Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . The Antinociceptive Agent SBFI-26 Binds to Anandamide Transporters FABP5 and FABP7 at Two Different Sites Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . Subst Abuse. 2017; 11: 1178221817711418. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . J Biol Chem. 2000 Sep 1;275(35):27045-54. 1FDQA/B2-132.1FE3oleic..1JJX.NMR5URA Acta Crystallogr D Biol Crystallogr. 2014 Feb;70(Pt 2):290-8. 4AZN,4AZO,4AZP,4AZQ,4AZM,4AZR1JJJ,1B56 Biochem J. 2002 Jun 15;364(Pt 3):725-37. 1JJJ,1B56(1-135,1-135) Chem Biol Drug Des. 2015;85(5): 534–540. 1FE3oleic,1FDQA/B2-132.1JJX.NMR5URA. Biochemistry, 1999, 38 (38), pp 12229–12239.1B56,3WBG(1-133.1-133) Abstract Human FABP5 and FABP7 are intracellular14-15 kDa lipid-binding proteīns as well as endocannabinoid transporters anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as fatty acid signalling, cell growth, regulation, differentiation, neurotransmitters and mediate a diverse set of physiological and psychological processes. SBFI-26 is an α-truxillic acid 1-naphthyl monoester that competitively inhibits the activities of FABP5 and FABP7 and produces antinociceptive and anti- inflammatory effects. Only (S)-SBFI-26 was present in the crystal structures. The substrate entry portal region binding at the canonical ligand-binding pocket in the crystal structures. Intracellular fatty acids-binding (FABPs) transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. The endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. FABP5–endocannabinoid interactions may be useful for future efforts in the development of small- molecule inhibitors to raise endocannabinoid levels. Cannabinoid receptors and the mechanisms by which derivatives of the Cannabis sativa plant bind to receptors and produce their physiological and psychological effects processes controlled by the central and peripheral nervous systems. Expression of brain fatty acid binding (B-FABP) is spatially and temporally correlated with neuronal differentiation during brain development. Human B-FABP clearly exhibits high affinity for the poly-unsaturated n-3 fatty acids α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid and for mono-unsaturated n-9 oleic acid (Kd from 28 - 53 nM) over poly-unsaturated n-6 fatty acids, linoleic acid and arachidonic acid (Kd from 115 - 206 nM). B-FABP has low binding affinity for saturated long chain fatty acids. Human B-FABP in complex with oleic acid shows that the oleic acid hydrocarbon tail assumes an "U-shaped" conformation while in the complex H H H

H H H H H H H H O H O H H H H O H 5ce4OLEATE.tgf H SBFI-26atcanonicalsiteofFAB5;SBFI-26atportalsiteofFAB5; HH H H H H SBFI-26atcanonicalsiteofFAB7 H H H H H H H O H H O H H H H H O arachidonate-2-glyceride H H 1FE3.PDB fabp7 H O H C with DHA docosahexaenoic acid C22:6 ω=3 the H N O O 2-AG C20:4 =6 hydrocarbon tail adopts a helical conformation 1FDQ. Anandamide The binding specificity is in part the result of eicosanoate amide of ethanol H H non-conserved amino acid Phe104, which interacts H O H endocannabinoid H with double bonds present in the lipid hydrocarbon tail H C H O and hydrogen bond to the Tyr131. H H H H H H The expression of B-FABP in glial cells and its H H H high affinity for docosahexaenoic acid, which is H HH DHA H H known to be an important component of neuronal H H H H docosahexaenoate C20:4 =6 H membranes, points towards a role for C22:6 =3 H B-FABP in supplying brain abundant fatty acids to the H H developing neuron. H H

1 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . Introduction 1. Endocannabinoids are signaling lipids that activate cannabinoid receptors in the central nervous system and peripheral tissues (Howlett et al., 2011 ▶). The best characterized endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are ethanolamine and glycerol derivatives of arachidonic acid, respectively. In contrast to hydrophilic neurotransmitters, endocannabinoids are not stored in vesicles. Instead, the magnitude and duration of endocannabinoid signaling is regulated through ‘on demand’ biosynthesis and prompt catabolism. AEA is principally hydrolyzed by fatty-acid amide hydrolase (FAAH), while 2-AG is inactivated by monoacylglycerol lipase (MAGL), ABHD6 and ABHD12 (Cravatt et al., 2001 ▶; Blankman et al., 2007 ▶; Deutsch & Chin, 1993 ▶). Owing to their limited solubility, endocannabinoids require carrier-assisted transport through the cellular cytoplasm. Fatty-acid-binding proteins (FABPs) as intracellular carriers that transport AEA from the plasma membrane to intracellular FAAH for hydrolysis (Kaczocha et al., 2009 ▶). FABPs are small ( 15 kDa) widely expressed intracellular lipid-binding proteins (Furuhashi & Hotamisligil, 2008 ▶) and bind a variety of lipophilic ligands including fatty acids, fatty-acid amides and xenobiotics (Velkov et al., 2005 ▶; Chuang∼et al., 2008 ▶; Kaczocha et al., 2012 ▶). Inhibition of FAAH or MAGL potentiates endocannabinoid-mediated antinociceptive and anti-inflammatory effects (Ahn et al., 2009 ▶; Long et al., 2009 ▶). Inhibition of intracellular endocannabinoid carriers such as FABPs may provide an alternative strategy to modulate endocannabinoid inactivation. FABP knockdown or inhibition with the selective inhibitor BMS-309403 (Sulsky et al., 2007 ▶) reduces AEA inactivation in cells (Kaczocha et al., 2009 ▶). FABP inhibitors augment endocannabinoid levels and produce beneficial anti-inflammatory and antinociceptive effects (Berger et al., 2012 ▶). Selective FABP inhibitors hinges upon understanding the bonding interactions between current-generation inhibitors and the FABP-binding pocket. Ten isoforms of FABP have been identified in various tissues at high-affinity binding of amphiphilic ligands, such as long-chain fatty acids, bile acids, retinoids and eicosanoids, with FABP4 mainly present in adipocytes and FABP5 in epidermis (Furuhashi & Hotamisligil, 2008 ▶). At the primary-sequence level, the conservation of FABP isoforms varies from low ( 15%) to very high ( 70%). The FABPs are conserved in three-dimensional structure: they form a ten-stranded β-barrel (Furuhashi & Hotamisligil, 2008 ▶; Hamilton, 2004 ▶). The β-barrel is comprised of two orthogonal five-∼stranded β-sheets: β-∼sheet 1 and β-sheet 2. One side of the β-barrel is capped by a helix–loop–helix motif and the other side by the (N) amino-terminal peptide. The structure is referred to as a β-clamshell, with the two β-sheets as a pair of valves (Sacchettini et al., 1989 ▶; Hodsdon & Cistola, 1997 ▶; Jenkins et al., 2002 ▶; Richieri et al., 1999 ▶). Endogenous fatty acids such as palmitic acid and oleic acid generally bind to FABPs in a similar manner, with their carboxylates binding to one or two conserved basic residues and their hydrocarbon chains nesting in the largely hydrophobic chambers (Furuhashi & Hotamisligil, 2008 ▶; Hohoff et al., 1999 ▶). Human FABP5 in complex with AEA or 2-AG and with the inhibitor BMS-309403. These two proteins are highly conserved, with 80% (FABP4 108/135 FABP5 residues) sequence identity. Tertiary structures found in this protein family show a highly conserved folding motif, i.e. a flbarrel consisting of two orthogonal fl-sheets with five anti-parallel fl-strands each and a helix-turn-helix domain partially covering the internal water-filled cavity [14]. The ligand is non-covalently bound inside the cavity, almost inaccessible to the external solvent. LBPs can be grouped according to , which is consistent with the ligand-binding characteristics [15]: (I) the intracellular retinoid-binding proteins; (II) the ileal lipid-binding protein (ILBP), which binds bile acid, and the liver-type FABP (‘L-FABP’), which binds two fatty acids; (III) intestinal-type FABP (I-FABP), which binds a single fatty acid in a linear conformation; and (IV) FABPs with the fatty acid bound in a highly bent or U-shaped conformation. The E-FABP examined in the present study belongs to the last group along with adipocyte-type FABP (A-FABP), brain-type FABP (‘B-FABP’), heart-type FABP (H- FABP) and myelin-type FABP (M-FABP), where the carboxylate end of the fatty acid is buried within the binding cavity and forms hydrogen bonds with highly conserved Tyr and Arg residues, either directly or through an ordered water molecule. The hydrocarbon tail of the fatty acid forms van der Waals interactions with hydrophobic residues in the binding cavity and with ordered water molecules that are in contact with polar residues inside the binding pocket [16]. The presence of six cysteine residues in the amino acid sequence of human E-FABP is highly unusual for LBPs. Four of the six cysteine residues are unique to the E-FABPs : Cys43, Cys47, Cys67 and Cys87. The cysteine residues Cys120 and Cys127 of E-FABP are partially conserved in some LBPs, but only the M-FABP sequence includes both (at positions 117 and 124 respectively). In the 3D structure of E-FABP, two cysteine residue pairs (Cys67/Cys87 and Cys120/Cys127) were identified by X-ray analysis to be close enough to allow disulphide bridge formation, but a disulphide bond was actually found only between Cys120 and Cys127 [17]. Since the exclusion of a disulphide bridge between Cys67 and Cys87 improved the Rfree factor of the crystallographical model, the existence of a covalent bond between these two side chains was considered unlikely.

