMI-1640 (NACALAI TESQUE, INC) supplemented with 10% fetal bovine serum (FBS; Biowest), and 1% penicillin–streptomycin (Gibco).


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Cell-free Assay for CD1d-lipid Binding.34 96-well micro-plates (Bio-one 96-well cell culture plate, Greiner) were coated with mouse CD1d:Ig Fusion protein (BD Biosciences) (0.25 g/well) in PBS (100 L) at 37 °C for 24 h. After washing with PBS, 100 nM of lipid ligands solution were added and incubated at 37 °C for 24 h. The above ligands solution was prepared by adding the ligands stock solution in DMSO to 0.5% Tween 20 and 0.9% NaCl in H2O, which was diluted with PBS containing 1% DMSO. The resulting solution included the 10-fold molar excess of lipid ligands to CD1d:Ig fusion protein used in the above coating process. After washing with PBS, 2E10 NKT hybridoma cells (2.5× 105 cells/well) were added and cultured at 37 °C for 48 h. IL-2 and IFN- release were measured by ELISA kit (Affymetrix).

Cytokine Secretion Assay Using 2E10 Hybridoma and RBL.CD1d Cells.34 RBL.CD1d (mouse CD1d) were used as antigen presenting cells (APC). RBL.CD1d (1.8×104 cells/well) were cocultured with 2E10 NKT hybridoma cells (1.8×104 cells/well) in the presence of various concentrations of glycolipids for 48 h. IL-2 and IFN- release were measured by ELISA kit (Affymetrix).

Cytokine Secretion Assay Using Mouse Splenocyte. Spleen cell suspension (from C57BL/6NCrSlc mice) was prepared in complete medium (RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), and 1% penicillin–streptomycin) and seeded into a 96 well plates (3.0×105 cells/well). Lipid ligands were added, and incubated at 37 °C for 48 h. IFN- and IL-4 release were measured by ELISA kit (Affymetrix).

General Molecular Modeling Work. For general molecular modeling work described below, the Maestro molecular modeling suite (Maestro version 10.4; Schrödinger, LLC: New York) with OPLS3 force field44 was used throughout. The same force field was used also for the WaterMap calculations and the MD simulations. The detailed methods for the calculations are described in Supporting information. ACS Paragon Plus Environment

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Supporting Information Supporting Information Available: This material is available free of charge via the Internet.

Acknowledgements We thank Profs. Iwabuchi (Kitasato University) and Kabayama (Osaka University) for providing RBL.CD1d cell lines and 2E10 hybridoma cells.

Funding Sources This work was supported by Grants-in-Aid for Scientific Research (Nos. JP26282211, JP26102732, JP26882036 and JP16H01162) from the Japan Society for the Promotion of Science, by ERATO Murata Lipid Active Structure Project, by Keio Gijuku Academic Development Funds, by Mizutani Foundation for Glycoscience, by Nagase Science Technology Foundation, and by Protein Research Foundation.

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