Human heart FABP3 IUCrJ. 2016 Jan 16;3(Pt 2):115-26.5CE4 J Biol Chem. 1992 Sep 15;267(26):18541-50.2HMB Bioorg Med Chem Lett. 2016;26(20):5092-5097.Fabp3 Human heart 5HZ9 4WBK,3WBG(1-133)3WBG(1-133.1-133) 2HMB,5CE4 Synchrotron Radiat. 2013 Nov 1; 20(Pt 6): 923–928. 3WBG(1-133.1-133) Angew Chem Int Ed Engl. 2015;54(5):1508-11. 4TJZ,4TKB,4TICH,4TKJ,3WVM 1HMR,1HMS,1HMT,5CE4(1-132.1-133) Structure. 1994 Jun 15;2(6):523-34.. 1HMSoleate,1HMTstearate,1HMRelaidate; muscle M-FABP Structure. 1994;2(6):523-34.1HMSoleate,1HMTstearate,1HMRelaidate Fig.1 Structural comparison of M-FABP in the presence of three C18 fatty acids and a C16 fatty acid. The backbone traces of M- FABP–oleate (green dark purple), M-FABP– stearate (green ligh magenta), M-FABP–elaidate (red cyan) and M-FABP–C16 (green red) are shown overlaid. The surface of the cavity is shown in light blue gray. Elaidic acid (magneta cyan) and the 13 ordered solvent molecules (blue yellow) within the cavity are also shown [produced by GRASP (A Nicholls, R Bharadwaz and B Honig, unpublished program)] M-FABP with three different C18 fatty acids — n -octadecanoate (more commonly called stearate), trans -Δ 9 -octadecenoate (or elaidate) and cis - Δ 9 -octadecenoate (or oleate). Angew Chem Int Ed Engl. 2015;54(5):1508-11. 4TJZ,4TKB,4TICH,4TKJ,3WVM Abstract Long-chain fatty acids (FAs) with low water solubility require fatty-acid-binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. Evaluating the affinity of FAs, sub-Angstrom X-ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. The heart FABP (FABP3) preferentially incorporates a U-shaped FA of C10–C18 using a lipid-compatible water cluster, and excludes longer FAs using a chain-length-limiting water cluster. Proteins recognize diverse lipids with different chain lengths.

a Figire S1. FA structures in co-crystals with proteins. (a) C16:0/yellow fever mosquito sterol carrier protein-2 like-3 (1PZ4). (b) 3-hydroxy C14:0-CoA/E. coli acyl carrier protein- UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase (4IHF), (c) C18:1 n-6c/maize nonspecific lipid-transfer protein showing a double conformation for FA molecule (1FK5,). FA c c alkyl chains are shown in spheres. Figure S2. Thermal fluctuation of FABP3-FA complex. (a) and (b) Fluctuations in the FABP3-C18:0 co-crystal structure obtained at room temperature shown with temperature factor (smaller in thinner and blue, and larger in thicker and red). (c) b Fluctuations of five SFAs bound to FABP3 in solution state deduced from 20 ns of MD simulations (C10:0 in violet, C12:0 in black, C14:0 in green, C16:0 in blue, and C18:0 in red). (e) Amino acid sequence of FABP3; Blue highlight: residues in contact with the FA; yellow: residues in contact with water in the pocket; pink: residues of a c hydrogen-bonded to the carboxylate of the FA.

S61 For energy production in the skeletal and heart muscle,1 the efficient cytosolic delivery of fuel such as long-chain fatty acids (LCFAs) is crucial. Mitochondrial metabolism prefers fatty acids (FAs) of a certain range of chain length. Thus, specific transporter and carrier proteins of the “fuel” FAs have been created as exemplified by the fatty-acid-binding proteins (FABPs).2, 3 FAs with flexible alkyl chains that do not exhibit a defined structure or noticeable electrostatic interactions. The human heart-type FABP (FABP3) identifies FAs not by exact matching but by broad recognition of fundamental structural similarities among numerous FAs. To date, more than 40 subtypes of FABPs have been identified,4 most of which share a highly conserved three-dimensional structure.3 FABP3, one LCFA molecule in a U-shape is accommodated in the binding cavity together with about 13 ordered water molecules.5 FABPs 4, 5, 7, and 8 in the binding sites of nonspecific lipid transporters universally expressed from bacteria to humans,6 the FAs are largely extended (Figure S1). The U-shape conformation of bound FA is critical for the incorporation of FAs with different chain lengths into the binding site of FABP3 and the other FABPs, and raise an intriguing question as to how the proteins do this by using a rigid β-clam architecture and ordered water molecules in the pocket. Protein Sci. 2002 Oct; 11(10): 2382–2392. 1G5W, Biochem J. 2001;354(Pt 2): 259–266.1G5W, Figure S4. Hydration sites identified by WaterMap in the binding site of FABP3 with SFAs. WatarMap analysis results for FABP3 complexed with C10:0–C24:0 (b– i) and its apo form (a). The structure analysis of the apo form a obtained by deleting the ligand from the C18:0/FABP3 complex identified 18 water molecules (27 hydration sites) within the binding site (see Figure 5a). For FABP3 complexed with C20:0–C24:0, whose X-ray structures are not available, the binding poses of SFAs are computationally modeled based on the binding pose of C18:0. Colour gradation of hydration sites is based on the free energy relative to bulk water with stable sites shown in green and unstable sites in red. In the binding site of the complex bound with C14:0, a dewetted cavity (surface area = 32.3 Å2, shown in blue mesh), a region where virtually no water density was found from the MD trajectory, was found. The terminal parts of the extended chains of C20:0 – C24:0 are predicted to intrude into the region occupied by Cluster 1 and disrupt the hydrogen bond network resulted in the formation of high energy hydration sites. Figure S5. Thermodynamic signature of the water molecules in Clusters 1 and 2 calculated by WaterMap. ΔH and –TΔS (relative to bulk solvent) of the hydration sites found in the binding site of FABP3 incorporating C10:0 are shown. The differences in their thermodynamic profiles for Clusters 1 and 2 are clearly seen. a) Entropy (– TΔS) of the hydration sites. b) Enthalpy of the hydration sites. Large –TΔS values of Cluster 1 mean translational and rotational freedom of the water molecules are heavily restricted. On the other hand, enthalpies of Cluster 1 water molecules are mostly similar to the value of bulk water. a b These observations strongly suggest the presence of a strong hydrogen bond network in Cluster 1. In contrast, the thermodynamic signature of Cluster 2 water molecules suggest that they are loosely hydrogen bonded to each other and have larger freedom of movement. The preferential displacement of Cluster 2 water molecules by the extended chain of SFAs is well explained by this difference in the thermodynamic profiles.

2 Figure S6. Examples of promiscuous recognition of lipids and hydrophobic ligands by rigid protein (A) and flexible proteins (B and C). Among other proteins, the above three lipid-binding proteins are focused; CERT is the relatively rigid proteins that bind various ceramides,[251 and cytochrome P450[26,27] and peroxisome proliferator-activated receptor (PPAR)[28-30] are known to recognize their ligands in an induced-fit manner. See Table S5 for detailed data. (A) CERT: Main chains of the apo- and C6-, C16-, and C18-ceramide bound structures of the CERT START domain, particularly near the binding cavity, are largely superimposed. Red, apo-form; green, [251 blue, and cyan, C6-, C16-, and C18-ceramide bound forms, respectively. The image was taken from reference[25] © The National Academy of Sciences of the United States of America. (B) Cytochrome P450 3A4: A superposition of the ketoconazole (hydrophobic ligand, not shown) complex (2J0C, dark colours) and the ligand-free structures (1TQN, light colours). Note that the structures of -helices F/F’, G/G’ and a C-terminus loop undergo large conformation changes by ligand binding as depicted by orange arrows. The images were taken from reference.[26] © The National Academy of Sciences of the United States of America.

(C) PPAR'y: The structures of ligand-binding cavities of PPAR'y with 5-hydroxyeicosapentaenoic acid (5-HEPA) and 4-hydroxydocosahexaenoic acid (4-HDHA). Note the differences in the conformation and orientation of the ligands and surrounding amino acid residues. The image was taken from reference[30] with permission. © Nature Publishing Group. Table S1. FABP3 binding parameters obtained from liposomal ITC experiments. a: FABP3 affinity and thermodynamic parameters for FAs* fatty acid K i (M) AFI (kJ/mol) T.\S (kJ/mol) SFA - C6:0 n.o.*** - - C7:0 5.08E-05 -62.04 36.52 08:0 4.61E-05 ± 3.33E-05 -79.46 ± 7.93 52.22 ± 6.38 C9:0 9.69E-07 -79.27 43.55 C10:0 1.07E-06 ± 3.64E-07 -69.14 ± 5.40 33.38 ± 6.11 C12:0 2.56E-06 ± 6.97E-07 -81.35 ± 5.81 47.93 ± 6.22 C14:0 1.84E-06 ± 7.64E-07 -79.30 ± 7.38 44.30 ± 9.12 C16:0 9.13E-07 ± 8.45E-08 -98.55 ± 22.98 62.63 ± 23.11 C18:0 1.95E-06 ± 6.12E-07 -96.63 ± 6.47 62.44 ± 6.38 C19:0 3.97E-06 -75.81 43.71 C20:0 2.37E-05 ± 5.40E-06 -91.89 ± 23.37 64.24 ± 23.12 C22:0 2.25E-05 ± 7.61E-06 -30.71 ± 11.94 2.64 ± 12.64 C24:0 2.70E-05 ± 8.84E-07 -8.91 ± 1.01 -18.21 ± 0.94 UFA C16:1 n-7c 4.39E-07 ± 1.62E-07 -137.91 ± 27.96 99.81 ± 28.65 C18:1 n-7c 1.21E-06 ± 3.14E-07 -80.77 ± 7.31 45.40 ± 7.59 018:1 n-7t 9.32E-07 ± 6.41E-08 -113.25 ± 10.89 77.39 ± 10.70 C18:1 n-9c 8.48E-07 ± 4.20E-08 -83.68 ± 5.68 47.59 ± 5.56 C18:1 n-9t 8.46E-07 ± 2.10E-07 -87.52 ± 7.53 51.23 ± 7.01 C18:1 n-12c 9.35E-07 ± 1.56E-07 -82.10 ± 4.02 45.21 ± 2.68 C18:2 n-6c 5.58E-07 ± 2.54E-07 -104.39 ± 9.12 66.71 ± 10.01

S10S133 C18:3 n-3c 7.36E-07 ± 2.15E-07 -103.54 ± 11.15 66.87 ± 10.60

*evaluated by liposomal ITC in 20 mM Tris-HCl pH8.0 and 100 mM NaCl at 310 K. Experiments were performed triplicate except for C7:0, C9:0 and C19:0.**For saturated FAs (C6:0-C20:0), another series of ITC measurements was carried out under the phosphate buffer conditions at 310 K, which provided similar thermodynamic parameters. ***not observed. b FABP3 affinity and thermodynamic parameters for FA analogues* ligand Kd (M) .OFF (kJimol) - TAS (1c.11mol) C10:0 1.63E-06 ± 2.63E-07 -80.66 ± 4.02 46.19 ± 4.47 016:0 8.85E-07 ± 4.29E-08 -44.86 ± 0.10 9.51 ± 0.83 retinoicacid n.o.** prostaglandin E2 5.45E-05 ± 2.62E-05 -66.81 ±14.80 41.32 ± 16.09 phytanic acid >5.00E-04 jasmonic acid 2A5E-05 ± 1.98E-07 -34.81 ± 0.46 7.65 ± 0.44

*evaluated by liposomal ITC in 20 mM K-phosphate pH7.0 and 100 mM NaCl at 310 K. ** not observed. Table S2. X-ray data processing and structure determination statistics for FABP3 in complex with the fatty acids. FA complexes Cl 0:0 complex C12:0 complex C14:0 complex C16:0 complex C18:0 complex Source Spring-8 BL44XU Spring-8 BL44XU SPring-8 BL44XU SPring-8 BL44XU Spring-8 BL44XU Wavelength (A) 0.80 0.80 0.80 0.80 0.80 Temperature (K) 100 100 100 100 100 Space group P212121 P212121 P212121 P212121 P212121 Unit-cell parameters (A) 54.5, 69.7, 33.9 54.4, 69.7, 33.8 54.6, 69.5, 33.8 54.7, 69.9, 33.7 54.6, 69.4, 33.8 a. b. c Resolution (A) 50 - 0.87 50 - 0.86 50 - 0.93 50 - 0.87 50 - 0.88 (High-resolution shell) (0.89 - 0.87) (0.87 - 0.86) (0.95 - 0.93) (0.89 - 0.87) (0.90 - 0.88) No. of reflections 1,061,542 1,298,725 997,728 1,021,456 1,249,690 Oscillation angle (°) 0.5 0.5 0.5 0.5 0.5 Total rotation angle 1°) 360 360 360 360 360 No. of unique reflections 104,970 107,896 86,123 106,214 100,773 Redundancy 10.1 (8.0) 12.0 (5.4) 11.6 (8.9) 9.6 (7.5) 12.4 (6.6) <110(!)) 10.1 (3.4) 9.8 (2.3) 10.1(3.5) 10.3 (3.7) 8.6 (2.5) Completeness (%) 98.6 (97.5) 98.0 (82.0) 98.9 (97.9) 99.5 (99.9) 98.6 (87.0) a 1?..4 (%) 7.1 (40.4) 6.6 (39.2) 7.9 (39.9) 6.8 (41.8) 6.6 (42.3) Mosaicity (°) 0.49 0.27 0.38 0.51 0.25 Refinement Resolution range (A) 42.94 - 0.87 42.94 - 0.86 42.91 - 0.93 43.08 - 0.87 42.89 - 0.88 No. of reflections 99,487 102.442 81,538 100,656 95,629 Data cutoff LS igma(Fo)] none none none none none Rmytb / Rfi,,C 0.10 / 0.11 0.10/0.12 0.11 / 0.12 0.10 / 0.12 0.10/0.11 No. of water molecules 193 194 165 189 174 II No. of fatty acids 1 1 1 1 1 RMSD bond length (A) 0023 0023 0.022 0.025 0.023 RMSD bond angle (°) 23 2.6 2.3 2.5 2.3 PDB ID 4TJZ 4TKB 4TICH 4TKJ 3WVM aRmerge = EhklEi |Ii(hkl) - |/ EhklEi Ii(hkl), where Ii(hkl) is the ith observed intensity of reflection hkl and is the average intensity over symmetry-equivalent measurements. Values in parentheses are for the highest resolution shell. bRcryst =E||Fo| - |Fc||/E|Fo| calculated from 95% of the data, which were used during the course of the refinement. cRfree =E||Fo| - |Fc||/E|Fo| calculated from 5% of the data, which were used during the course of the refinement. Bioorg Med Chem Lett. 2016;26(20):5092-5097.Fabp3 5HZ9 4WBK,3WBG(1-133)3WBG(1-133.1-133) 2HMB,5CE4. The FABP family comprises nine isoforms, which differ in their sequence and tissue distribution, but have remarkably similar structures.4 Our goal was to provide a dual FABP4/5 inhibitor selective against other FABP isoforms with good physicochemical and pharmacokinetic properties suitable as a tool for testing in models of metabolic diseases, angiogenesis or tumor growth. As selectivity filter, we chose activity against the structurally closely related FABP3 and the less similar FABP1. J Biol Chem. 1992 Sep 15;267(26):18541-50.2HMB Abstract The recombinant human muscle fatty acid-binding protein with a bound fatty acid within the interior core H-FABP3 contains 10 antiparallel beta-strands and two short alpha-helices which are arranged into two approximately orthogonal beta-sheets. The hydrocarbon tail of the fatty acid was found to be in a "U-shaped" conformation. Seven ordered water molecules were also identified within the interior of the protein in a pocket on the pseudosi face of the fatty acid's bent hydrocarbon tail. The methylene tail of the fatty acid forms van der Waals interactions with atoms from 13 residues and three ordered waters. The carboxylate of the fatty acid is located in the interior of the protein where it forms hydrogen bonds with the side chains of Tyr128 and Arg126 and two ordered water molecules. The distinct binding sites in order to satisfy different requirements within the