2 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . This is in agreement with the NMR data, where SYH resonances have been observed for the cysteine residues Cys43, Cys67 (tentative assignment) and Cys87, thus excluding the possibility of a second disulphide bridge in solution. PDB 4AZP(2-135,1-135), mouse FABP5 epidermal Results and discussion 3.3.1. Crystal structures of mouse FABP5 in complex with AEA AEA is an arachidonic acid derivative containing four double

bonds, with a chemical composition of C22H37NO2 and a mass of 347 Da (Fig. 1 ▶). AEA binds to FABP5. Figure 2. Interactions between AEA and mouse FABP5. (a) Cartoon view of the FABP5–AEA complex crystal structure. The secondary-structural elements, α- helices H1 and H2 and β-strands S1–S10, are labeled. The red letters ‘N’ and ‘C’ denote the amino- and carboxyl-termini of the protein, respectively. The bound AEA is shown as green sticks. (b) Electron density of AEA in the binding pocket of FABP5. The simulated OMIT map is contoured at the 2.5σ threshold and is shown as a blue mesh. (c) Detailed interactions between AEA (green sticks) and FABP5 (contacting residues shown as yellow sticks). The hydroxyl group of AEA forms a hydrogen bond to Tyr131 and a second, water-mediated, hydrogen bond to Arg129 (Fig. 2 ▶ c). The long lipophilic chain of AEA forms a loop that nests in the largely hydrophobic substrate pocket, with the nearest residues being Tyr22, Leu26, Leu32, Ala36, Pro41, Val60, Ala78 and Val118 at N-terminus. AEA complexed with FABP7 predicted a binding (Howlett et al., 2011 ▶). AEA to bind to FABP5 with an affinity of 1.3 µM, tenfold lower than its parent fatty acid arachidonic acid (Kaczocha et al., 2012 ▶). ∼

3.2. Crystal structure of mouse FABP5 in complex with 2-AG PDB entry 4azq. 2-AG to form five hydrogen bonds to the transporter (Fig. 3 ▶ d), between the carbonyl O atom of 2-AG and Arg129, between the carbonyl O atom of 2-AG and Tyr131, between Arg109 and one hydroxyl of 2-AG, which also makes a hydrogen bond to Cys43, and between the second hydroxyl group and Thr56. The looped lipophilic fragment of 2-AG is within 4 Å distance of FABP5 residues Phe19, Ala36, Val60, Ala78, Ile107 and Val118. Figure 3. Crystal structure of mouse FABP5 in complex with 2-AG. (a) Cartoon view of the structure. The secondary- structural elements, α-helices H1 and H2 and β-strands S1– S10, are labeled. The bound 2-AG is shown as green sticks. The red letters ‘N’ and ‘C’ denote the amino- and carboxyl- termini of the protein, respectively. (b) Electron density of 2- AG in the binding pocket of the FABP5–2-AG complex. The simulated OMIT map is contoured at the 2.5σ threshold and is displayed as a blue mesh. (c) Superposition of 2-AG with AEA in mFABP5. The two 2-AG hydroxyl groups insert deeper into the substrate chamber. (d) Detailed interactions between 2-AG and FABP5. Hydrogen bonds are shown as red dashes. Note that the viewing direction of (d) is 90° rotated from that in (b) to provide a different perspective. The FABP5 residues close to or in contact with 2-AG are shown as yellow∼ sticks. PDB 4AZQ (2-135,1-135), mouse FABP5 epidermal 2-AG

3 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . The two endocannabinoids both AEA and 2-AG may adopt multiple conformations at their respective termini in FABP5. Because of the lower position of AEA, the hydroxyl group at the tip can no longer form a hydrogen bond to Tyr131. Instead, the hydrophilic AEA head region rotates by 180° compared with the structure in monomeric mFABP5, such that the hydroxyl now forms a hydrogen bond to Arg109. The lowered AEA orients its carbonyl group towards Tyr131 and forms a hydrogen bond to the hydroxyl∼ of Tyr131. PDB human FABP5 4AZR Figure 6. The AEA binding mode in domain-swapped human FABP5. (a) Superposition of hFABP5–AEA with mFABP5–AEA. The H1–H2 cap in hFABP5 (cyan) moves left by >5 Å compared with that in the monomer (gray), significantly opening up the portal region. (b) The AEA in the domain-swapped hFABP5 (orange) enters 3.5 Å deeper into the substrate chamber compared with that in the mFABP5 monomer (gray). (c) Stereoview of the interaction of AEA with hFABP5. The hydrocarbon chain of AEA is surrounded by Met35 and Cys120 of one FABP5 (orange) and by Phe19, Tyr22, Met23 and Pro41 of the other FABP5 (cyan). The AEA hydroxyl forms a hydrogen bond to Arg109 as shown by the dashed red line (2.7 Å). The AEA carbonyl forms a hydrogen bond to Tyr131 (2.4 Å). In the FABP5 dimer structures, the N-terminal half of the first molecule (residues 1–59) forms a complete β-barrel with the C-terminal half of the second molecule (residues 60–134). In so doing, the loop (Glu57–Thr62) connecting the S3 and S4 β-strands in the monomeric structure is converted into a β-strand, and together with S3 and S4 forms an unusually long β-strand that connects the two domain-swapped monomers (Figs. 5 ▶ a and 5 ▶ b). The electron density in the connecting loop region between the S3 and S4 β-strands was impossible to model without swapping the N- and C-terminal subdomains of each monomer. Furthermore, a stretch of difference electron density appears if residues 57–62 from both chains are omitted during model building, clearly indicating the continuity of the β-strands across the two copies of the protein (Fig. 5 ▶ c). INTRODUCTION Brain tissue contains high amounts of poly-unsaturated fatty acids, such as arachidonic (AA), docosahexaenoic acids (DHA) and eicosapentaenoic acid (EPA), in their membrane phospholipids compared with other tissues (1, 2). The central nervous system uses DHA and other long-chain poly-unsaturated fatty acids during the early postnatal development (3) when , active synaptogenesis and photoreceptor membrane biogenesis take place (4-6). The critical role of DHA in the brain has been demonstrated by behavioral studies that have described n-3 deficiency (7). Arachidonic acid and DHA are synthesized by elongation, desaturation and β-oxidation steps of the dietary essential fatty acids 18:2(n-6) and 18:3(n-3), respectively (8). Deficiency of n-3 fatty acid leads to an altered electroretinogram (9), decreased visual activity (10), and impaired learning ability (11). In addition, DHA may be converted into neuroprostanes (F4-NPs) by free radical-catalyzed peroxidation (12-14) or may modulate the production of oxygenated metabolites from arachidonic acid with important roles in the physiology and pathology of the central nervous system. Similarly, oxidant-stress and peroxidation of lipids which have been implicated in the pathogenesis of a variety of human diseases, including atherosclerosis, cancer, and neurodegenerative disorders such as stroke, Alzheimer’s disease and Huntington’s diseases (15-21), oxidative injury of NPs could lead to impaired neuronal function (12). Fatty acid trafficking and transport are controlled in the bramin by expression of B-FABP in radial glia during the development of the central nervous system is strictly correlated with the differentiation and migration of neurons from these cells (22). The high level of expression of B-FABP during neurogenesis or neuronal migration has implicated B-FABP to play an important role during central nervous system development (23). The functions of a storage/delivery system for either DHA and EPA, or the precursors required for DHA production, assuring a steady supply of fatty acids to the developing central nervous system. Alternatively the protein may be involved in transport of fatty acids to specific sties of regulation, and may protect DHA from undergoing free radical-catalyzed peroxidation. Human B-FABP is a member of the intracellular 14-15 kDa lipid binding protein family. Members of this

4 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . protein family have high affinity for amphiphils such as fatty acids, eicosanoids, retinoids and bile acids. It has been proposed that these proteins are involved in the cellular uptake of lipids, their transport to metabolic pathways (24) and in regulation of lipid transport and metabolizing proteins (25,26). Although there is a wide range of variation in the amino acid sequence among members of the family (for example, human B-FABP shows 67% amino acid sequence identity to human H/M-FABP, 28% to human liver FABP), their three-dimensional structures are highly conserved, consisting of ten antiparallel β strands and two short α helices. The ten antiparalled β strands are arranged into two nearly orthogonal 5-stranded β sheets that surround the interior binding cavity. Some of the three dimensional structures reported to date are bovine myelin P2 (27), rat intestinal FABP (28), chicken liver basic FABP (29), bovine H-FABP (30), adipocyte lipid-binding protein (31), human M-FABP (32), Desert locust Schistocerca gregaria M-FABP (33), Manduca sexta FABP (34) and porcine Ileal lipid binding protein (35). The proteins can be grouped according to sequence homology, which is consistent with the ligand binding characteristics (36). Proteins 4 categories; (i) the intracellular retinoid binding proteins, (ii) the ileal lipid binding protein (which binds bile acid) and liver-FABP proteins that accommodate 2 fatty acids, (iii) intestinal-FABP which binds a single fatty acid in a linear conformation, and (iv) FABPs with the fatty acid bound in a highly bent or "U-shaped" conformation. Brain, adipocyte and the muscle/heart FABPs belong to the last group. Within grouping iv, the carboxylate moiety of the fatty acid is buried within the binding cavity and hydrogen bonds to conserved Tyr and Arg residues, either directly or through an ordered water molecule. The hydrocarbon tail of the fatty acid forms VDW interactions with hydrophobic residues that line the binding pocket and with ordered water molecules that are in contact with polar residues in the binding pockets (37). All of the FABPs studied to date exhibit a preference for long-chain saturated fatty acids (38). As B-FABP has a higher affinity for long chain poly-unsaturated fatty acids. The thermodynamic description of fatty acid binding.. Table 2: Binding parameters of recombinant human B-FABP for fatty acids . Fatty acid Kd a ∆H T∆S Bmax Kd Saturated [nM] [kJ/mol] [kJ/mol] [nM] Lauric acid (12:0) 443 ± 55 -37.3 ± 1.8 -0.4 ± 2.2 0.95 ± 0.34 Palmitic acid (16:0) 7100 Stearic acid (18:0) 13500 Monounsaturated n-9 Palmitoleic acid (16.1) 41.4 ± 1.1 -43.2 ± 2.3 -0.3 ± 2.2 0.92 ± 0.05 Oleic acid (18:1) 46.7 ± 1.4 -48.7 ± 3.0 -6.1 ± 2.9 0.99 ± 0.19 47 Polyunsaturated n-6 Linoleic acid (18:2) 115 ± 19 -51.1 ± 5.8 -10.8 ± 5.7 0.70 ± 0.11 Arachidonic acid (20:4) 207 ± 19 -60.3 ± 6.6 -21.6 ± 6.6 1.12 ± 0.17 Polyunsaturated n-3 α-Linolenic acid (18:3) 27.5 ± 1.3 -60.4 ± 6.0 -16.5 ± 5.8 0.98 ± 0.08 Eicosapentaenoic acid (20:5) 48.1 ± 21 -52.2 ± 2.4 -9.5 ± 3.3 0.95 ± 0.03 Docosahexaenoic acid (22:6) 53.4 ± 4.1 -53.2 ± 4.8 -10.9 ± 4.8 1.10 ± 0.15 15 all-trans retinoic acid 5900 Retinoic 9-cis retinoic acid 18500 Retinoic 13-cis retinoic acid 5000 Retinoic aMeasurements with long-chain fatty acids not possible due to diminished solubility under experimental conditions employed. Binding parameters derived from isothermal titration calorimetry (means ± S.D., n=3). bDissociation constants determined by Lipidex assay. Competition with [1-14C]OA and referenced to Kd=47 for OA as determined by isothermal titration calorimetry. B = Bmax F / (Kd + F), where Bmax is maximal number of binding sites, B is bound fatty acid concentration, F is unbound fatty acid concentration and Kd is dissociation constant. Table 3: Binding parameters of recombinant human H/M-FABP for fatty acids fatty acid Kd (nM) ∆H (kJ/mol) T∆S (kJ/mol) Bmax oleic acid (C18:1) 820 ± 10 -27.4 ± 0.8 -12.5 ± 5.9 0.94 ± 0.04 Linoleic acid (C18:2) 970 ± 8 -80.5 ± 1.7 -44.8 ± 1.8 0.93 ± 0.02 Eicosapentaenoic acid (C20:5) 3300 ± 10 -45.0 ± 4.3 -13.0 ± 4.3 0.93 ± 0.04 Docosahexaenoic acid (C22:6) 4100 ± 6 -60.1 ± 4.1 -28.6 ± 4.6 0.91± 0.01 All binding parameters were derived from isothermal titration calorimetry at 30°C as described under Experimental Procedures. Data are given as mean ± S.D., n=3.

5 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . Figure 1a. Ribbon representation of the structure of the human B-FABP:OA complex. The bound OA molecule found in the binding cavity is shown in ball and stick. Figure 1b. Ribbon representation of the structure of the human B-FABP:DHA complex. The bound DHA is shown in ball and stick. 1FDQ.pdb

Secondary structure elements and the corresponding residues are n-strand B1 (Cys5-Leu10), a-helix A1 (Phe16- Ala22), a-helix A2 (Gly26-Val35), n-strand B2 (Lys37-Glu45), n-strand B3 (Lys48-Ser55), n-strand B4 (Lys58- Gln65), n-strand B5 (Glu69-Thr74), n-strand B6 (Asp77-Asp87), n-strand B7 (Lys90-Trp97), n-strand B8 (Lys100- Ile108), n-strand B9 (Gly111-Phe119), n-strand B10 (Val122-Glu129). Figure 1c. Superposition of human B- FABP:DHA complex and human B-FABP:OA complex is shown using SPOCK (66) where the deviation between the Ca atoms is represented by the width of the worm. OA is shown in cyan and DHA is shown in orange. Deviation seen away from the cavity corresponds to the different crystal packing and that around the cavity is attributed to the differences caused by DHA. The general structure of human B-FABP is very similar to that of the other fatty acid binding proteins reported to date (53). Eighty seven of the 131 residues in human B-FABP form 10 antiparallel n-strands arranged into two perpendicular n-sheets and two short α-helices (A1, 7 residues, and A2, 10 residues) are located between n-strands B1 and B2. Backbone hydrogen bonds typical of antiparalled n-pleated sheets are found between all strands, with the exception of n-strands 4 and 5. These strands are separated and the space between these two strands is filled with protein side chain atoms. n-strands B1, B2, B3, B4, B5, and B6 form the first n-sheet and B6, B7, B8, B9, and B10 from the second n-sheet. An exaggerated bend in B6 allows it to act as a linker between two n-sheets, where the first 5 residues (Asp77-Lys81) of the n-strand hydrogen bond to the first n-sheet, while the remaining 6 residues (Ser82- Asp87) of B6 hydrogen bond to main chain atoms of B7, part of the second n- sheet. OA binding in U-shaped conformation. After the initial refinement with only atoms of the protein, Figure 3. Active site of the fatty acid (a) OA and (b) DHA are shown as found in the structures of their complexes with human B-FABP.Only important residues in the active site are shown. The twelve residues, which form VDW contacts, are Phe16, Val25, Thr29, Gly33, Ser55, Phe57, Lys58, Thr60, Ala75, Asp76, Phe104 and Met115 in OA complex. The twenty one residues which form VDW contacts are Phe16, Tyr19, Met20, Leu23, Val25, Thr29, Val32, Gly33, Thr36, Ser55, Thr60, Ile62, Glu72, Thr73, Thr74, Ala75, Asp76, Arg78, Gln95, Phe104 and Leu117 in DHA complex. 1FDQ.pdb O1 of the carboxylate of bound OA hydrogen bonds to the hydroxyl group of Tyr128 (2.8 Å) and to an ordered water molecule (3.0 Å) which in turn hydrogen bonds to the side chain oxygen of Thr53 and the guanidinium group of Arg106 (3.2 and 3.3 Å, respectively). The other oxygen is within direct hydrogen bonding distance to the guanidinium group of Arg126 (2.9 Å). Carbon atoms of the fatty acids’ aliphatic chain form VDW interactions with several residues of the protein (Figure 3a) and ordered water molecules. The phenyl ring of Phe16 appears to play a pivotal role in ligand binding as it is within 4.5 Å from