S14S84 tissues where they are expressed. Human muscle fatty acid binding protein (M-FABP)1 is primarily expressed in skeletal and cardiac muscle cells, where it represents 0.3-0.6% of the total cytosolic protein (Veerkamp et al., 1991). Its cDNA codes for a protein of 133 amino acids with a calculated molecular mass of 14,858 Da. The protein binds long chain fatty acids with a Kd of about 0.5 am, showing a slight preference for unsaturated fatty acids. The stoichiometry of binding is one molecule of fatty acid/molecule of protein. Synchrotron Radiat. 2013 Nov 1; 20(Pt 6): 923–928. 3WBG(1-133.1-133) Abstract Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteīns, revealed the detailed ANS-binding mechanism. Four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. H O The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with N O S O FABP3 and its crystal structure is almost the same as that of FABP3. FABP4 can bind with a higher affinity to ANS than FABP3. The orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS. Fig. 1. Chemical structure of 1-anilinonaphthalene-8-sulfonate (ANS) FABPs belong to the intracellular lipid-binding protein (iLBP) family and are involved in reversibly binding intracellular hydrophobic ligands such as FAs. FABPs are also essential for trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. To date, the main known FABPs are found in different cell types; for example, liver-type FABP (FABP1), intestinal-type FABP (FABP2), heart-type FABP (FABP3), adipocyte-type FABP (FABP4), epidermal-type FABP (FABP5), ileal-type FABP (FABP6), brain-type FABP (FABP7) and testis-type FABP (FABP9) (Offner et al., 1986 ▶; Coe & Bernlohr, 1998 ▶). Each FABP has its own sequence,shows 22–73% similarity in amino acid sequence, and exhibits distinct ligand preferences, although all of the FABPs share a common structure consisting of ten antiparallel β-strands that are capped by a pair of α-helices (Sacchettini et al., 1989 ▶; Scapin et al., 1992 ▶; LaLonde et al., 1994 ▶; Young et al., 1994 ▶; Zimmerman & Veerkamp, 2002 ▶; Zhang et al., 2003 ▶). IUCrJ. 2016 Jan 16;3(Pt 2):115-26.5CE4Abstract Heart fatty acid binding protein (H-FABP) in complex with oleic acid provided very detailed information about the cluster of water molecules and the bound oleic acid in the large internal cavity. The electrostatic potential in the fatty acid (FA) binding pocket used to study interactions involving the internal water molecules, the FA and the protein, showed H H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation⋯ and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface. Keywords: Neutron protein crystallography, high-resolution room-temperature X-ray crystallography, fatty acid binding protein, protein hydration layer, AIM topological properties Introduction 1. Water molecules are of the most importance for recognition between biological molecules. Many studies, extensively reviewed by Raschke (2006 ▸), have focused on hydration water molecules in protein surfaces, noting that they have slower correlation times than bulk water, in agreement with studies of water molecules in confined spaces. Due to contacts with the confining surfaces, the total number of water–water hydrogen bonds is reduced and their strength is reinforced, increasing the tetrahedrality and lowering the orientational dynamics and therefore the dielectric constant (Gilijamse et al., 2005 ▸). Furthermore, the lack of competing water molecules and the effect of environmental fluctuations in the confined space (Stanley et al., 2009 ▸) lower the diffusion coefficient and increase the viscosity (Chaplin, 2009 ▸). A comprehensive analysis of water in protein interfaces from crystal structures has shown a difference between biological and crystal-packing interfaces; the latter have 50% more water molecules than the former (Rodier et al., 2005 ▸), implying that water molecules are expelled when the biological interactions are completed. These results imply that the properties of water in hydration layers are very different from those of bulk water. These hydration layers play a role during biological interactions between macromolecules, but it is difficult to study

5 their atomic three-dimensional structures, as they are normally transient or affected by high thermal displacement parameters. This difficulty can be overcome by studying water clusters inside a protein. For this purpose, fatty acid binding proteins (FABPs), small proteins which act as intracellular lipid chaperones, are good models since they have a large internal cavity occupied by a fatty acid (FA) and a stable cluster of well ordered water molecules (Chmurzyńska, 2006 ▸). Unlike the biological interfaces between different proteins observed by X-ray crystallography (Rodier et al., 2005 ▸), the water cluster inside FABPs has more than one very ordered hydration layer. It can therefore be used as a probe to assess the general rules governing water structures in interfaces. Figure 1. A ribbon representation of the H-FABP structure determined in this work, with β-sheets in magenta and α-helices in cyan. The internal water cluster and the oleic acid are represented as spheres occupying the internal cavity (red = O atoms, yellow = C atoms, white = H or D atoms). FABPs coordinate lipid responses in cells and are also strongly linked to metabolic and inflammatory pathways (Haunerland & Spener, 2004 ▸; Chmurzyńska, 2006 ▸; Makowski & Hotamisligil, 2005 ▸; Coe & Bernlohr, 1998 ▸; Zimmerman & Veerkamp, 2002 ▸). FABPs are 14–15 kDa proteins that reversibly bind hydrophobic ligands, such as saturated and unsaturated long-chain FAs. All known FABPs share almost identical three-dimensional structures, including a ten- stranded antiparallel β-barrel (Chmurzyńska, 2006 ▸), which is formed by two orthogonal five-stranded β-sheets, as shown in Fig. 1 ▸. The binding pocket is located inside the β-barrel, the opening of which is framed by the N-terminal helix– loop–helix ‘cap’ domain, and FAs are bound to the interior cavity. Generally, conserved basic amino acid residues are required to bind the carboxylate head of an FA ligand in the binding pocket of an FABP (Chmurzyńska, 2006 ▸; Zimmerman & Veerkamp, 2002 ▸). The hydrocarbon tail of the ligand is lined on one side by hydrophobic amino acid residues and on the other side by ordered water molecules, which mediate the interaction between the protein and the ligand and contribute to differences in the enthalpic and entropic components of the ligand binding energy. X-ray structures of FABPs complexed with FAs reveal that the internal cavity accommodates both the ligand and water molecules (Wiesner et al., 1999 ▸; Sacchettini & Gordon, 1993 ▸). A recent study focusing on atomic resolution X-ray crystal structures of heart-FABP (H-FABP) complexed with FAs of varying alkyl chain lengths has shown that these water molecules in the binding pocket can be sorted into two distinct clusters exhibiting different stabilities (Matsuoka et al., 2015 ▸). The first cluster, studied in detail in the present work, is the more energetically stable and is composed of very ordered water molecules in both holo and apo H-FABP, as observed by NMR (Mesgarzadeh et al., 1998 ▸) and confirmed by molecular dynamics (MD) simulations (Bakowies & Gunsteren, 2002 ▸). The second cluster is made of less stable water molecules, which are expelled by FA alkyl chains longer than 12 carbon atoms. The conserved water cluster has been reviewed (Bottoms et al., 2006 ▸) and its function has been analysed (Lücke et al., 2002 ▸), proposing that these water molecules form a hydration shell that interacts with the bound ligand (Scapin et al., 1992 ▸; Kleywegt et al., 1994 ▸; LaLonde et al., 1994 ▸; Young et al., 1994 ▸). In holo intestinal-FABP [I-FABP, Protein Data Bank (PDB) code 2IFB], these water molecules are located at the concave face of the slightly bent FA ligand (Sacchettini et al., 1992 ▸), whereas in the holo forms of adipocyte-FABP (A-FABP, PDB code 1LIE) and heart- FABP (H-FABP, PDB code 1HMR), the water molecules are clustered beneath the pseudo-re face of the U-shaped FA (LaLonde et al., 1994 ▸). In these last two proteins, the surface of the binding cavity is divided into three sections, consisting of: (i) a cluster of hydrophobic side chains contacting the aliphatic chain of the ligand; (ii) a scaffold of polar and ionizable groups that interact with the bound cluster of water molecules; and (iii) a mixture of residue types near the entry portal. The purpose of this work was to obtain a complete atomic description of the ordered water cluster and its properties in the human H-FABP–oleic acid complex and to analyse interactions between the bound ligand, the water cluster and the protein residues. To achieve this, we used a combination of high-resolution X-ray crystallography and neutron protein crystallography (NPC) to determine the atomic positions (plus alternate conformations) for the water molecules, FA atoms and protein residues, including the positions of the hydrogen atoms (as deuterium). These experiments were conducted at room temperature, thus reflecting the actual in vivo conditions. The resulting X- ray/neutron structure has allowed the use of the charge-density distribution, expressed in terms of multipolar components (Hansen & Coppens, 1978 ▸). These components are obtained by transfer from the ELMAMII library (Domagała et al., 2012 ▸) for protein, FA and water molecule atom types. This ‘building blocks’ approach allows the accurate description of the continuous molecular electron density and the relevant derived properties of macromolecular

6 systems, without the need for fulfilling the stringent requirements of a complete multipole refinement (Liebschner et al., 2011 ▸). This transferred electron-density distribution was used to study the network of interactions formed by the protein, the ligand and the water cluster, on the basis of Bader’s quantum theory of atoms in molecules (QTAIM; Bader, 1994 ▸). As knowledge of the precise total charge distribution (nuclei positions and transferred aspherical electron density) allows the calculation of derived electrostatic properties, we performed calculations that determine the electrostatic potential being felt by the bound FA, and the electric field at the position of each internal ordered water molecule. Note that these measurements and the corresponding calculations are not biased by the experimental methods because we study a water cluster not involved in crystallographic symmetry contacts (as it is inside a cavity) and the experiments were conducted at room temperature. Therefore, the water properties observed should be close to those in vivo. Results and discussion 3. 3.1. Structure description Several FABP isoforms have been structurally investigated as isolated recombinant proteins by X-ray crystallography, NMR and other biochemical and biophysical techniques (Furuhashi & Hotamisligil, 2008 ▸). FABPs have an extremely wide range of sequence diversity, from 15 to 70% sequence identity between different members (Chmurzyńska, 2006 ▸). Analyses of the PDB entries 3rzy (A-FABP without FA) and 3p6c (A-FABP with citrate) (González & Fisher, 2015 ▸) show an internal water cluster which is well conserved, even when the FA molecule is not present. In this case the water in the space of the absent FA was not observed, probably due to disorder, as indicated by hydration site analysis of the apo form (Matsuoka et al., 2015 ▸). Figure 2. Cluster water molecules inside the cavity, with hydrogen- bond contacts indicated as yellow dashed lines (distances are given in Å). Water molecules with single occupancy and a close to tetrahedral conformation are indicated in green, and those with alternate conformations in magenta. O atoms in other water molecules are indicated in red.

In this work, the structure of perdeuterated human H-FABP was determined for the first time at room temperature with combined neutron and X-ray diffraction data to resolutions of 1.90 and 0.98 Å, respectively (for statistics of the data collection and refinement, see Tables S1–S3 in the supporting information). Analysis of the electron and nuclear scattering density maps showed that oleic acid (the FA naturally bound to H-FABP expressed in E. coli) does not occupy the whole internal cavity. As depicted in Fig. 1 ▸, the FA (yellow C atoms) is folded in the typical U- shaped conformation systematically observed in complexes between FABP and long-chain FAs (Smathers & Petersen, 2011 ▸; Zanotti, 1999 ▸). Its carboxylate head is in contact with the conserved Tyr128 and Arg126 side chains (Fig. 2 ▸) and, through a water bridge, with Arg106. Along with the FA, 14 water molecules fully occupy the rest of the cavity (Fig. 1 ▸), of which two are observed with double conformations. The water molecules are packed against the oleic acid and are connected with the external solvent through a narrow pore. These water molecules are very well ordered, even at room temperature (mean B factor for the O atoms = 15.6 Å2), more so than the oleic acid which presents a non-H-atom mean B factor of 32.5 Å2, ranging between 11.8 Å2 for the carboxyl group to 48 Å2 for the terminal methyl C atom. 3.2. Water cluster analysis 3.2.1. Hydrogen-bonding network Fig. 3 ▸ shows the electron (X-ray) and nuclear (neutron) scattering density maps for the internal water molecules, revealing the positions of their H atoms (as deuterium), and thus their orientation. We first use standard geometric criteria to locate hydrogen bonds involving water molecules in the ordered cluster. They are linked in a network showing mostly tetrahedral coordination (Fig. 2 ▸). The geometries of the hydrogen bonds in this network are described in detail in Table S4 in the supporting information. The hydrogen-bond distances between the oxygen and acceptor atoms of the ordered water molecules show a wide range of values between 2.68 and 3.05 Å, with donor group–acceptor angles systematically greater than 100°. However, these hydrogen bonds are on average rather short, with a mean O O distance of 2.83 Å, leading to a mean volume of 17.2 Å3

⋯ 7 per water molecule inside the cavity (the cavity volume was calculated with the program McVol; Till & Ullmann, 2010 ▸). This can be compared with a van der Waals water molecule volume between 16 and 18 Å3 and an average volume of 30 Å3 for bulk water at 24°C. Therefore, the water molecules are quite tightly packed in the binding cavity of H-FABP. All 14 of the water molecules in the cluster are involved in at least two hydrogen bonds as donors and two others as acceptors, corresponding to tetrahedral coordination with at least one chemical group from the protein. An additional three buried water molecules do not belong to the cluster. Among them, W1 is a resident water molecule conserved among nine different members of the FABP protein family and is presumed to play a structural role in the stabilization of the folded protein (Likić et al., 2000 ▸). It has a nearly flat coordination, being involved in one hydrogen bond as acceptor with the amide H atom of the Val84 main chain and in two hydrogen bonds as donor with the main chain O atoms of Val68 and Lys65. Two cluster water molecules (W13 and W24) are in contact with atom O2 of the FA carboxylate head, forming strong hydrogen bonds with O O distances of 2.68 and 2.76 Å, respectively. These are among the shortest hydrogen bonds involving water molecules, which is consistent with the fact that they are known to play a role in FA binding; they are indeed systematically found⋯ at quasi-identical positions in H-FABP and muscle- FABP (M-FABP) structures complexed with FAs [see PDB codes 3WVM, 4TKJ, 4TKH, 4TKB and 4TJZ (Matsuoka et al., 2015 ▸); 1HMR, 1HMS and 1HMT (Young et al., 1994 ▸)], and also at slightly displaced positions in other members of the FABP family (such as 4BVM; Ruskamo et al., 2014 ▸). Figure 3. Cluster water molecules inside the cavity, with electron and nuclear scattering density maps. Cyan: 2F o − F c neutron map contoured at 1.7 r.m.s.; magenta: 2F o − F c electron density map contoured at 2.0 r.m.s.. Tetrahedral water molecules with single occupancy and a close to tetrahedral conformation are indicated in green. O atoms in other water molecules are indicated in red. Dashed lines indicate hydrogen bonds (distances are given in Å)