6 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . carbons C4, C7, C12 and C13 of OA’s aliphatic chain. In addition, the side chains of residues Tyr19, Met20, Leu23, Thr36, Pro38, Val40, Thr53, and Arg78 are positioned around the outer surface of the binding site forming a set of interactions that appear to stabilize the folded conformation of the fatty acid. The carboxylate moiety of DHA binds to the hydroxyl group of Tyr128 (3.1 Å) and the guanidinium group of Arg126 (2.6 Å) in a fashion similar to that observed for hydrogen bonding for OA. The double bonds in DHA form extensive t-t interactions with side chains of the protein. For example, t-t interactions are formed between the C4-C5 double bond and the benzene ring of Phe104 as well as the sulfur of Met115. It is important to note that residues Phe104 and Met 115 are structurally replaced by Leu104 and Leu115 in M-FABP, and Ile104 and Val115 in ALBP. In addition, double bonds C4-C5, C7-C8, C10-C11 and C13-C14 are arranged around Phe16 to permit additional t stacking interactions. Fatty Acid Binding Studies. Representative titrations of human B-FABP with DHA and arachidonic acid are shown in Figure 4. The binding isotherm was derived from integration of the peaks and fitting to a model assuming one binding site. Dissociation constants, binding enthalpies and calculated entropies obtained for B- FABP with different ligands are shown in Table 2. The binding stoichiometry in all cases was very close to 1:1 and the binding entropies near zero, indicating that binding was primarily an enthalpy driven event. As with other FABPs, medium chain fatty acids such as lauric acid bind with relatively low affinity. Moreover, the binding of longer saturated fatty acids could not be accurately assayed by titration calorimetry, due to their inherent insolubility, so a second type of binding assay was used (see below). Mono-unsaturated n-9 fatty acids bound to human B-FABP with high affinity (Kd 41-47 nM), as did the long chain poly-unsaturated n-3 fatty acids (27-53 nM). The protein also showed a preference for n-3 poly-unsaturated fatty acids compared to those of the n-6 group (Kd 115-206 nM). DISCUSSION A unique feature of the human B-FABP:DHA complex is t-t interactions which occur between Phe104 and the C4-C5 double bond of DHA. Residue Phe104 is conserved in human B-FABP and chicken retina FABP. In murine (44,52,54,55) and rat (46) B-FABP the comparable residue is a Cys. The sulfur in Cys104 of murine and rat B-FABP may also form sulfur-t interaction with the C4-C5 double bond of DHA in a similar fashion to the known sulfur-t interactions as observed in eye lens protein (57). A comparison of the binding sites of human B-FABP:DHA, human B-FABP:OA, H/M-FABP:OA, ALBP:palmitate and ALBP:arachidonate shows that fatty acids of different length were all found to fit within the confines of the binding cavity. Interestingly, the volume does not adjust significantly in response to fatty acid length or degree of saturation. The binding cavity is about 14 Å wide at the widest point between residues 75 and 117 in all FABPs. The volume of the cavity of the B-FABP:DHA complex (after removal of the fatty acid and ordered waters) is approximately 914 Å3. This is about 2 Å3 larger than the B-FABP:OA complex, and the cavity for the M-FABP:OA complex is about 25 Å3 more spacious than B-FABP:DHA complex. Therefore, the amount of space within the binding cavity appears to be constant and thus, has little effect of fatty acid specificity. While M-FABP, B-FABP and ALBP have highly conserved residues within their respective fatty acids binding sites, only B-FABP has a phenylalanine residue, Phe104, in close proximity to the C4-C5 double bond found in both the DHA structure and a model we have constructed of B-FABP:EPA, using Insight II (Molecular Simulations). As discussed above, we believe that the t-t interactions between the side chain of Phe104 and the C4-C5, is a primary determinate for binding specificity. The comparable residue to Phe104, in M/H-FABP and ALBP is Leu and Ile respectively, which do not have a propensity to form t interactions and this may explain why M-FABP and ALBP do not show strong affinity for EPA and DHA (Table 2). However, all members of the group have a comparable residue to Phe16, which is in a location that could support t-t interactions with the C9-C10 double bonds found in OA, linoleic and a-linolenic acid. It is important to note that these fatty acids all bind with high affinity to the group iv FABPs. Thermodynamics of Binding. The binding of fatty acids to human B-FABP is driven mainly by the enthalpic term. Similar results have been reported for the binding of OA to locust muscle FABP (59) and of (1,8)-anilinonaphtalenesulfonate to intestinal FABP (60). Enthalpic contributions are attributable to non-covalent bonding interactions such as electrostatic and hydrogen bonding interactions which are observed in the B-FABP crystal structures between residues Thr53, Arg106, Arg126 and Tyr128 and the carboxylate group of DHA and OA. Despite the significant difference in conformation of DHA and OA, the enthalpic components to the binding energy are similar. The enthalpic contribution of binding observed for OA, linoleic acid and α-linolenic acid is comparable but the entropic contribution decreases with the number of double bonds. This may be due to decreased flexibility caused by additional double bonds. We have previously proposed that the affinity of a given fatty acid for an FABP can be classified into 3 primary components; 1) the energetic strain imposed on the ligand in the bound state, 2) the specific interactions of the protein with the bound ligand, and 3) the desolvation of the binding cavity and the ligand upon binding (55). The relatively low entropic contribution to fatty acid binding that have been observed in several studies of different FABPs has lead to the

7 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . suggestion that desolvation may be less important (61). The structural and thermodynamic analysis of B-FABP suggests that this protein has a binding pocket that is well suited to bind n-3 fatty acids by creating an environment within the cavity that permits binding of these fatty acids in a low energy conformation, and provides specific interactions of the protein with conserved double bonds in the fatty acid. Comparison of B-FABP and M-FABP indicates the number and positions of the double bonds appear to be a primary determinant of binding affinity. For example, while M-FABP’s binding cavity is large enough to accommodate DHA in its low energy conformation and the atoms that compose the binding cavities are nearly identical, the presence of Phe104 in B-FABP permits additional π-π interactions and therefore the binding of DHA to B-FABP (Kd=53 nM) is significantly tighter than to M-FABP (Kd=4100 nM). DISCUSSION PDB 1JJJ,1B56(1-135,1-135) The solution structure of human E-FABP has been determined by high-resolution NMR spectroscopy on the basis of sequence-specific 'H and '5N resonance assignments. Spin-system heterogeneities were observed for several amino acid residues, in some cases indicating separate backbone conformational states around the portal region. However, even though a weak electron density in the X-ray data of human E-FABP [17] suggested a less well- defined structure in the turn between β-strands βC and βD (comprising residues 58–61 as part of the portal region), no spin-system heterogeneities were observed for these particular residues in the solution structure, thus excluding separate long-lived conformational states as reported for H-FABP [45]. On the other hand, a more ‘diffuse’ structure in the βC–βD turn, together with a lack of the otherwise highly conserved Phe57 ring as a portal lid, might possibly explain the relatively low oleic acid-binding affinity of E-FABP compared with other FABPs in the LBP subfamily IV [47]. Furthermore, on the basis of the proton chemical shift assignment, the presence of a second disulphide bridge in human E-FABP can definitely be ruled out. The cystine bond, which has been detected between Cys120 and Cys127 by the previous X-ray study [17], is unique in the LBP family. Even though biochemical studies have indicated that the respective residues Cys117 and Cys124 in M-FABP are disulphide linked as well [48], their SY positions are too far apart (4.5 Å ) in the crystal structure for the presence of a disulphide bridge [49]. A comparison with the E-FABP structure provides no obvious explanation for this structural discrepancy observed between these two members of LBP subfamily IV, except maybe for the lower atomic resolution (2.7 Å ) of the M-FABP data. Still, the high structural homology of this cysteine residue pair strongly suggests the presence of a cystine bond in M-FABP as well, and therefore poses the question why this is not seen in the crystal structure. The overall structural fold of human E-FABP in solution is very similar to the solution structures of other members of the LBP family, such as H-FABP [23,45], ILBP [21], I-FABP [50,51], as well as cellular retinoic acid- binding protein type II [52] and cellular retinol-binding protein types I and II [53,54]. The presence of an N- terminal helical loop, however, is a unique attribute of the LBP subfamily IV. The loop usually consists of four residues, starting with a hydrophobic amino acid, followed by one or two hydrophilic residues and a highly con- served phenylalanine residue (substituted by a leucine residue only in the case of E-FABP) in the last position. The non-polar residues in the first and last position are part of the hydrophobic cluster at the bottom of the protein cavity, whereas the hydrophilic residues are accessible to the external solvent. This additional structural feature in LBP subfamily IV might therefore contribute to the overall stability of the β-barrel fold. Marked differences in conformational stability and binding affinity for fatty acids have been reported for paralogous FABPs of LBP subfamily IV [47,55]. In all types of this subgroup, the fatty acid inside the cavity is bound in a U-shaped conformation with hydrogen-bond formation between the carboxylate group and a triad of protein side chains consisting of two arginine residues (one via an ordered water molecule) and one tyrosine residue (E-FABP numbering Arg109, Arg129 and Tyr131). Among these FABP types, E-FABP displays the lowest conformational stability in the presence of urea, in spite of the existence of a unique disulphide bridge. Furthermore, E-FABP shows the second lowest binding affinity for oleic acid after A-FABP. In contrast, H-FABP exhibits a very stable conformation and strong ligand binding. A cluster of hydrophobic side chains, which closes the end of the β-barrel structure that is located opposite to the helix-turn-helix domain, might play a significant role in both ligand binding and protein stability. Several members of this hydrophobic cluster are substituted in E-FABP (relative to H-FABP) by residues with different hydrophobicities: Leu7 (Phe4), Phe65 (Ile62), Cys67 (Phe64, Cys87 (Val84) and Phe89(Leu86). This might, in part, explain the decreased conformational stability of E-FABP, whereas other substitutions, like Leu60 (Phe57) and Val118 (Leu115), could be responsible for the weaker binding of fatty acids to E-FABP. Significant differences in the backbone dynamics between bovine H-FABP and porcine ILBP have been reported previously on the basis of hydrogen/deuterium exchange [21,23] and 15N relaxation experiments [38]. The extremely slow amide–proton-exchange behaviour observed for H-FABP indicated a clear distinction in the stability of the hydrogen-bonding network between these two β-barrel proteins. Hence, both the hydrogen/ deuterium exchange behaviour and the microdynamic parameters may provide valuable information about the inβuence of molecular dynamic processes on the functional aspects of different LBPs. The backbone dynamics data of human E-FABP were, for this purpose, compared with both bovine H-FABP and porcine ILBP.

8 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . The dynamics behaviour of E-FABP contrasts, for example, with that of ILBP [38], which shows a larger spread in the S2 values and several non-terminal residues, with S2 values well below 0.7. Yet, other members of the LBP family also exhibit distinct patterns of backbone dynamics. It has been reported, for example, that A-FABP has a greater backbone mobility than H-FABP, especially in the portal region [56]. This is due to the fact that A- FABP has lower S2 values in the portal region, whereas H-FABP shows higher S2 values distributed uniformly throughout the amino acid sequence. For I-FABP, on the other hand, a very high mobility around the portal region has been described [57], even though the S2 values for some residues in this region were abnormally low. Summarizing the results on protein dynamics obtained in the present study, it can be concluded that the different LBP family members E-FABP, H-FABP and ILBP are characterized by varying stabilities in the protein backbone structures. Hydrogen/ deuterium exchange experiments presented significant differences in the chemical exchange with the solvent, for the backbone amide protons belonging to the hydrogen-bonding network in the β- sheets. The β-barrel structure of H-FABP appears to be the most rigid, with exchange processes presumably slower than the millisecond-to-microsecond time range. ILBP, on the other hand, shows the fastest hydrogen exchange as well as a significant number of R8x terms, implying a decreased stability in the β-sheet structure. Finally, E-FABP appears to rank between these two proteins on the basis of the hydrogen/deuterium exchange, with R8x terms in the β-strands indicating millisecond-to-microsecond exchange processes like in ILBP. According to biochemical data [47], the conformational stabilities of the human paralogues H-FABP, ILBP and E-FABP decrease in this order, which is partially in contrast with the results described in the present study. However, both studies agree that, within LBP subfamily IV, the H-FABP has a much more rigid structure than E- FABP. Differences in the arrangement of the hydrophobic cluster inside the protein cavity, in particular the replacement of Phe4 (H-FABP) by Leu7 (E-FABP), may be responsible for this distinction. Moreover, the higher conformational stability of H-FABP may also be related to the tighter binding of fatty acid ligands to H-FABP relative to E-FABP [47]. Possibly, there is a correlation between protein stability and ligand-binding affinity, if a more βexible structure allows the bound ligand to leave the binding cavity more easily. On the other hand, the lack of the highly conserved phenylalanine portal lid (Phe57 in H-FABP) in E-FABP could be the predominant factor for the lower fatty acid-binding affinities of the latter protein. Future site-directed mutagenesis studies on human E-FABP may provide definite answers to the questions posed above. Moreover, additional NMR investigations on the dynamics of other intracellular LBPs will be needed for a more concise interpretation of the distinctions in binding affinity and specificity. Finally, the determination of the solution structure of human E-FABP now permits further studies on intermolecular interactions, in particular with S100A7 in regard to . This work was supported in part by the grant SP 135/10-1 from the Deutsche Forschungsgemeinschaft to F.S. We wish to thank David Fushman (University of Maryland) for kindly providing MATLAB routines used in relaxation data analysis. L.H.G.-G. was recipient of a scholarship from the German Academic Exchange Service (DAAD). Footnotes 1 Present address: Department of Immunology and Biochemistry, St. George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, U.K. ↵2 Present address: Institut für Rechtsmedizin, Universitätsklinikum Münster, Röntgenstrasse 23, D-48149 Münster, Germany. The↵ 1H and 15N resonance assignments for recombinant human E-FABP have been deposited at the BioMagResBank (http://www.bmrb.wisc.edu) under the accession number BMRB-5083. The atom co-ordinates of the 20 conformers representing the solution structure of human E-FABP have been deposited at the Research Collaboratory for Structural Bioinformatics (‘RCSB’) (http://www.rcsb.org) under the accession number 1JJJ. Abbreviations used: FABP, fatty acid-binding protein; A-FABP, adipocyte-type FABP; E-FABP, epidermal-type FABP; H-FABP, heart-type FABP; M-FABP, myelin-type FABP; I-FABP, intestinal-type FABP; ILBP, ileal lipid- binding protein; LBP, lipid-binding protein; NOE, nuclear Overhauser effect; HSQC, heteronuclear single-quantum coherence; RMSD, root-mean-square deviation; 2D, two-dimensional; 3D, three-dimensional. The Biochemical Society, London ©2002

1FDQX-ray2.10A/B2-132[»]1FE3X-ray2.80A2-132[»]1JJXNMR-A2-132[»] 5URAX-ray1.85A/B/C/D1-132[»] FABP7 Human brain 5URA_A(-1-132,1-132) FABP7 Human brain 8KS 4(C28 H22 O4) FABP7 Human brain inhibitor (1S,2S,3S,4S)-3- -{[(NAPHTHALEN-1-YL)OXY]CARBONYL}-2,4-DIPHENYLCYCLOBUTANE-1-CARBOXYLIC ACID SO4 SULFATE ION 8(O4 S 2-) 17 HOH *579(H2 O) HELIX 1AA1SERA-1ALAA 41 6 HELIX 2AA2ASNA 16GLYA 251 10

9 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . HELIX 3AA3GLYA 27THRA 371 11 SHEET 1AA110THRA 61 PHEA 65 0 SHEET 2AA110LYSA 49 LEUA 55-1 N VALA 50 O PHEA 65 SHEET 3AA110THRA 40 GLUA 46-1 N ILEA 42 O ARGA 53 SHEET 4AA110ALAA 7 GLNA 15-1 N TRPA 9 O VALA 41 SHEET 5 AA110 VAL A 123 LYS A 131 -1 O VAL A 126 N GLN A 15 SHEET 6 AA110 LYS A 113 PHE A 120 -1 N MET A 116 O ARG A 127 SHEET 7 AA110 LYS A 101 LYS A 110 -1 N ASN A 104 O THR A 119 SHEET 8AA110LYSA 91 TRPA 98-1 N HISA 94 O PHEA105 SHEET 9AA110ASNA 80 ASPA 88-1 N SERA 86 O VALA 93 SHEET 10AA110PHEA 71 THRA 74-1 N GLUA 73 O CYSA 81 SITE 1AC116PHEA 17 META 21 GLYA 34 THRA 37 SITE 2AC116PROA 39 THRA 54 SERA 56 ASPA 77 SITE 3 AC1 16 PHE A 105 MET A 116 LEU A 118 ARG A 127 SITE 4 AC1 16 TYR A 129 HOH A 312 HOH A 327 HOH A 432 SITE 1AC2 2PHEA 28 ARGA 31 SITE 1AC3 6THRA 37 LYSA 38 SERA 56 THRA 57 SITE 2AC3 6HOHA311 HOHA407 1FE3,1FDQA/B(1-132)2-132.1FE3oleic.1JJX.5URA FABP7 Human bramin H H H