Only three other cluster water molecules (W51, W7 and W31) interact, through weak C—H O hydrogen bonds, with the H atoms bound to atoms C3, C5, C6 and C18 of the FA. W31, located at the top of the U-shape of the FA, acts as a C—H O hydrogen-bond acceptor in three interactions, with H O distances of 3.46 (donor group⋯ is C5—H52), 3.38 (C3—H31) and 2.55 Å (C18—H181). This way, the conserved W31 water molecule bridges both extremities of the FA, clearly⋯ contributing to stabilizing its folded ‘U’ conformation. Another⋯ C—H O interaction links non-cluster W28 (located on the other side of the U formed by the ligand) and the C14—H141 hydrogen atom of the FA. To summarize, it appears that, from a hydrogen-bond geometry perspective, the FA alkyl chain forms⋯ few interactions with the water cluster. This observation agrees with the recent finding by Matsuoka and co-workers, who have shown in a convincing way that the energetic stability of the water molecules in this cluster prevents long FAs from folding correctly in the binding pocket (Matsuoka et al., 2015 ▸). Hence, this cluster presents an intrinsic stability independent of the formation of strong interactions with the aliphatic chain of the FA. 3.2.2. Alignment between electric field and water molecule dipoles As expected, the water molecules buried in this cavity are exposed to strong electric fields, ranging between 6.6 (7) and 21.4 (8) GV m−1 when computed using the vacuum dielectric constant. We studied the relation between the electric field and the orientations of the water molecule dipole moments, defined in terms of the two angles α and β (§2.4). For the 17 water molecules included in the analysis (14 in the cluster and three buried ones), it appears that both measured angles are significantly smaller than 90°, with average values of 20° and 23° for α and β, respectively (Fig. 4 ▸, Table 1 ▸). There are no examples, even taking into account the estimated uncertainties on these angles, where both vectors show inverted directions (angles larger than 90°), meaning there is a clear correlation between them. Water molecules hydrogen-bonded in a tetrahedral coordination are expected to be located in a close dipolar environment, where positive charges correspond to H atoms interacting with the water O atom, and negative charges to electronegative atoms accepting hydrogen bonds with the water protons. However, surprisingly, the most favourable cases, i.e. where both angles are close to zero, do not necessarily correspond to ideal tetrahedral coordination of the water molecules. For example, this is the case for a water molecule (W28) that is in contact, on its oxygen side, with the oleic acid molecule and located in the narrow pore connecting the binding pocket with the external solvent molecules. This water molecule is involved (as an acceptor) in only one clear hydrogen bond

8 (with the Arg126 side chain) but nevertheless presents a nearly perfect alignment with the external electric field (Table 1 ▸). This implies that the alignment is not driven by the local environment alone but by the overall electric field. Figure 4. Partial view of the water cluster filling the binding pocket along with the FA. Water molecule dipole moments are represented as thin red arrows, with the scale 1 Å = 2 debye. Electric field vectors computed at the water molecules’ centres of mass are represented as green arrows, using the scale 1 Å = 0.1 e Å−2 = 14.4 GV m−1. The oleic acid ligand can be seen at the bottom of the picture.

Table 1 Electric field magnitudes, and angles between the electric field and the water molecule dipole moments, measured at the water molecules’ centres of mass See §2.4 for the definition of the angles α and β and for the estimation of uncertainty values. The raw angle is that between the water molecule dipole moment and the electric field vector. Water molecule labelElectric field magnitude (GV m−1)Raw angle (°)α angle (°)β angle (°)Water O-atom B factor (Å2) 1 16.6 (7) 31 (4) 26 (4) 15 (3) 10.6 3 19.6 (6) 9 (2) 8 (4) 3 (2) 12.4 6 11.3 (6) 50 (5) 17 (6) 45 (5) 11.5 7 11.5 (7) 27 (4) 7 (7) 26 (4) 11.7 8 8.9 (7) 45 (5) 13 (6) 42 (5) 12.8 13 21.4 (8) 62 (3) 58 (3) 14 (4) 11.6 17 11.1 (7) 58 (5) 40 (5) 34 (4) 14.3 20 18.6 (9) 15 (3) 5 (5) 14 (3) 17.8 24 16.3 (7) 51 (5) 39 (5) 27 (3) 14.5 26 13.7 (7) 22 (6) 5 (8) 21 (6) 22.8 28 18.3 (7) 11 (5) 9 (7) 6 (4) 24.2 30 18.2 (5) 31 (5) 12 (6) 29 (4) 20.4 31 6.6 (7) 78 (7) 60 (6) 27 (5) 13.4 38 16.2 (7) 41 (4) 32 (5) 23 (4) 15.5 51 16.0 (8) 17 (4) 15 (7) 8 (4) 19.4 67 18 (1) 19 (5) 2 (5) 19 (5) 29.7 103 17.4 (7) 35 (3) 0 (5) 35 (3) 28.2 Furthermore, water molecules that are subjected to a stronger external electric field present a better alignment between their dipole and the field vector computed at their centre of mass (Table 1 ▸). Conversely, a weaker electric field corresponds to a larger observed angle. This trend can be observed for all the studied water molecules except for W13: excluding W13, the correlation coefficient between the electric field/dipole moment raw angle and the corresponding electric field values reaches 0.76, but it drops to 0.56 if W13 is included in the statistics (Fig. S2 in the supporting information). However, this can be explained by the peculiarities of the W13 environment. This conserved water molecule is tightly packed between the negatively charged carboxylate head of the FA and the basic side chain of Arg106, and is hence located in a region of strong external electric field. Moreover, it makes the shortest donor hydrogen-bond interactions of all considered water molecules (with the O atom of W20, O O = 2.69 Å, and with atom O2 of the fatty acid, O O = 2.68 Å) and among the shortest as hydrogen-bond acceptor (with the Arg106 and Thr40 side chains; Table S4 in the supporting information). This may indicate that the formation⋯ of strong hydrogen bonds, especially as hydrogen-bond⋯ donor, can overcome the torque effect of a misalignment with the external electric field. The opposite phenomenon can be illustrated by the case of water molecule W3, which is also subjected to a strong electric field but forms comparatively weaker donor hydrogen bonds (with the main-chain O atoms of Leu104 and Leu91, O O = 2.99 and 2.85 Å, respectively). Consequently, W3 shows a good alignment of its dipole moment with the electric field. It has⋯ been already shown by molecular dynamics simulations that the average reorientation time of water molecules located within 7 Å of the protein surface is significantly longer than that of bulk water (Rocchi et al., 1998 ▸). Hence, even if the orientation of water molecules depends on many factors, such as steric constraints or hydrogen bonding, the reorientation time may be increased by the restriction of the rotational freedom of interfacial water

9 molecules by the dipole/field alignment effect we characterize in this study. This result can be linked to the decrease in the relative dielectric constant of such water clusters when compared with bulk water. The relative weights of the hydrogen-bonding and dipole-alignment effects might vary according to each case, as shown by the comparison between W3 and W13, and it seems that, when a water molecule is not constrained by the formation of strong hydrogen bonds like W13, its tendency to align according to the felt electric field appears stronger. 3.2.3. Electrostatic interaction energies The intrinsic stability of the embedded water cluster was studied by evaluating the electrostatic interaction energies with their environment. In order to characterize the relative contribution of the ligand charge distribution to these energies, the computations were performed in two stages, with and without its contribution. A comparison of the electrostatic interaction energies (Table 2 ▸) in both situations confirms the stability of the water cluster. As expected, the two water molecules W13 and W24, which are strongly hydrogen-bonded and in close contact with the negatively charged carboxylate head of the FA, undergo a significant destabilization in the −1 −1 −1 absence of the ligand (ΔE elec = 11 and 16 kcal mol , respectively; 1 kcal mol = 4.184 kJ mol ). All the other water −1 molecules in the cluster show either a weak destabilization (largest ΔE elec = 2 kcal mol for W51) or a weak −1 stabilization (largest ΔE elec = −2 kcal mol for W20). Contrary to the cases of W13 and W24, whose destabilization appears to be clearly significant, the electrostatic interaction energies for the 12 other water molecules in the cluster vary by amounts that are lower than, or of the same order of magnitude as, the estimated errors on these quantities. These results agree with the cluster observed in the atomic resolution apo form of an adipocyte FABP4 (PDB code 3rzy; González & Fisher, 2015 ▸), which shows water molecules at similar positions to W3, W6, W7, W8, W17, W31, W26 and W51 (Fig. S3 in the supporting information) observed in the present study. Hence, it appears that, apart from W13 and W24, the water cluster is inherently stable, and from an electrostatic interaction energy perspective the presence of the FA does not significantly influence its stability. Again, this confirms that this water cluster is an inherent part of the H- FABP structure, and apart from the formation of hydrogen bonds with the polar head of the ligand, its role may be limited to an exclusion factor for ligands whose alkyl chain is too long (Matsuoka et al., 2015 ▸). Table 2 Electrostatic interaction energies (kcal mol−1) of the 14 water molecules in the cluster with their environment, computed with (left column) and without (right column) the FA charge-density contribution, along with their estimated uncertainties in parentheses Water E elec with E elec without molecule FA FA label contribution contribution 3 −29 (1) −30 (1) 6 −25 (3) −26 (2) 7 −26 (1) −27 (1) 8 −17 (1) −18 (1) 13 −33 (2) −22 (2) 17 −19 (2) −19 (2) 20 −29 (2) −31 (2) 24 −23 (2) −7 (2) 26 −20 (2) −21 (2) 30 −35 (1) −34 (2) 31 −7 (1) −8 (1) 51 −29 (2) −27 (2) 67 −17 (2) −16 (1) 103 −24 (3) −24 (3) 3.3. Ligand binding 3.3.1. Electrostatic environment of the fatty acid The electrostatic environment of the bound FA was also analysed. As expected for a negatively charged ligand, the electrostatic potential generated by the whole hydrated protein at the surface of the ligand is globally electropositive (Fig. 5 ▸). A clear electrostatic complementarity is observed, where the negatively charged FA carboxylate group interacts with the electropositive potential of the basic Arg126 and Arg106 side chains. Ruskamo and co-workers reported that, for their 0.93 Å resolution X-ray structure of A-FABP in complex with palmitate, Arg106 appeared unprotonated on one amine group, leading to a neutral side chain (Ruskamo et al., 2014 ▸). We do not observe the same phenomenon here: both arginine side chains are clearly protonated and contribute to a strong electropositive potential. As mentioned above, one side of the hydrocarbon U-shaped tail of the FA is in contact with the side chains of hydrophobic residues, where the electrostatic complementarity is less obvious, as the slightly positive charges of side-chain H atoms are in contact with similarly charged H atoms of the FA. These residues contribute to a weaker but still electropositive potential, nevertheless accommodating the low positive charges of the FA hydrocarbon H atoms. The sole exception occurs for the slightly electronegative environment of the ligand terminal methyl group. This is due to the nearby proximity of the

10 main-chain carbonyl O atoms of Thr53 and Lys58, which are involved in C—H O hydrogen bonds with the FA methyl group. In the ELMAMII modelling, methyl H atoms are slightly more positively charged than H atoms of CH2 types (partial charges are 0.041 and 0.037 e, respectively), resulting in a weak positive-charge⋯ accumulation at the terminal methyl group of the alkyl chain. Figure 5. 0.01 e Å−3 total electron-density isosurface of the FA in the binding pocket, mapped by the electrostatic potential (e/Å) generated by the whole protein, including explicit water molecules.

To summarize, we observe then a very fine electrostatic complementarity between the charge distribution characterizing long-chain FAs and various regions of the binding pocket. The complementarity observed in the methyl part of the FA may be linked to the better affinity of FABPs for ligands which are long enough to allow the ‘U-shaped’ conformation, bringing the terminal methyl to an electrostatically favourable region. 3.3.2. Topological analysis Intra- and intermolecular interactions can be precisely characterized and quantified by performing a topological analysis of electron-density distribution in the framework of the QTAIM approach, developed by Bader (1994 ▸). Studying bonding interactions in this approach consists of analysing the topology of the total electron density by searching ridges, termed bond paths, of maximal value between nuclei, mirroring lines of maximally negative potential energy density (Bader, 1998 ▸). On such an interatomic (actually internuclei) bond path lies a point of special importance, named the bond critical point (BCP), where the electron density displays a saddle- type curvature, i.e. is minimal along the bond path. It has been shown by Bader and coworkers, and exploited in numerous studies (Matta, 2007 ▸), that the existence of a bond path bridging atoms, and an associated BCP, is a ‘universal indicator of chemical bonding of all kinds: weak, strong, closed-shell, and open-shell interactions’ (Matta, 2 2007 ▸). Indeed, values of the electron density ρ(r cp) and of its Laplacian ρ(r cp)(i.e. the sum of its second derivatives) on the BCP allow one to determine the type of interaction and quantify its strength. For instance, covalent bonds are characterized by a negative value of the Laplacian, while in closed-shell∇ bonding (e.g. hydrogen bonds) the depletion of the electron density in the interatomic region leads to a positive Laplacian. The strengths of various types of closed-shell interactions, measured in terms of electron-density properties at the BCP, have been extensively studied by