H H H H H H H H O H O H H H H O H 5ce4OLEATE.tgf H HH H H H H H H H H H H H H H H OLA OLEIC ACID C18 H34 O2 H 5CE4 HELIX 11ASNA15GLYA241 10 HELIX 22GLYA26THRA361 11 SHEET 1 A10THRA 60 PHEA 64 0 SHEET 2 A10LYSA 48 LEUA 54-1 N VALA 49 O PHEA 64 SHEET 3 A10THRA 39 GLUA 45-1 O THRA 39 N LEUA 54 SHEET 4 A10THRA 7 GLNA 14-1 N TRPA 8 O VALA 40 SHEET 5 A10VALA122 LYSA130-1 N VALA125 O GLNA 14 SHEET 6 A10LYSA112 PHEA119-1 N META113 O TYRA128 SHEET 7 A10LYSA100 LYSA109-1 O ASNA103 N THRA118 SHEET 8 A10LEUA 91 TRPA 97-1 O LEUA 91 N ARGA106 SHEET 9 A10ASNA 79 LEUA 86-1 O LYSA 81 N LYSA 96 SHEET 10 A10PHEA 70 THRA 73-1 O PHEA 70 N SERA 82 SITE 1AC1 9GLYA 33 PROA 38 VALA 40 THRA 53 SITE 2AC1 9SERA 55 LYSA 58 ASPA 76 ARGA126 SITE 3AC1 9TYRA128 O C O

docosahexaenoate C22:6 =3

1FDQA/B(1-132)2-132)1FE3oleic.1JJX.5URA FABP7 Human bramin HXA DOCOSA-4,7,10,13,16,19-HEXAENOIC ACID 2(C22 H32 O2)5 HOH *96(H2 O) HELIX 11VALA 1CYSA 55 5 HELIX 22ASNA15LEUA231 9 HELIX 33GLYA26THRA361 11 SHEET 1 A10ILEA 62 PHEA 64 0 SHEET 2 A10LYSA 48 LEUA 54-1 N VALA 49 O PHEA 64 SHEET 3 A10THRA 39 GLUA 45-1 O THRA 39 N LEUA 54 SHEET 4 A10ALAA 6 GLNA 14-1 O ALAA 6 N ILEA 42

10 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . SHEET 5 A10VALA122 LYSA130-1 O VALA125 N GLNA 14 SHEET 6 A10LYSA112 PHEA119-1 N META113 O TYRA128 SHEET 7 A10LYSA100 LYSA109-1 O ASNA103 N THRA118 SHEET 8 A10LYSA 90 TRPA 97-1 N LEUA 91 O ARGA106 SHEET 9 A10ASNA 79 ASPA 87-1 N LYSA 81 O LYSA 96 SHEET 10 A10PHEA 70 THRA 73-1 O PHEA 70 N SERA 82 SITE 1AC113PROA 38 VALA 40 THRA 53 THRA 60 SITE 2AC113ILEA 62 GLUA 72 THRA 74 ALAA 75 SITE 3 AC1 13 ASP A 76 MET A 115 ARG A 126 TYR A 128 SITE 4 AC1 13 HOH A 409 10 20 30 40 50 MVEAFCATWK LTNSQNFDEY MKALGVGFAT RQVGNVTKPT VIISQEGDKV 60 70 80 90 100 VIRTLSTFKN TEISFQLGEE FDETTADDRN CKSVVSLDGD KLVHIQKWDG 110 120 130132 KETNFVREIK DGKMVMTLTF GDVVAVRHYE KA FABP7 Human brain 4AZNX-ray2.51A/B1-135[»]4AZOX-ray2.33A1-135[»]4AZPX-ray2.10A1-135[»]4AZQX-ray2.00A1-135[»]FABP5 mouse epidermal H O Anandamide arachidonate amide of athanol O C N H endocannabinoid C20:4 =6

4AZP(2-135,1-135) FABP5 mouse epidermal eicosanoate amide of ethanol O

O C

N H O O H A9M N-(2-HYDROXYETHYL)ICOSANAMIDE C22 H45 N O2 CL CHLORIDE ION CL 1- ; 4 HOH *84(H2 O) HELIX 11LEUA 4GLUA 85 5 HELIX 22GLYA18GLYA271 10 HELIX 33GLYA29ALAA391 11 SHEET 1 AA10THRA 62 ASNA 68 0 SHEET 2 AA10ASNA 51 GLUA 57-1 O ILEA 52 N CYSA 67 SHEET 3 AA10ASPA 42 ASPA 48-1 O ASPA 42 N GLUA 57 SHEET 4 AA10GLYA 9 HISA 17-1 O GLYA 9 N ILEA 45 SHEET 5 AA10ALAA125 LYSA133-1 O THRA128 N HISA 17 SHEET 6 AA10 LYS A 115 MET A 122 -1 O MET A 116 N TYR A 131 SHEET 7 AA10 LYS A 103 LYS A 112 -1 O THR A 106 N VAL A 121 SHEET 8 AA10ALAA 93 TRPA100-1 O LEUA 94 N ARGA109 SHEET 9 AA10LYSA 82 GLNA 90-1 O GLUA 84 N GLNA 99 SHEET 10 AA10PHEA 73 THRA 76-1 O PHEA 73 N THRA 85 SITE 1AC112META 23 LEUA 26 ALAA 36 VALA 60 SITE 2AC112LYSA 61 ALAA 78 ASPA 79 ILEA107 SITE 3 AC1 12 VAL A 118 ARG A 129 TYR A 131 HOH A2032 SITE 1AC2 1THRA 76 4AZNX-ray2.51A/B1-135[»]4AZOX-ray2.33A1-135[»]4AZPX-ray2.10A1-135[»]4AZQX-ray2.00A1-135[»]FABP5 mouse epidermal O H O C Anando-2-Glyceride H C 1 C O O C C20:4 =6 + C3 20 4AZQ(1-135,1-135) FABP5 mouse epidermal G2A 2-HYDROXY-1-(HYDROXYMETHYL)ETHYL ICOSANOATE C23 H46 O4