Mata et al. (2010 ▸). In particular, they showed that their dissociation energies D e can be estimated from the value of the electronic potential energy density V(r cp) at the BCP (Espinosa & Molins, 2000 ▸), which is accessible from the 2 values of ρ(r cp) and ρ(r cp) using the Abramov formula (Abramov, 1997 ▸). In this study, the knowledge of an accurate total charge distribution, made up of precise nuclei positions (including H atoms)∇ and the transferred aspherical electron density, definitely makes the QTAIM approach the method of choice to analyse, at an atomic level, intermolecular interactions in the H-FABP complex. Hence, a topological analysis of the transferred electron density was performed to search for interatomic interactions between atoms of the bound ligand and its environment, i.e. of the protein and water molecules. All interactions found by locating a saddle BCP and an associated bond path are summarized in Table 3 ▸. We can distinguish four main categories of interatomic contacts involving the FA: (i) hydrogen bonds with carboxylate O atoms as acceptors; (ii) C—H O hydrogen bonds between FA H atoms and water O atoms; (iii) a C—H π hydrogen bond involving the π electrons of the oleic acid C=C double bond (oleic acid presents a single unsaturation at the ω9 position); and⋯ (iv) 35 contacts between H atoms, including two intramolecular ones. All ⋯hydrogen bonds shown by geometric criteria were confirmed by the localization of a bond path and a saddle critical point. 2 Residue atom FA atom Distance Å ρ(r cp) ρ(r cp) Inter molecularH H contacts Table 3 Summary of interactions involving the FA and ∇ ⋯ 11 HD2 Phe57 H131 1.91 0.08 (1) 1.0 (1) their topological properties: distances between HG22 Thr53 H182 2.12 0.055 (3) 0.87 (2) interacting atoms (Å), and values of electron density (in HB3 Ala75 H112 2.21 0.049 (3) 0.58 (3) e Å−3) and Laplacian (in e Å−5) at the corresponding HG21 Val25 H9 2.22 0.041 (3) 0.48 (4) bond critical point HG3 Pro38 H152 2.23 0.049 (3) 0.56 (4) Values in parentheses are standard errors obtained as HZ Phe16 H42 2.29 0.033 (4) 0.37 (4) described in the supplementary information. HD11 Leu117 H22 2.34 0.036 (2) 0.36 (2) HE2 Tyr19 H62 2.38 0.030 (2) 0.34 (3) HB1 Ala33 H122 2.43 0.032 (3) 0.39 (3) HB2 Lys58 H172 2.49 0.026 (2) 0.27 (3) HD12 Leu23 H72 2.59 0.022 (3) 0.29 (4) HB2 Lys58 H183 2.61 0.021 (3) 0.30 (3) HZ Phe16 H121 2.65 0.017 (2) 0.18 (2) HG2 Met20 H9 2.69 0.017 (1) 0.23 (1) HG21 Thr53 H162 2.72 0.014 (1) 0.19 (1) HG21 Thr36 H142 2.74 0.015 (1) 0.15 (1) HE1 Phe16 H41 2.75 0.0140 (9) 0.23 (2) HG11 Val25 H10 2.76 0.014 (1) 0.13 (2) HZ Phe16 H151 2.79 0.0141 (9) 0.16 (1) HB1 Ala33 H141 2.82 0.011 (1) 0.16 (2) HG22 Thr29 H10 2.82 0.0116 (4) 0.127 (8) HE2 Phe57 H111 2.83 0.012 (1) 0.123 (7) HG23 Thr60 H183 2.89 0.011 (1) 0.15 (2) HA Ala33 H141 2.91 0.009 (1) 0.13 (1) HE1 Phe16 H71 2.91 0.0107 (7) 0.139 (1) HD13 Leu117 H41 2.91 0.012 (1) 0.133 (8) HB3 Ser55 H171 2.93 0.0096 (6) 0.105 (5) HB3 Pro38 H171 2.95 0.0100 (6) 0.112 (4) HB1 Ala75 H132 3.18 0.0054 (5) 0.079 (4) HB Thr74 H61 3.24 0.0077 (3) 0.064 (2) HB Thr36 H141 3.30 0.0046 (2) 0.057 (2) HG3 Lys58 H111 3.34 0.0050 (4) 0.048 (3) HD23 Leu104 H32 3.56 0.0026 (1) 0.040 (2) Intra molecul ar H H contacts H162 Ola133 H31 2.57 0.021 (2) 0.27 (2) H21 Ola133 H151⋯ 3.08 0.007 (1) 0.082 (6) C-H π hydro gen bond HB3 Asp76 C9 2.79 0.039 (2) 0.38 (2) Hydroge⋅⋅⋅ n⋅ bonds with FA carboxylat e group acceptor atom HH Tyr128 O1 1.73 0.31 (3) 1.8 (2) HE Arg126 O1 1.85 0.229 (5) 2.03 (2) H2 W24 O2 1.86 0.25 (2) 1.62 (7) H2 W13 O2 1.90 0.20 (2) 2.01 (5) HH21 Arg126 O1 2.23 0.086 (5) 1.10 (7) HD23 Leu115 O1 3.12 0.0168 (3) 0.283 (7) C—H O hydro gen bond s involvingalkyl chainatoms O W7 H62 2.55 0.050 (5) 0.82 (6) O ⋅⋅ W⋅ 31 H181 2.55 0.054 (6) 0.7 (1) O W51 H51 2.63 0.049 (2) 0.80 (3) O Lys58 H183 2.82 0.033 (2) 0.56 (3) OD1 Asp76 H82 2.86 0.026 (2) 0.36 (3) O Thr53 H182 2.98 0.023 (2) 0.26 (2) O W31 H31 3.38 0.0074 (5) 0.117 (6) O W31 H52 3.46 0.0057 (3) 0.101 (6) O W28 H141 3.47 0.006 (1) 0.09 (1)

12 The carboxylate head of the FA accepts a total of six hydrogen bonds, whose bond paths and critical points are depicted in Fig. 6 ▸. Carboxylate atom O1 acts as acceptor in highly bifurcated hydrogen bonds, combining the very strong O—H O1 bond with the Tyr128 hydroxyl group, two N—H O1 bonds with the guanidinium group of Arg126, and a weak C—H O1 interaction with an H atom of the Leu115 side chain. On the other side, atom O2 interacts only with W24 and⋯W13 through O—H O2 hydrogen bonds. Three of these⋯ interactions present H O (1, 2) distances between 1.73 and⋯ 1.9 Å, reflecting the strength of the FA carboxylate-group binding in the FABP cavity. Using the relationship between the dissociation⋯ energy (D e) of a hydrogen bond and the electron density⋯ and Laplacian values at the corresponding BCP (Espinosa et al., 1998 ▸; Mata et al., 2010 ▸), the total D e of the interactions involving the FA polar head reaches 35 kcal mol−1. The enthalpy gain upon oleic acid binding by H-FABP, measured by calorimetric methods (Matsuoka et al., 2015 ▸), reaches ΔH = −20.9 kcal mol−1; even if this value cannot be directly compared with the estimated total D e, their relative magnitudes indicate clearly the preeminent contribution to the protein–ligand binding affinity of these few interactions involving the polar head of the FA. Figure 6. Bond critical points and associated bond paths (pictured in green) of hydrogen bonds involving the O atoms of the FA carboxylate head.

For such a molecule containing numerous CH groups, the formation of the C—H O hydrogen bonds commonly encountered in proteins is favoured, especially with water O atoms, and therefore could be expected to be a significant contributor to the stabilization of the FA alkyl chain (Sarkhel & Desiraju, 2004 ▸). However,⋯ this is not the case here, as the ligand alkyl chain is mostly in contact with the H atoms of the hydrophobic residues pointing into the binding pocket. This is the case for all FA C atoms except C1–C6, which line the cavity occupied by the water cluster, and methyl atom C18, which ends up near W31 due to the U-shaped fold of the alkyl chain bringing the FA tail close to its head. As a consequence, the FA tail forms only nine interatomic contacts of C—H O type located by the mean of a bond path and a saddle BCP (Table 3 ▸). Among these nine contacts, three involving the O atoms of W31 and W28 present long H O distances (H O > 3.3 Å) and consequently low electron-density⋯ values at the corresponding BCP −3 [ρ(r cp) < 0.008 e Å ]. However, it must be noted that these weak contacts were located, with similar electron-density values at the BCP,⋯ in all the perturbed⋯ models accounting for uncertainties on atomic coordinates and charge-density parameters generated to estimate the standard error on the electron-density derived properties (see §2.4). For this reason, even if they can hardly be defined as true C—H O hydrogen bonds, they can nevertheless be considered as actual water–ligand weak stabilizing interactions. Using the potential energy density, which can be estimated from ρ(r cp) and 2 −1 ρ(r cp) at their BCP, each of these weak contacts⋯ presents a bond energy of 1 kcal mol , i.e. about 30% of that of a standard C—H O hydrogen bond. The other six C—H O interactions display H O distances between 2.55 and 2.86 Å,∇ so they satisfy the distance criteria defining C—H O hydrogen bonds (Sarkhel∼ & Desiraju, 2004 ▸). Three of these interactions involve⋯ the H atoms of the FA methyl group,⋯ interacting with, respectively,⋯ the Lys58 main chain, the Asp76 side chain and the W31 O atom. Six of the nine⋯ C—H O hydrogen bonds involve water H atoms, and for this category of interaction the role of W31 appears noteworthy. This sole water molecule is in fact responsible for three of the nine C—H O contacts and half of those involving water⋯ O atoms. W31 is indeed located, and properly oriented, at a position allowing it to interact with both ends of the FA alkyl chain: two weak C—H O contacts with FA atoms H31 and H52 bound⋯ to, respectively, atoms C3 and C5 (i.e. located near the polar head), and a C—H O hydrogen bond with atom H181 of the FA methyl terminal group. Hence, it appears that W31 is ideally positioned⋯ to stabilize the U-shaped conformation of the FA, by bridging the tail of the molecule to a position located near its head, as⋯ seen in Fig. 7 ▸(a).

13 Figure 7. (a) Bond critical points and associated bond paths of H H (light blue), C—H O (red) and C—H π (green) hydrogen bonds. For⋯ the sake of clarity, only⋯ protein atoms involved⋯ in the interactions are represented (as grey spheres for H atoms and red spheres for O atoms). Hydrogen bonds involving the carboxylate group of the FA are represented in Fig. 6 ▸ and thus omitted from this picture. (b) Bond critical points and associated bond paths of H H bonds between the FA and Phe16 side chain (light blue), and (a) (b) FA intramolecular⋯ H H bonds (red). The most striking feature of the topological analysis of interactions between the FA and the protein is the presence of 35 C—Hδ+ δ+H—C intermolecular interactions between the H atoms of the oleic acid molecule⋯ and of the hydrophobic side chains, as well as two intramolecular ones linking pairs of H atoms located at both ends of the alkyl chain (Table 3 ▸). These⋯ interactions form an intricate network, visible through their curved associated bond paths, represented along their corresponding critical points in Fig. 7 ▸(a). These 35 H H contacts involve 27 of the 33 H atoms of the FA, meaning that some of them present a bifurcated geometry (H9, H10, H41, H111, H171 and, again, H183, which is also involved in contacts with the Lys58 main-chain O atom), and⋯ a trifurcated one for H141. However, they are distributed evenly along the alkyl chain, from H22 on atom C2 to those of the C18 terminal methyl group. Obviously, the presence of these contacts is a direct consequence of the packing of the FA on the hydrophobic side of the binding pocket. As expected, a large majority (27) of the 35 H H contacts shown by the presence of a bond path display internuclei distances larger than the sum of the H atoms’ van der Waals radii (r H 1.2 Å; Bondi, 1964 ▸). −3 2 These interactions are characterized by ρ(r cp) values between 0.002⋯ and 0.03 e Å , while the ρ(r cp) values lie in the range 0.04–0.4 e Å−5. Again, the low range in electron density value appears very small, but≃ these interactions were found topologically in each of the models used to represent the degrees of uncertainty of the atomic∇ coordinates. Such contacts, whose internuclei distance is larger than twice the H-atom van der Waals radius, can be classified as weak stabilizing van der Waals interactions (Wolstenholme & Cameron, 2006 ▸). Even if, individually, each of the weak H H interactions contributes only moderately, they may collectively have a significant impact on the H-FABP–FA binding energy. This is in agreement with the observation by Matsuoka et al. (2015 ▸) that the enthalpic gain upon FA binding⋯ by H-FABP tends to increase with the size of the alkyl chain, up to a chain-length limit imposed by the stable water cluster. The shortest H H contact (H H = 2.57 Å) falling within this category is intramolecular, between atoms H31 and H161 located near, respectively, the head and the tail of the FA. Hence, similar to the C—H O interactions with the W31 O atom, this H⋯H interaction⋯ could contribute to stabilizing the closed conformation of the fatty acid. The Phe16 residue forms the largest number of such ‘long’ H H bonds with the FA tail (Fig. 7 ▸ b).⋯ Its side chain points to the top of the pseudo-si⋯ face of the FA, perpendicular to the plane formed by atoms C1–C16 defining the U conformation. The FA wraps around a line going through the⋯ Phe16 CG and CZ atoms, locating its HZ and HE1 atoms at less than 3 Å from the H atoms on atoms C4, C7, C12 and C15 of the FA. This structural arrangement allowed the suggestion by Zanotti and co-workers that Phe16 ‘may be a key determinant in FA specificity and affinity in M-FABP’ (Zanotti et al., 1992 ▸). This was later confirmed by directed mutagenesis experiments, where Phe16 was mutated into tyrosine, serine (which are less prone to forming H H bonds due to the presence of the polar hydroxyl H atom) or valine (which is significantly less bulky than phenylalanine) residues, resulting in all cases in a significant drop in the oleic acid binding activity (Volkov et al., 2004 ▸). In⋯ the present study, the observed H H bonds are favoured by the orientation of the Phe16 side chain with respect to the U-shaped FA, as shown by the BCPs and bond paths represented in Fig. 7 ▸(b). Again, the position of the Phe16 side chain favouring the formation of several⋯ H H bonds with both sides of the FA alkyl chain may be an explanation, at a detailed atomic level, of the important role of Phe16 in FA binding in the FABP binding pocket. ⋯ Among these H H interactions listed in Table 3 ▸, ten are especially noteworthy as they present internuclear distances lower than the sum of the van der Waals radii of the interacting H atoms, so that they could be considered as ‘steric non-bonded repulsive’,⋯ while being counterbalanced by the other stabilizing H H contacts which appear, in this structure, to be more numerous. However, such H H interactions (not to be confused with dihydrogen bonding or hydride bonds) have already been studied by means of the AIM theory. Matta and co-workers⋯ showed that these ⋯

14 interactions, where two H atoms bearing the same or similar weak positive charges (typically C—H hydrogen atoms) come in close proximity to allow the formation of a bond path, lead to a local stabilizing contribution to the molecular energy (Matta et al., 2003 ▸). This stabilizing contribution has also been shown by other studies. Wolstenholme & Cameron (2006 ▸) compared the topological properties of H H bonds with those of conventional hydrogen bonds, and classified them as weak favourable interactions (Koch & Popelier, 1995 ▸). The relationship existing between the number of H H bonds formed between branched alkanes and⋯ their corresponding boiling points has been shown (Monteiro & Firme, 2014 ▸). In the present study, H H bonds found by topological analysis of the total electron density fall within⋯ the stabilizing interactions shown by Matta et al., with internuclear distances less than 2.4 Å, and 2 −3 −5 electron density ρ(r cp) and Laplacian ρ(r cp) at the⋯ BCPs greater than 0.03 e Å and 0.3 e Å , respectively. From this point of view, one could consider that these short H H bonds contribute to stabilizing the FA conformation, and consequently also to favouring its binding∇ with H-FABP. Conclusions 4. ⋯ In this study we have used both X-ray and neutron diffraction data to determine the structure of an H-FABP–oleic acid complex at room temperature. The use of a tiny perdeuterated crystal (0.05 mm3) allowed us to locate the deuterium atoms of the ordered water molecule cluster bound inside an internal pocket, together with the FA. On the basis of this structure, we have then performed electrostatic calculations and electron-density topological analysis using a transferred aspherical charge distribution to analyse the internal water cluster and the interactions between the bound FA, the water molecules and the protein atoms. From this analysis, we can extract three main conclusions: (i) The internal cluster of 14 water molecules presents an inherent stability and seems to contribute moderately to the stabilization of the FA binding by the formation of a few weak C—H O interactions. This agrees with recent results (Matsuoka et al., 2015 ▸) suggesting that the role of this cluster is to discriminate between correctly sized and too long FAs, or too rigid ligands, rather than a stabilizing one. However, the⋯ structurally conserved water molecule W31 is ideally positioned to interact with both ends of the FA, presumably contributing to stabilizing its U-shaped conformation. (ii) On the basis of the transferred charge distribution, we observed a striking electrostatic complementarity between the binding pocket and the bound FA, especially for the carboxylate head and the terminal methyl group. The aliphatic tail of the FA is mostly in contact with hydrophobic residues, allowing the formation of numerous intermolecular H H bonds as well as two intramolecular ones, revealed by the presence of BCPs and bond paths. Most of these H H bonds can be classified as weak van der Waals interactions and together they contribute collectively to the stabilization of⋯ the observed FA conformation. (iii)⋯ Within the cluster, the positions and orientations of the water molecules are strongly determined by the alignment of the water dipoles along the electrostatic field of the hydrated protein. The hydration layers around proteins fulfil multiple roles and can have several states, which are difficult to study in three dimensions because of the inherent disorder in the transition to bulk water. By focusing on the internal water cluster of H-FABP, we have been able to observe in high detail the alignment of the water dipoles with the surrounding electrostatic field. This point might possibly be extrapolated to the ensemble of the hydration layers, explaining the observation that the mobility of water molecules in these layers is strongly restricted and therefore significantly different from bulk water, in which there is no defined orientation (the mean dipole moment is zero). The alignment of the water dipoles along the electrostatic field could give particular properties to protein hydration layers, extending and eventually modulating the electrostatic properties of the protein surface. Note that this should in particular be the case during the formation of protein complexes, since hydration water molecules become confined in the interface between the protein surfaces, and therefore should have properties similar to those observed in the internal cavity of the FABPs. Such strong alignment implies a much lower dielectric constant, and gives a structural basis to the longer-range electrostatic interactions necessary for the formation of protein complexes. IUCrJ. 2016 Jan 16;3(Pt 2):115-26.5CE4

1G5WNMR-A2-133[»] FABP3Humanheart 1HMRX-ray1.40A2-133[»]1HMSX-ray1.40A2-133[»]1HMTX-ray1.40A2-133[»]2HMBX-ray2.10A2-133[»] 3RSWX-ray2.60A/B1-133[»]3WBGX-ray2.15A/B/C/D1-133[»]3WVMX-ray0.88A1-133[»]3WXQX-ray1.60A1-133[»] 4TJZX-ray0.87A1-133[»]4TKBX-ray0.86A1-133[»]4TKHX-ray0.93A1-133[»]4TKJX-ray0.87A1-133[»] 4WBKX-ray1.37A1-133[»] 5B27X-ray1.02A1-133[»]5B28X-ray0.90A1-133[»]5B29X-ray1.28A2-133[»] 5CE4Other0.98A1-132[»]5HZ9X-ray2.30A/B/C/D/E/F/G/H1-133[»][»] FABP3 Human heart 3WVM(0-132,1-133)3WXQX-ray1.60A1-133[»] FABP3 Human heart

15 O O O C O

STE STEARIC ACID C18 H36 O2 ; 5 HOH *174(H2 O) P6G HEXAETHYLENE GLYCOL 2(C12 H26 O7) POLYETHYLENE GLYCOL PEG400 HELIX 11VALA 1LEUA 55 5 HELIX 22ASNA15LEUA231 9 HELIX 33GLYA26SERA341 9 SHEET 1 A10THRA 60 LYSA 65 0 SHEET 2 A10ILEA 48 HISA 54-1 N LEUA 49 O PHEA 64 SHEET 3 A10THRA 39 ASNA 45-1 N THRA 39 O HISA 54 SHEET 4 A10GLYA 6 LYSA 14-1 N TRPA 8 O THRA 40 SHEET 5 A10ALAA122 GLUA131-1 O GLUA131 N THRA 7 SHEET 6 A10LYSA112 HISA119-1 N LEUA115 O ARGA126 SHEET 7 A10GLNA100 ILEA109-1 N GLUA107 O ILEA114 SHEET 8 A10LYSA 90 TRPA 97-1 N GLNA 95 O THRA102 SHEET 9 A10LYSA 79 ASPA 87-1 N THRA 85 O VALA 92 SHEET 10 A10PHEA 70 THRA 73-1 N PHEA 70 O SERA 82 SITE 1AC1 7THRA 53 LYSA 58 LEUA115 ARGA126 SITE 2 AC1 7 TYR A 128 HOH A1083 HOH A1094 SITE 1AC212VALA 11 SERA 34 META 35 LYSA 37 SITE 2AC212PHE A 70 ASPA 71 GLYA 120 THR A121 SITE 3 AC2 12 HOH A1026 HOH A1101 HOH A1102 HOH A1106 SITE 1AC3 6GLNA 31 META 35 ASPA 47 LYSA 65 SITE 2 AC3 6 GLY A 111 HOH A1153 5CE4(1-132.1-133),2IFB,1T8V,1SA8,1IFB,1DC9,1A57,1ICM(2-132.1-132) FABP3 Human heart H H H

H H H H H H H H O H O H H H H O H

H HH H H H H H H H H H H H H H H OLA OLEIC ACID C18 H34 O2 H 3 HOH *179(H2 O) HELIX 1AA1VALA 1LEUA 55 5 HELIX 2AA2ASNA15LEUA231 9 HELIX 3AA3GLYA 26META 351 10 SHEET 1AA110THRA 60 PHEA 64 0 SHEET 2AA110ILEA 48 HISA 54-1 N LEUA 49 O PHEA 64 SHEET 3AA110THRA 39 ASNA 45-1 N THRA 39 O HISA 54 SHEET 4AA110GLYA 6 LYSA 14-1 N TRPA 8 O THRA 40 SHEET 5 AA110 ALA A 122 LYS A 130 -1 O THR A 127 N VAL A 11 SHEET 6 AA110 LYS A 112 HIS A 119 -1 N LEU A 115 O ARG A 126 SHEET 7 AA110 GLN A 100 ILE A 109 -1 N GLU A 107 O ILE A 114 SHEET 8AA110LYSA 90 TRPA 97-1 N TRPA 97 O GLNA100 SHEET 9AA110LYSA 79 ASPA 87-1 N THRA 85 O VALA 92 SHEET 10AA110PHEA 70 THRA 73-1 N PHEA 70 O SERA 82 SITE 1AC1 7THRA 53 LYSA 58 LEUA115 ARGA126 SITE 2 AC1 7 TYR A 128 HOH A 323 HOH A 350

16 O C O O 4TJZ(0-132,1-133) DKA DECANOIC ACID C10 H20 O2 FABP3 Human heart P6G HEXAETHYLENE GLYCOL POLYETHYLENE GLYCOL PEG400 2(C12 H26 O7) HELIX 1AA1VALA 1LEUA 55 5 HELIX 2AA2ASNA15LEUA231 9 HELIX 3AA3GLYA26SERA341 9 SHEET 1AA110THRA 60 LYSA 65 0 SHEET 2AA110ILEA 48 HISA 54-1 N LEUA 49 O PHEA 64 SHEET 3AA110THRA 39 ASNA 45-1 N THRA 39 O HISA 54 SHEET 4AA110GLYA 6 LYSA 14-1 N TRPA 8 O THRA 40 SHEET 5AA110ALAA122 GLUA131-1 O GLUA131 N THRA 7 SHEET 6 AA110 LYS A 112 HIS A 119 -1 N LEU A 115 O ARG A 126 SHEET 7 AA110 GLN A 100 ILE A 109 -1 N GLU A 107 O ILE A 114 SHEET 8AA110LYSA 90 TRPA 97-1 N TRPA 97 O GLNA100 SHEET 9AA110LYSA 79 ASPA 87-1 N ILEA 83 O LEUA 94 SHEET 10AA110PHEA 70 THRA 73-1 N PHEA 70 O SERA 82 SITE 1 AC1 5 LEU A 115 ARG A 126 TYR A 128 HOH A 430 SITE 2AC1 5HOHA442 SITE 1AC213VALA 11 SERA 34 META 35 THRA 36 SITE 2AC213LYSA 37 PHEA 70 ASPA 71 GLYA120 SITE 3 AC2 13 THR A 121 HOH A 324 HOH A 334 HOH A 344 SITE 4 AC2 13 HOH A 462 SITE 1AC3 5GLNA 31 ASPA 47 LYSA 65 GLYA111

O C O O O O 4TKB(0-132,1-133) FABP3 Human heart DAO LAURIC ACID C12 H24 O2 P6G HEXAETHYLENE GLYCOL POLYETHYLENE GLYCOL PEG400 2(C12 H26 O7) HELIX 1AA1VALA 1LEUA 55 5 HELIX 2AA2ASNA15LEUA231 9 HELIX 3AA3GLYA26SERA341 9 SHEET 1AA110THRA 60 LYSA 65 0 SHEET 2AA110ILEA 48 HISA 54-1 N LEUA 49 O PHEA 64 SHEET 3AA110THRA 39 ASNA 45-1 N THRA 39 O HISA 54 SHEET 4AA110GLYA 6 LYSA 14-1 N TRPA 8 O THRA 40 SHEET 5 AA110 ALA A 122 LYS A 130 -1 O THR A 127 N VAL A 11 SHEET 6 AA110 LYS A 112 HIS A 119 -1 N LEU A 113 O TYR A 128 SHEET 7 AA110 GLN A 100 ILE A 109 -1 N GLU A 107 O ILE A 114 SHEET 8AA110LYSA 90 TRPA 97-1 N TRPA 97 O GLNA100 SHEET 9AA110LYSA 79 ASPA 87-1 N ILEA 83 O LEUA 94 SHEET 10AA110PHEA 70 THRA 73-1 N PHEA 70 O SERA 82 SITE 1AC1 9THRA 29 PHEA 57 LYSA 58 ALAA 75 SITE 2 AC1 9 LEU A 115 ARG A 126 TYR A 128 HOH A 449 SITE 3AC1 9HOHA454 SITE 1AC213VALA 11 SERA 34 META 35 THRA 36 SITE 2AC213LYSA 37 PHEA 70 ASPA 71 GLYA120 SITE 3 AC2 13 THR A 121 HOH A 319 HOH A 325 HOH A 342 SITE 4 AC2 13 HOH A 472 SITE 1AC3 6GLNA 31 ASPA 47 LYSA 65 GLYA111 SITE 2AC3 6HOH A386 HOHA413

17 O

O O C 4TKH(0-132,1-133) MYR MYRISTIC ACID C14 H28 O2 FABP3 Human heart P6G HEXAETHYLENE GLYCOL POLYETHYLENE GLYCOL PEG400 C12 H26 O7 HELIX 1AA1VALA 1LEUA 55 5 HELIX 2AA2ASNA15LEUA231 9 HELIX 3AA3GLYA 26META 351 10 SHEET 1AA110THRA 60 PHEA 64 0 SHEET 2AA110ILEA 48 HISA 54-1 N LEUA 49 O PHEA 64 SHEET 3AA110THRA 39 ASNA 45-1 N THRA 39 O HISA 54 SHEET 4AA110GLYA 6 LYSA 14-1 N TRPA 8 O THRA 40 SHEET 5AA110ALAA122 GLUA131-1 O GLUA131 N THRA 7 SHEET 6 AA110 LYS A 112 HIS A 119 -1 N LEU A 115 O ARG A 126 SHEET 7 AA110 GLN A 100 ILE A 109 -1 N GLU A 107 O ILE A 114 SHEET 8AA110LYSA 90 TRPA 97-1 N LEUA 91 O ARGA106 SHEET 9AA110LYSA 79 ASPA 87-1 N THRA 85 O VALA 92 SHEET 10AA110PHEA 70 THRA 73-1 N PHEA 70 O SERA 82 SITE 1 AC1 5 LEU A 115 ARG A 126 TYR A 128 HOH A 426 SITE 2AC1 5HOHA433 SITE 1AC211VALA 11 SERA 34 LYSA 37 PHEA 70 SITE 2 AC2 11 ASP A 71 GLY A 120 THR A 121 HOH A 313 SITE 3 AC2 11 HOH A 321 HOH A 338 HOH A 411 FABP3 Human heart

O

O O O

4TKJ(0-132,1-133) FABP3 Human heart PLM PALMITIC ACID C16 H32 O2;P6G HEXAETHYLENE GLYCOL 2(C12 H26 O7) GLP GLUCOSAMINE 6-PHOSPHATE C6 H14 N O8 P ; P6G POLYETHYLENE GLYCOL PEG400 HELIX 1AA1VALA 1LEUA 55 5 HELIX 2AA2ASNA15LEUA231 9 HELIX 3AA3GLYA26SERA341 9 SHEET 1AA110THRA 60 LYSA 65 0 SHEET 2AA110ILEA 48 HISA 54-1 N LEUA 49 O PHEA 64 SHEET 3AA110THRA 39 ASNA 45-1 N THRA 39 O HISA 54 SHEET 4AA110GLYA 6 LYSA 14-1 N GLYA 6 O ILEA 42 SHEET 5 AA110 ALA A 122 LYS A 130 -1 O THR A 127 N VAL A 11 SHEET 6 AA110 LYS A 112 HIS A 119 -1 N LEU A 115 O ARG A 126 SHEET 7 AA110 GLN A 100 ILE A 109 -1 N GLU A 107 O ILE A 114 SHEET 8AA110LYSA 90 TRPA 97-1 N TRPA 97 O GLNA100 SHEET 9AA110LYSA 79 ASPA 87-1 N THRA 85 O VALA 92 SHEET 10AA110PHEA 70 THRA 73-1 N PHEA 70 O SERA 82 SITE 1AC1 7THRA 53 LYSA 58 LEUA115 ARGA126 SITE 2 AC1 7 TYR A 128 HOH A 448 HOH A 454 SITE 1AC2 9VALA 11 SERA 34 LYSA 37 ASPA 71 SITE 2 AC2 9 GLY A 120 HOH A 309 HOH A 335 HOH A 387 SITE 3AC2 9HOHA480 SITE 1AC3 6GLNA 31 ASPA 47 LYSA 65 GLYA111 SITE 2AC3 6LYS A130 HOHA308 SITE 1AC413VALA 11 ASPA 12 LYSA 14 LYSA 58