11 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . O C C O C O 20 O C 1

CL CHLORIDE ION CL 1- ; 4 HOH *68(H2 O) HELIX 11SERA 3GLUA 85 6 HELIX 22GLYA18GLYA271 10 HELIX 33GLYA29META381 10 SHEET 1 AA10THRA 62 ASNA 68 0 SHEET 2 AA10ASNA 51 GLUA 57-1 O ILEA 52 N CYSA 67 SHEET 3 AA10ASPA 42 ASPA 48-1 O ASPA 42 N GLUA 57 SHEET 4 AA10GLYA 9 HISA 17-1 O GLYA 9 N ILEA 45 SHEET 5 AA10ALAA125 LYSA133-1 O THRA128 N HISA 17 SHEET 6 AA10 LYS A 115 MET A 122 -1 O MET A 116 N TYR A 131 SHEET 7 AA10 LYS A 103 LYS A 112 -1 O THR A 106 N VAL A 121 SHEET 8 AA10ALAA 93 TRPA100-1 O LEUA 94 N ARGA109 SHEET 9 AA10LYSA 82 GLNA 90-1 O GLUA 84 N GLNA 99 SHEET 10 AA10PHEA 73 THRA 76-1 O PHEA 73 N THRA 85 SITE 1AC112META 23 ALAA 36 CYSA 43 THRA 56 SITE 2AC112VALA 60 ALAA 78 ASPA 79 ARGA109 SITE 3 AC1 12 MET A 116 ARG A 129 TYR A 131 HOH A2027 SITE 1AC2 1THRA 76 10 20 30 40 50 60 704AZR MASLKDLEGK WRLMESHGFE EYMKELGVGL ALRKMAAMAK PDCIITCDGN NITVKTESTV KTTVFSCNLG 80 90 100 110 120 130135 EKFDETTADG RKTETVCTFQ DGALVQHQQW DGKESTITRK LKDGKMIVEC VMNNATCTRV YEKVQFABP5 mouse epidermal 1B56X-ray2.05A1-135[»]1JJJNMR-A1-135[»] 4AZMX-ray2.75A/B1-135[»]4AZRX-ray2.95A/B1-135[»]4LKPX-ray1.67A1-135[»]4LKTX-ray2.57A/B/C/D1-135[»] 5HZ5X-ray1.40A2-135[»]5UR9X-ray2.20A/B/C/D/E/F/G/H1-135[»] 5UR9,5HZ5,4LKP,4LKT,4AZR,4AZM,1JJJ,1B56(1-135,1-135) FABP5 Human epidermal 8KS (1S,2S,3S,4S)-3-{[(NAPHTHALEN-1-YL)OXY]CARBONYL}-2,4- -DIPHENYLCYCLOBUTANE-1-CARBOXYLIC ACID 8(C28 H22 O4) SO4 SULFATE ION 15(O4 S 2-) MYR MYRISTIC ACID 4(C14 H28 O2) 1PE PENTAETHYLENE GLYCOL PEG400 2(C10 H22 O6) 38 HOH *344(H2 O) HELIX 1AA1THRA 3LEUA 75 5 HELIX 2AA2GLYA 18GLYA 271 10 HELIX 3AA3GLYA 29ALAA 391 11 SHEET 1AA110LYSA 61 THRA 68 0 SHEET 2AA110ASNA 51 SERA 58-1 N THRA 56 O THRA 63 SHEET 3AA110ASPA 42 CYSA 47-1 N THRA 46 O THRA 53 SHEET 4AA110GLYA 9 LYSA 17-1 N GLYA 9 O ILEA 45 SHEET 5 AA110 VAL A 125 GLU A 135 -1 O ILE A 130 N VAL A 14 SHEET 6 AA110 LYS A 115 MET A 122 -1 N VAL A 118 O ARG A 129 SHEET 7 AA110 LYS A 103 LYS A 112 -1 N LYS A 110 O VAL A 117 SHEET 8AA110ALAA 93 TRPA100-1 N GLNA 98 O SERA105 SHEET 9AA110LYSA 82 THRA 90-1 N ASNA 88 O VALA 95 SHEET 10AA110PHEA 73 THRA 76-1 N PHEA 73 O THRA 85 SITE 1AC1 9THRA 56 LYSA 61 THRA 62 THRA 63 SITE 2AC1 9ALAA 78 ARGF 12 VALF 14 LYSH 55 SITE 1AC2 4ARGA 12 GLUA135 LYSE 24 ARGE 33 SITE 1AC3 2ILEA 30 ARGA 33 SITE 1AC4 5SERA 16 LYSA 17 GLYA 18 PHEA 19 SITE 2AC4 5ASPA 20 SITE 1AC5 7PHEA 19 META 23 GLYA 36 CYSA120 SITE 2 AC5 7 ARG A 129 TYR A 131 HOH A 326 5HZ5,4LKP,4LKT,4AZR,4AZM,1JJJ,1B56(1-135,1-135) FABP5 Human epidermal DMSDIMETHYLSULFOXIDEC2H6OS SO4SULFATEIONO4S2- 65X 6-CHLORO-4-PHENYL-2-(PIPERIDIN-1-YL)-3-(1H-TETRAZOL-5-YL)QUINOLINE C21 H19 CL N6 FORMUL 2 DMS

12 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . FORMUL 3 SO4 FORMUL 4 65X FORMUL 5 HOH *99(H2 O) HELIX 1AA1THRA 3GLUA 85 6 HELIX 2AA2GLYA 18LEUA 261 9 HELIX 3AA3GLYA 29ALAA 391 11 SHEET 1AA110THRA 63 THRA 68 0 SHEET 2AA110ASNA 51 GLUA 57-1 N LEUA 52 O CYSA 67 SHEET 3AA110ASPA 42 ASPA 48-1 N ILEA 44 O LYSA 55 SHEET 4AA110GLYA 9 LYSA 17-1 N TRPA 11 O CYSA 43 SHEET 5 AA110 VAL A 125 VAL A 134 -1 O ILE A 130 N VAL A 14 SHEET 6 AA110 LYS A 115 MET A 122 -1 N VAL A 118 O ARG A 129 SHEET 7 AA110 LYS A 103 LYS A 112 -1 N LYS A 110 O VAL A 117 SHEET 8AA110ALAA 93 TRPA100-1 N GLNA 96 O ILEA107 SHEET 9AA110LYSA 82 THRA 90-1 N ASNA 88 O VALA 95 SHEET 10AA110PHEA 73 THRA 76-1 N PHEA 73 O THRA 85 SSBOND 1CYSA 120 CYSA 127 1555 1555 2.05 SITE 1AC1 7LYSA 72 PHEA 73 GLUA 74 THRA108 SITE 2 AC1 7 GLU A 119 VAL A 121 HOH A 336 SITE 1AC2 8SERA 16 LYSA 17 GLYA 18 PHEA 19 SITE 2AC2 8ASPA 20 HOHA333 HOHA377 HOHA382 SITE 1AC317PHEA 19 LEUA 32 ALAA 39 PROA 41 SITE 2AC317THRA 56 ALAA 78 ILEA107 ARGA109 SITE 3 AC3 17 VAL A 118 CYS A 120 ARG A 129 TYR A 131 SITE 4 AC3 17 HOH A 311 HOH A 341 HOH A 350 HOH A 378 SITE 5 AC3 17 HOH A 392 4LKP,4LKT,4AZR,4AZM,1JJJ,1B56(1-135,1-135) FABP5 Human epidermal CL CHLORIDE ION CL 1- DMS DIMETHYL SULFOXIDE 2(C2 H6 O S) NH4 AMMONIUM ION H4 N 1+ SO4 SULFATE ION O4 S 2- HELIX 11THRA 3GLUA 85 6 HELIX 22GLYA18GLYA271 10 HELIX 33GLYA29ALAA391 11 SHEET 1 A10THRA 62 THRA 68 0 SHEET 2 A10ASNA 51 GLUA 57-1 N THRA 56 O THRA 63 SHEET 3 A10ASPA 42 ASPA 48-1 N ILEA 44 O LYSA 55 SHEET 4 A10GLYA 9 LYSA 17-1 N TRPA 11 O CYSA 43 SHEET 5 A10VALA125 LYSA133-1 O ILEA130 N VALA 14 SHEET 6 A10LYSA115 META122-1 N VALA118 O ARGA129 SHEET 7 A10LYSA103 LYSA112-1 N LYSA110 O VALA117 SHEET 8 A10ALAA 93 TRPA100-1 N GLNA 98 O SERA105 SHEET 9 A10LYSA 82 THRA 90-1 N ASNA 88 O VALA 95 SHEET 10 A10PHEA 73 THRA 76-1 N PHEA 73 O THRA 85 SSBOND 1CYSA 120 CYSA 127 1555 1555 2.04 SITE 1AC1 2LYSA110 LYSA112 SITE 1AC2 7LYSA 24 THRA 46 ASPA 48 LEUA 52 SITE 2AC2 7THRA 53 HOHA316 HOHA356 SITE 1AC3 7LYSA 72 PHEA 73 GLUA 74 THRA108 SITE 2 AC3 7 GLU A 119 VAL A 121 HOH A 346 SITE 1AC4 3LYSA 17 GLYA 18 SO4A205 SITE 1AC5 8SERA 16 LYSA 17 GLYA 18 PHEA 19 SITE 2AC5 8ASPA 20 NH4A204 HOHA340 HOHA375 4LKT,4AZR,4AZM,1JJJ,1B56(1-135,1-135) FABP5 Human epidermal

O O O O C EIC 9,12-LINOLEIC ACID 4(C18 H32 O2) TAR D(-)-TARTARIC ACID 3(C4 H6 O6) GOL GLYCEROL GLYCERIN; PROPANE-1,2,3-TRIOL 9(C3 H8 O3) SO4 SULFATE ION 3(O4 S 2-) CL CHLORIDE ION CL 1- CIT CITRIC ACID C6 H8 O7 NH4 AMMONIUM ION 3(H4 N 1+) FORMUL 29 HOH *69(H2 O) HELIX 11THRA 3LEUA 75 5