18 SITE 2AC413ASNA 59 THRA 73 THRA 74 ALAA 75 SITE 3 AC4 13 ASP A 77 HOH A 313 HOH A 323 HOH A 368 SITE 4 AC4 13 HOH A 378 FABP3 Human heart 3WVM(0-132.1-133) FABP3 Human heart O O O C O

STE STEARIC ACID C18 H36 O2 P6G HEXAETHYLENE GLYCOL POLYETHYLENE GLYCOL PEG400 2(C12 H26 O7) HELIX 11VALA 1LEUA 55 5 HELIX 22ASNA15LEUA231 9 HELIX 33GLYA26SERA341 9 SHEET 1 A10THRA 60 LYSA 65 0 SHEET 2 A10ILEA 48 HISA 54-1 N LEUA 49 O PHE A 64 SHEET 3 A10THRA 39 ASNA 45-1 N THRA 39 O HIS A 54 SHEET 4 A10GLYA 6 LYSA 14-1 N TRPA 8 O THR A 40 SHEET 5 A10 ALA A 122 GLU A 131 -1 O GLUA131 N THRA 7 SHEET 6 A10 LYS A 112 HIS A 119 -1 N LEU A 115 O ARG A 126 SHEET 7 A10 GLN A 100 ILE A 109 -1 N GLU A 107 O ILE A 114 SHEET 8 A10LYSA 90 TRPA 97-1 N GLNA 95 O THR A 102 SHEET 9 A10LYSA 79 ASPA 87-1 N THRA 85 O VAL A 92 SHEET 10 A10PHEA 70 THRA 73-1 N PHEA 70 O SER A 82 SITE 1AC1 7THRA 53 LYSA 58 LEUA115 ARGA126 SITE 2 AC1 7 TYR A 128 HOH A1083 HOH A1094 SITE 1AC212VALA 11 SERA 34 META 35 LYSA 37 SITE 2AC212PHE A 70 ASPA 71 GLYA 120 THR A121 SITE 3 AC2 12 HOH A1026 HOH A1101 HOH A1102 HOH A1106 SITE 1AC3 6GLNA 31 META 35 ASPA 47 LYSA 65 SITE 2 AC3 6 GLY A 111 HOH A1153 3WBG(1-133)3WBG(1-133.1-133) 2HMB,5CE4(2-133.1-133) FABP3 Human heart 2AN ABCD 201 8-ANILINO-1-NAPHTHALENE SULFONATE 4(C16 H13 N O3 S) FABP3 Human heart

H O N O S O

9 HOH *174(H2 O) HELIX 11META 0LEUA 55 6 HELIX 22ASNA15LEUA231 9 HELIX 33GLYA26THRA361 11 SHEET 1 A10THRA 60 PHEA 64 0 SHEET 2 A10ILEA 48 HISA 54-1 N LEUA 49 O PHE A 64 SHEET 3 A10THRA 39 ASNA 45-1 N THRA 39 O HIS A 54 SHEET 4 A10GLYA 6 LYSA 14-1 N TRPA 8 O THR A 40 SHEET 5 A10 ALA A 122 LYS A 130 -1 O GLUA129 N LYSA 9 SHEET 6 A10 LYS A 112 HIS A 119 -1 N LEU A 115 O ARG A 126 SHEET 7 A10 GLN A 100 ILE A 109 -1 N GLU A 107 O ILE A 114 SHEET 8 A10LYSA 90 TRPA 97-1 N LEUA 91 O ARG A 106 SHEET 9 A10LYSA 79 ASPA 87-1 N ASPA 87 O LYS A 90 SHEET 10 A10PHEA 70 THRA 73-1 N PHEA 70 O SER A 82 SITE 1AC1 8TYRA 19 LEUA 23 ALAA 33 THRA 36 SITE 2AC1 8SERA 55 ASPA 76 HOHA334 HOHA336

19 O O C O

1HMT(1-131,2-133)STE STEARIC ACID C18 H36 O2 FABP3 Human heart HELIX 1AIPHEA16LEUA231 8 HELIX 2AIIPHEA27VALA321 6 SHEET 1 A4LEUA 5 LYSA 14 0 SHEET 2 A4PROA 38 LYSA 44-1 N THRA 40 O TRP A 8 SHEET 3 A4LEUA 49 HISA 54-1 N LYSA 52 O ILE A 41 SHEET 4 A4ASNA 59 PHEA 64-1 N ILEA 62 O LEU A 51 SHEET 1 B6GLUA 69 THRA 73 0 SHEET 2 B6ARGA 78 ASPA 87-1 N VALA 80 O GLU A 72 SHEET 3 B6LEUA 91 LYSA 96-1 N VALA 92 O THR A 85 SHEET 4 B6GLUA101 LEUA108-1 N LEUA104 O HIS A 93 SHEET 5 B6LEUA113 THRA118-1 N THRA116 O VAL A 105 SHEET 6 B6VALA123 GLUA131-1 N ARGA126 O LEU A 115 SITE 1AC1 8ALAA 33 SERA 55 LYSA 58 LEUA115 SITE 2 AC1 8 ARG A 126 TYR A 128 HOH A 139 HOH A 140 O O O

O

1HMS(1-131,2-133) OLA OLEIC ACID C18 H34 O2 FABP3 Human heart HELIX 1AIPHEA16LEUA231 8 HELIX 2AIIPHEA27VALA321 6 SHEET 1 A4LEUA 5 LYSA 14 0 SHEET 2 A4PROA 38 LYSA 44-1 N THRA 40 O TRP A 8 SHEET 3 A4LEUA 49 HISA 54-1 N LYSA 52 O ILE A 41 SHEET 4 A4ASNA 59 PHEA 64-1 N ILEA 62 O LEU A 51 SHEET 1 B6GLUA 69 THRA 73 0 SHEET 2 B6ARGA 78 ASPA 87-1 N VALA 80 O GLU A 72 SHEET 3 B6LEUA 91 LYSA 96-1 N VALA 92 O THR A 85 SHEET 4 B6GLUA101 LEUA108-1 N LEUA104 O HIS A 93 SHEET 5 B6LEUA113 THRA118-1 N THRA116 O VAL A 105 SHEET 6 B6VALA123 GLUA131-1 N ARGA126 O LEU A 115 SITE 1AC1 7ALAA 33 LYSA 58 LEUA115 ARGA126 SITE 2 AC1 7 TYR A 128 HOH A 152 HOH A 174

O

O O C O 1HMR(1-131,2-133)ELA 9-OCTADECENOIC ACID C18 H34 O2 FABP3 Human heart HELIX 1AIPHEA16LEUA231 8 HELIX 2AIIPHEA27VALA321 6 SHEET 1 A4LEUA 5 LYSA 14 0 SHEET 2 A4PROA 38 LYSA 44-1 N THRA 40 O TRP A 8 SHEET 3 A4LEUA 49 HISA 54-1 N LYSA 52 O ILE A 41

20 SHEET 4 A4ASNA 59 PHEA 64-1 N ILEA 62 O LEU A 51 SHEET 1 B6GLUA 69 THRA 73 0 SHEET 2 B6ARGA 78 ASPA 87-1 N VALA 80 O GLU A 72 SHEET 3 B6LEUA 91 LYSA 96-1 N VALA 92 O THR A 85 SHEET 4 B6GLUA101 LEUA108-1 N LEUA104 O HIS A 93 SHEET 5 B6LEUA113 THRA118-1 N THRA116 O VAL A 105 SHEET 6 B6VALA123 GLUA131-1 N ARGA126 O LEU A 115 SITE 1AC1 7ALAA 33 THRA 53 LYSA 58 ARGA126 SITE 2 AC1 7 TYR A 128 HOH A 152 HOH A 174 10 20 30 40 50 60 70 80 MVDAFLGTWK LVDSKNFDDY MKSLGVGFAT RQVASMTKPT TIIEKNGDIL TLKTHSTFKN TEISFKLGVE FDETTADDRK 90 100 110 120 130133 VKSIVTLDGG KLVHLQKWDG QETTLVRELI DGKLILTLTH GTAVCTRTYE KEA FABP3 Human heart 2F73X-ray2.50A/B/C/D/E/F/G/H1-127[»]2L67NMR-A2-127[»]2L68NMR-A2-127[»]2LKKNMR-A2-127[»]2PY1NMR-A1-127[»] 3B2HX-ray1.55A2-127[»]3B2IX-ray1.86A2-127[»]3B2JX-ray2.00A2-127[»]3B2KX-ray1.73A2-127[»]3B2LX-ray2.25A2-127[»] 3STKX-ray1.55A2-127[»]3STMX-ray2.22X2-127[»]3STNX-ray2.60A2-127[»]3VG2X-ray2.40A2-127[»]3VG3X-ray2.22A2-127[»] 3VG4X-ray2.50A2-127[»]3VG5X-ray2.00A2-127[»]3VG6X-ray2.22A2-127[»]3VG7X-ray1.44A2-127[»] FABPL Human liver 3STK-N-M(2-127.1-127) pH=6.8,1.8 Å FABPL Human liver PLM 130 131 PALMITIC ACID 2(C16 H32 O2) HELIX 11ASNA14GLYA231 10 HELIX 22PROA25LYSA331 9 SHEET 1 A9LYSA 57 THRA 64 0 SHEET 2 A9HISA 47 ALAA 54-1 N ALAA 54 O LYSA 57 SHEET 3 A9VALA 38 ASNA 44-1 N VALA 42 O LYSA 49 SHEET 4 A9GLYA 5 GLUA 13-1 N GLYA 5 O ILEA 41 SHEET 5 A9ILEA118 ARGA126-1 O ILEA123 N GLNA 10 SHEET 6 A9ILEA108 LEUA115-1 N META113 O PHEA120 SHEET 7 A9ILEA 98 ASNA105-1 N ASNA105 O ILEA108 SHEET 8 A9LYSA 90 PHEA 95-1 N LEUA 91 O THRA102 SHEET 9 A9GLNA 84 LEUA 85-1 N GLNA 84 O VALA 92 SHEET 1 B2GLUA 68 GLUA 72 0 SHEET 2 B2LYSA 78 VALA 82-1 O THRA 81 N CYSA 69 SITE 1AC1 8SERA 39 ILEA 52 GLUA 72 THRA102 SITE 2 AC1 8 ASN A 111 ARG A 122 PLM A 131 HOH A 227 SITE 1AC2 5ALAA 54 SERA 56 ASNA111 ARGA122 SITE 2AC2 5PLMA130 10 20 30 40 50 MSFSGKYQLQ SQENFEAFMK AIGLPEELIQ KGKDIKGVSE IVQNGKHFKF 60 70 80 90 100 TITAGSKVIQ NEFTVGEECE LETMTGEKVK TVVQLEGDNK LVTTFKNIKS 110 120 127 VTELNGDIIT NTMTLGDIVF KRISKRI FABPL Human liver 2V1K(2-154.1-154) pH=6.8 HEM PROTOPORPHYRIN IX CONTAINING FE [DIHYDROGEN 3,7,12,17-TETRAMETHYL-8,13- DIVINYL-2,18-PORPHINEDIPROPIONATO(2-)]IRON C34 H32 FE N4 O4 SO4 2(O4 S 2-) GOL 2(C3 H8 O3) HELIX 11SERA 3ASPA201 18 HELIX 22ASPA20HISA361 17 HELIX 33PROA37PHEA435 7 HELIX 44THRA51SERA581 8 HELIX 55SERA58LYSA771 20 HELIX 66HISA82LYSA961 15 HELIX 7 7PROA100HISA1191 20 HELIX 8 8GLYA124GLYA1501 27 LINK FE HEMA1154 NE2HISA 93 1555 1555 2.14 SITE 1AC123THRA 39 LYSA 42 PHEA 43 LYSA 45 SITE 2AC123HISA 64 VALA 68 LEUA 89 SERA 92 SITE 3AC123HISA 93 HISA 97 ILEA 99 TYRA103 SITE 4 AC1 23 HIS A 113 HIS A 116 GLN A 128 PHE A 138 SITE 5 AC1 23 GOL A1158 HOH A2182 HOH A2183 HOH A2184 SITE 6 AC1 23 HOH A2185 HOH A2186 HOH A2193

21 SITE 1AC2 5LYSA 50 THRA 51 GLUA 52 HOHA2187 SITE 2 AC2 5 HOH A2188 SITE 1 AC3 3 GLY A 1 HOH A2190 HOH A2191 SITE 1AC4 8LYSA 45 HISA 64 HISA113 SERA117 SITE 2 AC4 8 HEM A1154 GOL A1159 HOH A2088 HOH A2192 SITE 1AC5 7ARGA 31 LYSA 45 HISA113 SERA117 SITE 2 AC5 7 GOL A1158 HOH A2088 HOH A2193 2V1K(2-154.1-154) pH=6.8 ,MBO(2-154.1-154) 1.8 ANGSTROMS HEM PROTOPORPHYRIN IX CONTAINING FE C34 H32 FE N4 O4 OXY OXYGEN MOLECULE O2 HELIX 1ASERA 3GLUA181 16 HELIX 2BASPA20SERA351 16 HELIX 3CHISA36LYSA421 7 HELIX 4DTHRA51ALAA571 7 HELIX 5ESERA58LYSA771 20 HELIX 6FLEUA86THRA951 10 HELIX 7 GPROA100ARGA1181 19 HELIX 8 HGLYA124LEUA1491 26 LINK NE2HISA 93 FE HEMA155 1555 1555 2.07 LINK FE HEMA155 O1 OXYA555 1555 1555 1.83 SITE 1AC1 4SERA 58 GLUA 59 ASPA 60 HOHA227 SITE 1AC222THRA 39 LYSA 42 PHEA 43 ARGA 45 SITE 2AC222HISA 64 VALA 68 LEUA 89 SERA 92 SITE 3AC222HISA 93 HISA 97 ILEA 99 TYRA103 SITE 4 AC2 22 LEU A 104 HOH A 163 HOH A 217 HOH A 229 SITE 5 AC2 22 HOH A 234 HOH A 269 HOH A 337 HOH A 469 SITE 6 AC2 22 HOH A 472 OXY A 555 SITE 1AC3 5PHEA 43 HISA 64 VALA 68 HEMA155 SITE 2AC3 5HOHA341 10 20 30 40 50 MGLSDGEWQQ VLNVWGKVEA DIAGHGQEVL IRLFTGHPET LEKFDKFKHL 60 70 80 90 100 KTEAEMKASE DLKKHGTVVL TALGGILKKK GHHEAELKPL AQSHATKHKI 110 120 130 140 150 PIKYLEFISD AIIHVLHSKH PGDFGADAQG AMTKALELFR NDIAAKYKEL 154 GFQG 1LFO,2JU3-7-8(1-127,1-127), 3GSH, 1LIP, (1-91=27-117,1-117), ASY (12E)-10-OXOOCTADEC-12-ENOIC ACID C18 H32 O3 ZN NA TFA TRIFLUOROACETIC ACID HELIX 11ASNA 2LYSA111 10 HELIX 22CYSA13GLNA181 6 HELIX 33SERA24ALAA381 15 HELIX 44SERA40ILEA581 19 HELIX 55ASNA62ASNA741 13 SSBOND 1CYSA 3 CYSA 50 1555 15552.02 SSBOND 2CYSA 13 CYSA 27 1555 15552.05 SSBOND 3CYSA 28 CYSA 73 1555 15552.04 SSBOND 4CYSA 48 CYSA 87 1555 15552.02 LINK OD2ASPA 7 C9 ASYA302 1555 1555 1.45 LINK OD2ASPB 7 C9 ASYB303 1555 1555 1.45 LINK OD1ASNA 29 ZN ZNA103 1555 1555 2.22 LINK OD2ASPA 33 ZN ZNA103 1555 1555 2.06 LINK NE2HISA 35 ZN ZNA101 1555 1555 2.06 LINK O LEUA63 NA NAA201 1555 15552.73 LINK O ILEA81 NA NAA201 1555 15552.69 LINK OD2ASPA 86 NA NAA203 1555 1555 2.73 LINK ZN ZNA101 O2 ASY B 303 1555 1555 1.97 LINK ZN ZNA101 O1 ASYB303 1555 1555 2.31 LINK ZN ZNA102 O2 ASY A 302 1555 1555 1.95 LINK ZN ZNA102 O1 ASYA302 1555 1555 2.20 LINK ZN ZNA101 O HOHB448 1555 1555 1.77 LINK ZN ZNA101 O HOHA392 1555 1555 2.20 LINK ZN ZNA102 O HOHA397 1555 1555 1.98

22 LINK ZN ZNA102 O HOHA376 1555 1555 2.37 LINK ZN ZNA103 O HOHA401 1555 1555 1.98 LINK NA NAA201 O HOHA320 1555 1555 2.65 LINK ZN ZNB104 O HOHA392 1555 1555 2.54 LINK ZN ZNA103 O HOHA329 1555 1555 2.68 CISPEP 1GLYA 22 PROA 23 0 -4.90 CISPEP 2GLYB 22 PROB 23 0 -0.18 SITE 1AC1 6HISA 35 HOHA392 ZNB104 ASYB303 SITE 2AC1 6HOH B440 HOHB448 SITE 1AC2 4ASY A302 HOHA376 HOHA 397 HIS B 35 SITE 1AC3 4ASNA 29 ASPA 33 HOHA329 HOHA401 SITE 1AC4 5LEUA 63 ALAA 67 ILEA 81 SERA 82 SITE 2AC4 5HOHA320 SITE 1AC5 3ASPA 86 ASPB 84 HOHB434 SITE 1AC6 9LEUA 34 HISA 35 ALAA 38 VALA 47 SITE 2AC6 9VALA 77 PROA 78 TYRA 79 HOHA358 SITE 3AC6 9HOHA368 SITE 1AC710ASPA 7 META 10 GLYA 53 ILEA 54 SITE 2AC710ILEA 58 ZNA102 HOHA397 HISB 35 SITE 1AC8 6ASPA 84 ZNA101 HOHA392 ARGB 56 SITE 1AC9 6ASPA 84 ARGB 56 ZNB104 HOHB440 SITE 1BC213HISA 35 ASNA 76 ZNA101 ASPB 7 1MID,3GSH, 1LIP, (1-91=27-117,1-117), LAP [2-((1-OXODODECANOXY-(2-HYDROXY-3-PROPANYL))-PHOSPHONATE-OXY)-ETHYL]- -TRIMETHYLAMMONIUM L-ALFA-LYSOPHOSPHATIDYLCHOLINE, LAUROYL 3(C20 H43 N O7 P 1+) HELIX 11ASNA 2LYSA111 10 HELIX 22CYSA13GLNA181 6 HELIX 33SERA24ALAA381 15 HELIX 44SERA40GLYA571 18 HELIX 55ASNA62CYSA731 12 HELIX 66ASPA86ILEA905 5 SSBOND 1CYSA 3 CYSA 50 1555 15552.03 SSBOND 2CYSA 13 CYSA 27 1555 15552.02 SSBOND 3CYSA 28 CYSA 73 1555 15552.04 SSBOND 4CYSA 48 CYSA 87 1555 15552.03 CISPEP 1GLYA 22 PROA 23 0 0.08 SITE 1AC1 5ALAA 55 VALA 77 SERA 82 PROA 83 SITE 2AC1 5HOHA110 SITE 1AC2 2META 10 ILEA 54 SITE 1AC3 8ARGA 44 ALAA 66 ASNA 76 VALA 77 SITE 2AC3 8PRO A 78 HOHA110 HOHA 123 HOH A149 10 20 30 40 50 MARAQVLLMA AALVLMLTAA PRAAVALNCG QVDSKMKPCL TYVQGGPGPS 60 70 80 90 100 GECCNGVRDL HNQAQSSGDR QTVCNCLKGI ARGIHNLNLN NAASIPSKCN 110 117 VNVPYTISPD IDCSRIY 2N2Z(1-93=30-122,1-122) HELIX 11THRA 2ALAA111 10 HELIX 22CYSA13ARGA181 6 HELIX 33PROA26ALAA401 15 HELIX 44THRA42ILEA601 19 HELIX 55ASNA64GLYA761 13 SSBOND 1CYSA 3 CYSA 52 1555 15552.11 SSBOND 2CYSA 13 CYSA 29 1555 15551.99 SSBOND 3CYSA 30 CYSA 75 1555 15552.03 SSBOND 4CYSA 50 CYSA 89 1555 15552.11 10 20 30 40 50 MGVSRACFVV MVVVYMVVAA TPNVKLAEAL TCGQVTGALA PCLGYLRTAG 60 70 80 90 100 SVPVPLTCCN GVRGLNNAAR TTIDRRTACN CLKQTANAIA DLNLNAAAGL 110 120 122 PAKCGVNIPY KISPSTDCNR VV

23 1GH1,1BWO,1CZ2(1-90=24-113,1-113) 1BWO,1CZ2(1-90=24-113,1-113) HELIX 11HISA 5GLNA181 14 HELIX 22SERA24ALAA381 15 HELIX 33SERA40ARGA561 17 HELIX 44ASNA62GLYA741 13 SSBOND 1CYSA 3 CYSA 50 1555 15552.02 SSBOND 2CYSA 13 CYSA 27 1555 15552.02 SSBOND 3CYSA 28 CYSA 73 1555 15552.01 SSBOND 4CYSA 48 CYSA 87 1555 15552.02 CISPEP 1GLYA 22 PROA 23 1 0.79 CISPEP 2GLYA 22 PROA 23 2 0.32 CISPEP 3GLYA 22 PROA 23 3 3.50 CISPEP 4GLYA 22 PROA 23 4 3.87 CISPEP 5GLYA 22 PROA 23 5 3.29 CISPEP 6GLYA 22 PROA 23 6 1.17 CISPEP 7GLYA 22 PROA 23 7 5.87 CISPEP 8GLYA 22 PROA 23 8 -0.82 CISPEP 9GLYA 22 PROA 23 9 3.72 CISPEP 10GLYA 22 PROA 23 10 2.65 CISPEP 11GLYA 22 PROA 23 11 -3.05 CISPEP 12GLYA 22 PROA 23 12 1.34 CISPEP 13GLYA 22 PROA 23 13 3.28 CISPEP 14GLYA 22 PROA 23 14 3.88 CISPEP 15GLYA 22 PROA 23 15 -8.19 LPC [1-MYRISTOYL-GLYCEROL-3-YL]PHOSPHONYLCHOLINE 4(C22 H47 N O7 P 1+) HELIX 1H1CYSA 3VALA171 15 HELIX 2H2GLYA25GLNA371 13 HELIX 3H3GLNA41ALAA551 15 HELIX 4H4GLUA63CYSA731 11 SSBOND 1CYSA 3 CYSA 50 1555 15552.03 SSBOND 2CYSA 13 CYSA 27 1555 15552.04 SSBOND 3CYSA 28 CYSA 73 1555 15552.03 SSBOND 4CYSA 48 CYSA 87 1555 15552.04 CISPEP 1GLYA 22 PROA 23 0 0.43 SITE 1AC111HISA 35 ARGA 44 HISA 59 ASNA 60 SITE 2AC111ALAA 66 LEUA 77 TYRA 79 SERA 82 SITE 3AC111LEUA 83 VALA 90 HOHA133 SITE 1AC216ASPA 7 VALA 10 ARGA 11 LEUA 14 SITE 2AC216ILEA 54 GLYA 57 ILEA 58 HISA 59 SITE 3AC216PROA 78 HOHA165 ASPB 64 ASPB 86 SITE 1AC312ARGA 39 SERA 40 GLNA 41 ARGA 44 SITE 1AC4 8HOHA159 ASPB 7 VALB 10 ARGB 11 10 20 30 40 50 AQVMLMAVAL VLMLAAVPRA AVAIDCGHVD SLVRPCLSYV QGGPGPSGQC 60 70 80 90 100 CDGVKNLHNQ ARSQSDRQSA CNCLKGIARG IHNLNEDNAR SIPPKCGVNL 110 113 PYTISLNIDC SRV 1FK1,1AFH,(1-93,1-120) 1FK1 DAOLAURICACID C12H24 O2 FMT FORMIC ACID 3(C H2 O2) 1FK2 MYRMYRISTICACID C14H28 O2 FMT FORMIC ACID 3(C H2 O2) 1FK3 PAM PALMITOLEIC ACID C16 H30 O2 FMT FORMIC ACID 3(C H2 O2) 1FK4 STESTEARICACID C18H36 O2 FMT FORMIC ACID 3(C H2 O2) 1FK5 OLAOLEICACID C18H34 O2 FMT FORMIC ACID 3(C H2 O2) 1FK6 LNL ALPHA-LINOLENIC ACID C18 H30 O2 FMT FORMIC ACID 3(C H2 O2) 1FK7 RCL RICINOLEIC ACID C18 H34 O3 FMT FORMIC ACID 3(C H2 O2) 1MZM PLMPALMITICACID C16H32 O2 FMT FORMIC ACID 3(C H2 O2) 1BE2 ,(1-91,1-120) PLMPALMITICACID C16H32 O2 HELIX 11CYSA 4ALAA181 15 HELIX 22ALAA27ALAA391 13 HELIX 33THRA43ALAA581 16 HELIX 44ALAA65LYSA741 10 SSBOND 1CYSA 4 CYSA 52 1555 15552.16

24 SSBOND 2CYSA 14 CYSA 29 1555 15552.18 SSBOND 3CYSA 30 CYSA 75 1555 15552.22 SSBOND 4CYSA 50 CYSA 89 1555 15552.21 SITE 1AC1 5VALA 33 ASNA 37 ARGA 46 LEUA 53 SITE 2AC1 5ILEA 83 SITE 1AC2 7ARGA 47 ALAA 48 ASNA 51 GLYA 62 SITE 2AC2 7LEUA 63 HOHA127 HOHA155 SITE 1AC3 4ALAA 69 PROA 72 THRA 82 ILEA 83 SITE 1AC4 6ASNA 51 LYSA 54 ASNA 64 GLYA 66 SITE 2AC4 6THRA 87 CYSA 89 1FK3 SSBOND 1CYSA 4 CYSA 52 1555 15552.03 SSBOND 2CYSA 14 CYSA 29 1555 15552.05 SSBOND 3CYSA 30 CYSA 75 1555 15552.04 SSBOND 4CYSA 50 CYSA 89 1555 15552.03 SITE 1AC1 7ILEA 11 ILEA 15 ALAA 18 VALA 33 SITE 2AC1 7ARGA 46 TYRA 81 HOHA513 SITE 1AC2 7ASNA 51 LYSA 54 ASNA 64 ALAA 65 SITE 2AC2 7GLYA 66 THRA 87 CYSA 89 1FK4 SSBOND 1CYSA 4 CYSA 52 1555 15552.05 SSBOND 2CYSA 14 CYSA 29 1555 15552.04 SSBOND 3CYSA 30 CYSA 75 1555 15552.01 SSBOND 4CYSA 50 CYSA 89 1555 15552.03 SITE 1AC1 9ILEA 15 VALA 33 ARGA 46 ALAA 57 SITE 2AC1 9TYRA 81 ILEA 83 HOHA120 HOHA123 SITE 3AC1 9HOHA147 SITE 1AC2 6ARGA 47 ALAA 48 ASNA 51 GLYA 62 SITE 2AC2 6LEUA 63 HOHA155 SITE 1AC3 8ASNA 51 LYSA 54 ASNA 64 ALAA 65 SITE 2AC3 8GLYA 66 THRA 87 ASPA 88 CYSA 89 1FK5 SSBOND 1CYSA 4 CYSA 52 1555 15552.04 SSBOND 2CYSA 14 CYSA 29 1555 15552.02 SSBOND 3CYSA 30 CYSA 75 1555 15552.02 SSBOND 4CYSA 50 CYSA 89 1555 15552.04 SITE 1AC119VALA 33 LEUA 36 ASNA 37 ALAA 40 SITE 2AC119ARGA 46 ALAA 49 CYSA 50 LEUA 53 SITE 3AC119ILEA 79 TYRA 81 ILEA 83 VALA 92 SITE 4 AC1 19 HOH A 351 HOH A 360 HOH A 361 HOH A 383 SITE 5 AC1 19 HOH A 457 HOH A 535 HOH A 564 SITE 1AC2 6SERA 61 THRA 87 ASPA 88 ARGA 91 SITE 2AC2 6HOH A326 HOHA349 SITE 1AC310ASNA 51 LYSA 54 ASNA 64 ALAA 65 SITE 2AC310GLYA 66 THRA 87 ASPA 88 CYSA 89 SITE 3 AC3 10 HOH A 320 HOH A 703 SITE 1AC4 4ASNA 51 CYSA 89 HOHA366 HOHA703 1FK6 SSBOND 1CYSA 4 CYSA 52 1555 15552.06 SSBOND 2CYSA 14 CYSA 29 1555 15552.02 SSBOND 3CYSA 30 CYSA 75 1555 15552.01 SSBOND 4CYSA 50 CYSA 89 1555 15552.03 SITE 1AC1 8ASNA 37 ALAA 40 ARGA 46 ALAA 49 SITE 2AC1 8ALAA 57 TYRA 81 HOHA201 HOHA223 SITE 1AC2 4ALAA 48 ASNA 51 GLYA 62 LEUA 63 SITE 1AC3 8ASNA 51 LYSA 54 ASNA 64 ALAA 65 SITE 2AC3 8GLYA 66 THRA 87 CYSA 89 HOHA208 1FK7 SSBOND 1CYSA 4 CYSA 52 1555 15552.03 SSBOND 2CYSA 14 CYSA 29 1555 15552.02 SSBOND 3CYSA 30 CYSA 75 1555 15552.04 SSBOND 4CYSA 50 CYSA 89 1555 15552.05 SITE 1AC110ALAA 18 VALA 33 ARGA 46 ALAA 49