13 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . HELIX 22GLYA18GLYA271 10 HELIX 33GLYA29ALAA391 11 SHEET 1 A10THRA 62 THRA 68 0 SHEET 2 A10ASNA 51 GLUA 57-1 N THRA 56 O THRA 63 SHEET 3 A10ASPA 42 ASPA 48-1 N ASPA 42 O GLUA 57 SHEET 4 A10GLYA 9 LYSA 17-1 N GLYA 9 O ILEA 45 SHEET 5 A10VALA125 LYSA133-1 O ILEA130 N VALA 14 SHEET 6 A10LYSA115 META122-1 N VALA118 O ARGA129 SHEET 7 A10LYSA103 LYSA112-1 N LYSA110 O VALA117 SHEET 8 A10ALAA 93 TRPA100-1 N GLNA 98 O SERA105 SHEET 9 A10LYSA 82 THRA 90-1 N ASNA 88 O VALA 95 SHEET 10 A10PHEA 73 THRA 76-1 N PHEA 73 O THRA 85 CISPEP 1ALAB 0 METB 1 0 1.62 SITE 1AC1 6META 23 LEUA 60 ARGA109 ARGA129 SITE 2AC1 6TYRA131 HOHA720 SITE 1AC2 2ILEA 30 ARGA 33 SITE 1AC3 4PHEA 65 SERA 66 GLUA 74 THRA 76 SITE 1AC4 6ALAA 39 LYSA 40 SERA 58 THRA 59 SITE 1AC5 6THRA 63 GLUA 75 THRA 76 THRA 77 SITE 2AC5 6GLNA 96 ARGA109 SITE 1AC6 6SERA 16 LYSA 17 GLYA 18 PHEA 19 SITE 2AC6 6ASPA 20 GLUA 21 O H Anandamide C O N C20:4 =6 H

4AZR,4AZM,1JJJ,1B56(1-135,1-135) FABP5 Human epidermal H H H H H H H H H H H O H H H H H H H Anandamide H O H H H H N O H H HH C H O C H H H H N H H H H H CL CHLORIDE ION CL 1- H 5 HOH *32(H2 O) A9M N-(2-HYDROXYETHYL)ICOSANAMIDE 2(C22 H45 N O2) HELIX 11GLYA18GLYA271 10 HELIX 22GLYA29META381 10 SHEET 2 BA10ASNA 51 THRA 59-1 O LEUA 52 N CYSB 67 SHEET 3 BA10LYSA 40 ASPA 48-1 O LYSA 40 N THRA 59 SHEET 4 BA10GLYA 9 LYSA 17-1 O GLYA 9 N ILEA 45 SHEET 1 AA10THRA 62 THRA 68 0 SHEET 5 AA10VALA125 LYSA133-1 O THRA128 N LYSB 17 SHEET 6 AA10 LYS A 115 MET A 122 -1 O LEU A 116 N TYR A 131 SHEET 7 AA10 LYS A 103 LYS A 112 -1 O THR A 106 N VAL A 121 SHEET 8 AA10ALAA 93 TRPA100-1 O LEUA 94 N ARGA109 SHEET 9 AA10LYSA 82 THRA 90-1 O GLNA 84 N GLUA 99 SHEET 10 AA10PHEA 73 THRA 76-1 O PHEA 73 N THRA 85 CISPEP 1HISA 0 META 1 0 4.06 SITE 1AC1 7ARGA109 ARGA129 TYRA131 PHEB 19 SITE 1AC2 7PROA 41 CYSA 43 HOHA2005 ARGB109

N H N O O

O BMS-309403 4AZM,1JJJ,1B56(1-135,0-134) FABP5 Human epidermal T4B ((2'-(5-ETHYL-3,4-DIPHENYL-1H-PYRAZOL-1-YL)-3-BIPHENYLYL)OXY)ACETIC ACID

14 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. .

BMS-309403

N N

H

O O BMS-309403 O 2(C31 H26 N2 O3) GOL GLYCEROL GLYCERIN; PROPANE-1,2,3-TRIOL C3 H8 O3 5 HOH *44(H2 O) HELIX 11THRA 3GLUA 85 6 HELIX 22GLYA18GLYA271 10 HELIX 33GLYA29ALAA391 11 SHEET 1 AA18PHEA 73 THRA 76 0 SHEET 2 AA18LYSA 82 THRA 90-1 O THRA 83 N GLUA 75 SHEET 3 AA18ALAA 93 TRPA100-1 O ALAA 93 N THRA 90 SHEET 4 AA18 LYS A 103 LYS A 112 -1 O LYS A 103 N TRP A 100 SHEET 5 AA18 LYS A 115 MET A 122 -1 O LYS A 115 N LYS A 112 SHEET 6 AA18 VAL A 125 LYS A 133 -1 O VAL A 125 N MET A 122 SHEET 10 AA18ASNA 51 THRA 68-1 O LEUA 52 N CYSB 67 SHEET 11 AA18ASPA 42 ASPA 48-1 O ASPA 42 N GLUA 57 SHEET 12 AA18GLYA 9 LYSA 17-1 O GLYA 9 N ILEA 45 SITE 1AC115PHEA 19 META 23 LEUA 26 GLYA 36 SITE 2AC115PROA 41 THRA 56 HOHA2009 GLNB 64 SITE 1AC213GLNA 64 ASPA 79 ARGA 81 ARGA109 SITE 2 AC2 13 VAL A 118 CYS A 120 ARG A 129 TYR A 131 SITE 1AC3 4GLYA 29 ILEA 30 LYSA 34 ASPB 79 1JJJ,1B56(1-135,1-135) FABP5 Human epidermal HELIX 11THRA 3GLUA 85 6 HELIX 22GLYA18GLYA271 10 HELIX 33GLYA29ALAA391 11 SHEET 1 A10THRA 62 THRA 68 0 SHEET 2 A10ASNA 51 GLUA 57-1 N THRA 56 O THRA 63 SHEET 3 A10ASPA 42 CYSA 47-1 N ILEA 44 O LYSA 55 SHEET 4 A10GLYA 9 LYSA 17-1 N TRPA 11 O CYSA 43 SHEET 5 A10THRA126 LYSA133-1 O GLUA132 N ARGA 12 SHEET 6 A10LYSA115 VALA121-1 N VALA118 O ARGA129 SHEET 7 A10LYSA103 LYSA112-1 N LYSA112 O LYSA115 SHEET 8 A10ALAA 93 TRPA100-1 N GLNA 98 O SERA105 SHEET 9 A10LYSA 82 THRA 90-1 N GLNA 84 O GLUA 99 SHEET 10 A10LYSA 72 THRA 76-1 N PHEA 73 O THRA 85 SSBOND 1CYSA 120 CYSA 127 1555 1555 2.63 1B56(1-135,1-135) FABP5 Human epidermal PLM PALMITIC ACID C16 H32 O2 3 HOH *39(H2 O) HELIX 11VALA 4LEUA 75 4 HELIX 22PHEA19LEUA261 8 HELIX 33ILEA30META381 9 SHEET 1 A9THRA 62 THRA 68 0 SHEET 2 A9ASNA 51 GLUA 57-1 N THRA 56 O THRA 63 SHEET 3 A9ASPA 42 ASPA 48-1 N ASPA 48 O ASNA 51 SHEET 4 A9GLYA 9 ASPA 15-1 N TRPA 11 O CYSA 43 SHEET 5 A9VALA125 LYSA133-1 N GLUA132 O ARGA 12 SHEET 6 A9LYSA115 META122-1 N META122 O VALA125 SHEET 7 A9LYSA103 LYSA112-1 N LYSA112 O LYSA115 SHEET 8 A9ALAA 93 TRPA100-1 N TRPA100 O LYSA103 SHEET 9 A9VALA 86 THRA 90-1 N THRA 90 O ALAA 93 SHEET 1 B2PHEA 73 THRA 76 0 SHEET 2 B2LYSA 82 THRA 85-1 N THRA 85 O PHEA 73 SSBOND 1CYSA 120 CYSA 127 1555 1555 2.32 SITE 1AC110META 35 GLYA 36 ASPA 79 ARGA109 SITE 2 AC1 10 VAL A 118 ARG A 129 TYR A 131 HOH A 213

15 Biochemistry. 2017 Jul 11;56(27):3454-3462. 5URA (1-132,1-132) 1FDQ,1FE3oleic..1JJX.NMR. . SITE 3 AC1 10 HOH A 214 HOH A 236 10 20 30 40 50 MATVQQLEGR WRLVDSKGFD EYMKELGVGI ALRKMGAMAK PDCIITCDGK 60 70 80 90 100 NLTIKTESTL KTTQFSCTLG EKFEETTADG RKTQTVCNFT DGALVQHQEW 110 120 130 135 DGKESTITRK LKDGKLVVEC VMNNVTCTRI YEKVE FABP5 Human epidermal

16