25 SITE 2AC110LEUA 53 ALAA 57 ALAA 68 TYRA 81 SITE 3AC110VAL A 92 HOHA464 SITE 1AC2 6SERA 61 THRA 87 ASPA 88 ARGA 91 SITE 2AC2 6HOH A351 HOHA451 1MZM SSBOND 1CYSA 4 CYSA 52 1555 15552.03 SSBOND 2CYSA 14 CYSA 29 1555 15552.05 SSBOND 3CYSA 30 CYSA 75 1555 15552.03 SSBOND 4CYSA 50 CYSA 89 1555 15552.04 SITE 1AC1 6ILEA 15 ALAA 18 ARGA 46 TYRA 81 SITE 2AC1 6HOH A131 HOHA197 SITE 1AC2 8ASNA 51 LYSA 54 ASNA 64 ALAA 65 SITE 2AC2 8GLYA 66 CYSA 89 HOHA119 HOHA159 1BE2 HELIX 11GLYA 4GLYA191 16 HELIX 22GLUA26GLNA371 12 HELIX 33SERA41ARGA561 16 HELIX 44LEUA63CYSA731 11 SSBOND 1CYSA 3 CYSA 50 1555 15552.01 SSBOND 2CYSA 13 CYSA 27 1555 15552.02 SSBOND 3CYSA 28 CYSA 73 1555 15552.02 SSBOND 4CYSA 48 CYSA 87 1555 15552.02 CISPEP 1GLYA 22 PROA 23 1 1.79 CISPEP 2GLYA 22 PROA 23 2 -2.72 CISPEP 3GLYA 22 PROA 23 3 -8.07 CISPEP 4GLYA 22 PROA 23 4 -3.94 CISPEP 5GLYA 22 PROA 23 5 7.81 CISPEP 6GLYA 22 PROA 23 6 3.50 CISPEP 7GLYA 22 PROA 23 7 5.21 CISPEP 8GLYA 22 PROA 23 8 -11.26 CISPEP 9GLYA 22 PROA 23 9 12.22 CISPEP 10GLYA 22 PROA 23 10 2.77 SITE 1PLB16LYSA 9 META 10 CYSA 13 LEUA 14 SITE 2PLB16CYSA 27 LEUA 34 HISA 35 ARGA 44 SITE 3PLB16GLNA 45 VALA 47 CYSA 48 LEUA 51 SITE 4PLB16ILEA 69 TYRA 79 ILEA 81 ILEA 85 SITE 1AC114LYSA 9 META 10 CYSA 13 LEUA 14 SITE 2AC114TYRA 16 VALA 17 VALA 31 ARGA 44 SITE 3AC114VALA 47 CYSA 48 LEUA 51 ILEA 69 SITE 4AC114TYRA 79 ILEA 81 10 20 30 40 50 MARTQQLAVV ATAVVALVLL AAATSEAAIS CGQVASAIAP CISYARGQGS 60 70 80 90 100 GPSAGCCSGV RSLNNAARTT ADRRAACNCL KNAAAGVSGL NAGNAASIPS 110 120 KCGVSIPYTI STSTDCSRVN 2ALG,2B5S(0,1-91,1-91), DAO LAURIC ACID 2(C12 H24 O2) HELIX 11THRA 2ALAA111 10 HELIX 22CYSA13GLYA201 8 HELIX 33PROA24ALAA381 15 HELIX 44THRA40VALA581 19 HELIX 55ASNA62CYSA731 12 HELIX 66ASNA86VALA905 5 SSBOND 1CYSA 3 CYSA 50 1555 15552.04 SSBOND 2CYSA 13 CYSA 27 1555 15552.04 SSBOND 3CYSA 28 CYSA 73 1555 15552.04 SSBOND 4CYSA 48 CYSA 87 1555 15552.04 SITE 1AC1 6ALAA 26 ASNA 29 HOHA209 HOHA255 SITE 2AC1 6THRB 2 GLYB 4 SITE 1AC2 8ARGA 44 ALAA 47 CYSA 48 SERA 55 SITE 2AC2 8LYSA 80 ILEA 81 SERA 82 HP6A204 SITE 1AC3 3ILEA 31 LEUA 69 DAOA202

26 SITE 1AC4 6SERA 8 SERB 7 SERB 8 VALB 17 SITE 2AC4 6ARGB 18 ILEB 81 SITE 1AC5 6ASNA 29 ARGA 32 ASNA 36 VALA 75 SITE 2AC5 6METB 0 GLNB 5 DAO LAURIC ACID 2(C12 H24 O2) HELIX 11THRA 2ALAA111 10 HELIX 22CYSA13GLYA201 8 HELIX 33PROA24ALAA381 15 HELIX 44THRA40VALA581 19 HELIX 55ASNA62CYSA731 12 HELIX 66ASNA86VALA905 5 SSBOND 1CYSA 3 CYSA 50 1555 15552.04 SSBOND 2CYSA 13 CYSA 27 1555 15552.05 SSBOND 3CYSA 28 CYSA 73 1555 15552.04 SSBOND 4CYSA 48 CYSA 87 1555 15552.04 SITE 1AC1 6ALAA 26 ASNA 29 HOHA226 THRB 2 SITE 2AC1 6GLYB 4 HOHB250 SITE 1AC2 6SERA 8 SERB 7 LEUB 10 VALB 17 SITE 2AC2 6ARGB 18 HOHB247 SITE 1AC3 9ARGA 44 ALAA 47 CYSA 48 LYSA 52 SITE 2AC3 9SERA 55 LYSA 80 ILEA 81 SERA 82 SITE 3AC3 9HP6A204 SITE 1AC4 3ILEA 31 ILEA 77 DAOA202 10 20 30 40 50 ITCGQVSSAL APCIPYVRGG GAVPPACCNG IRNVNNLART TPDRQAACNC 60 70 80 90 91 LKQLSASVPG VNPNNAAALP GKCGVHIPYK ISASTNCATV K 2R09,2R0D(63-399,54-396A,54-394B,1-399) MSE SELENOMETHIONINE 16(C5 H11 N O2 SE) 4IP INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE 2(C6 H16 O18 P4) PGE TRIETHYLENE GLYCOL C6 H14 O4 PE5 3,6,9,12,15,18,21,24-OCTAOXAHEXACOSAN-1-OL-(2-{2-[2-(2-{2-[2-(2-ETHOXY-ETHOXY)-ETHOXY]- ETHOXY}- -ETHOXY)-ETHOXY]-ETHOXY}-ETHOXY)-ETHANOL, POLYETHYLENE GLYCOL PEG400 C18 H38 O9 HELIX 11THRA63ASPA801 18 HELIX 22ASPA80ASNA911 12 HELIX 3 3SERA 97GLYA1081 12 HELIX 4 4ASNA112GLUA1221 11 HELIX 5 5ASPA124LEUA1371 14 HELIX 6 6ASNA144LEUA1531 10 HELIX 7 7GLUA161ASNA1801 20 HELIX 8 8SERA186ASNA2061 21 HELIX 9 9THRA214ASNA2221 9 HELIX 1010PROA232GLUA2461 15 HELIX 1111ASPA257THRA2615 5 HELIX 1212SERA364ARGA3811 18 HELIX 1313ASPA382ALAA3961 15 SHEET 1 A9SERA314 VALA318 0 SHEET 2 A9CYSA326 TYRA330-1 O GLUA328 N ARGA316 SHEET 3 A9TYRA358 SERA361-1 O ILEA360 N PHEA327 SHEET 4 A9ARGA267 LEUA274-1 N LEUA272 O SERA361 SHEET 5 A9TRPA281 THRA289-1 O PHEA286 N GLYA269 SHEET 6 A9CYSA292 PHEA296-1 O TYRA294 N ILEA287 SHEET 7 A9GLYA306 PROA309-1 O ILEA308 N LEUA293 SHEET 8 A9CYSA342 THRA344-1 O CYSA342 N ILEA307 SHEET 9 A9VALA350 GLUA352-1 O VALA351 N LYSA343 LINK C ALAA71 N MSEA72 1555 15551.34 LINK C MSEA72 N GLYA73 1555 15551.33 LINK C ASNA78 N MSEA79 1555 15551.33 LINK C MSEA79 N ASPA80 1555 15551.33 LINK C ARGA167 N MSEA168 1555 1555 1.33 LINK C MSEA168 N MSEA169 1555 1555 1.33 LINK C MSEA169 N GLUA170 1555 1555 1.33 LINK C ILEA198 N MSEA199 1555 1555 1.34 LINK C MSEA199 N LEUA200 1555 1555 1.34

27 LINK C THRA220 N MSEA221 1555 1555 1.34 LINK C MSEA221 N ASNA222 1555 1555 1.33 LINK C TRPA371 N MSEA372 1555 1555 1.33 LINK C MSEA372 N LYSA373 1555 1555 1.33 LINK C ASPA386 N MSEA387 1555 1555 1.33 LINK C MSEA387 N LEUA388 1555 1555 1.33 SITE 1AC1 7HISA 57 HISA 58 HISA 60 HOHA695 SITE 2 AC1 7 LEU B 272 LYS B 323 PRO B 324 SITE 1 AC2 7 GLN A 147 ARG A 150 SER A 242 GLN A 337 SITE 2 AC2 7 HOH A 448 HOH A 498 HOH A 534 SITE 1 AC3 9 ARG A 210 HOH A 440 HOH A 517 HOH A 741 SITE 2AC3 9HOHA760 HOHA799 HISB 55 HISB 56 SITE 3AC3 9HISB 57 SITE 1 AC4 5 ARG A 217 HOH A 557 HOH A 599 HOH A 788 SITE 2AC4 5HISB 60 SITE 1 AC5 3 LYS A 282 ARG A 284 HOH A 693 SITE 1 AC6 5 ASP A 301 LYS A 302 GLU A 303 HOH A 569 SITE 2AC6 5HOHA774 SITE 1 AC7 6 TRP A270 PRO A363 GLYB 54 HOHB 535 SITE 2 AC7 6 HOH B578 HOH B616 SITE 1 AC8 24 LYS A 273 GLY A 275 GLY A 276 ARG A 277 SITE 2 AC8 24 VAL A 278 THR A 280 LYS A 282 ARG A 284 SITE 3 AC8 24 TYR A 295 ARG A 305 LYS A 343 ASN A 354 SITE 4 AC8 24 HIS A 355 HOH A 409 HOH A 415 HOH A 418 SITE 5 AC8 24 HOH A 430 HOH A 432 HOH A 442 HOH A 450 SITE 6 AC8 24 HOH A 513 HOH A 556 HOH A 651 HOH A 744 SITE 1 AC9 3 ASN A 208 PHE A 263 HOH A 684 SITE 1 BC1 12 ALA A 196 MSE A 221 ASN A 222 ARG A 223 SITE 2 BC1 12 THR A 289 ASP A 290 TYR A 385 LEU A 388 SITE 3 BC112 ALA A389 LYS A392 SERB 62 GLNB 65 2R0D(63-399,54-396A,54-394B,1-399) 4IP INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE 2(C6 H16 O18 P4) PEG DI(HYDROXYETHYL)ETHER 3(C4 H10 O3) HELIX 11THRA63ASPA801 18 HELIX 22ASPA80ASNA911 12 HELIX 3 3SERA97GLYA1081 12 HELIX 4 4ASNA112GLYA1211 10 HELIX 5 5ASPA124LEUA1371 14 HELIX 6 6ASNA144LEUA1531 10 HELIX 7 7GLUA161ASNA1801 20 HELIX 8 8SERA186ASNA2061 21 HELIX 9 9THRA214ASNA2221 9 HELIX 1010PROA232GLUA2461 15 HELIX 1111ASPA257THRA2615 5 HELIX 1212SERA364ARGA3811 18 HELIX 1313ASPA382ALAA3961 15 SHEET 1 A9SERA314 VALA318 0 SHEET 2 A9CYSA326 TYRA330-1 O GLUA328 N ARGA316 SHEET 3 A9TYRA358 SERA361-1 O ILEA360 N PHEA327 SHEET 4 A9ARGA267 LEUA274-1 N LEUA272 O SERA361 SHEET 5 A9TRPA281 THRA289-1 O LEUA288 N ARGA267 SHEET 6 A9CYSA292 PHEA296-1 O TYRA294 N ILEA287 SHEET 7 A9GLYA306 PROA309-1 O ILEA308 N LEUA293 SHEET 8 A9CYSA342 THRA344-1 O CYSA342 N ILEA307 SHEET 9 A9VALA350 GLUA352-1 O VALA351 N LYSA343 SITE 1 AC1 9 GLN A 147 ARG A 150 SER A 242 GLN A 337 SITE 2 AC1 9 HOH A 450 HOH A 540 HOH A 576 HOH A 594 SITE 3AC1 9HOHA762 SITE 1AC2 8HOHA596 HISB 55 HISB 56 HISB 57 SITE 2 AC2 8 HOH B 410 HOH B 504 HOH B 512 HOH B 530 SITE 1AC3 6HISA 57 HISA 58 HISA 60 LEUB272 SITE 2AC3 6LYS B323 PROB324 SITE 1AC4 4ARG A217 HOHA525 HOHA 716 HIS B 60 SITE 1 AC5 24 LYS A 107 LYS A 273 GLY A 275 GLY A 276 SITE 2 AC5 24 ARG A 277 VAL A 278 THR A 280 LYS A 282 SITE 3 AC5 24 ARG A 284 TYR A 295 ARG A 305 LYS A 343

28 SITE 4 AC5 24 ASN A 354 HIS A 355 HOH A 409 HOH A 412 SITE 5 AC5 24 HOH A 414 HOH A 431 HOH A 488 HOH A 498 SITE 6 AC5 24 HOH A 538 HOH A 567 HOH A 572 HOH A 752 SITE 1 AC7 4 TYR A 385 LEU A 388 LYS A 392 HOH A 749 SITE 1 AC8 5 GLY A 269 TRP A 270 ARG A 283 TRP A 285 1U2B 4IP (261-387,1-399) HELIX 1 1SERA365ASPA3831 19 SHEET 1 A9SERA315 GLUA318 0 SHEET 2 A9CYSA327 TYRA331-1 O GLUA329 N ARGA317 SHEET 3 A9VALA358 SERA362-1 O ILEA361 N PHEA328 SHEET 4 A9ARGA267 LEUA274-1 N LEUA272 O SERA362 SHEET 5 A9TRPA282 THRA290-1 O LEUA289 N ARGA267 SHEET 6 A9CYSA293 PHEA297-1 O TYRA295 N ILEA288 SHEET 7 A9GLYA307 PROA310-1 O ILEA309 N LEUA294 SHEET 8 A9CYSA343 THRA345-1 O CYSA343 N ILEA308 SHEET 9 A9VALA351 GLUA353-1 O VALA352 N LYSA344 SITE 1 AC1 6 LYS A 273 LYS A 283 ARG A 285 TYR A 296 SITE 2AC1 6ARG A306 SO4A502 SITE 1 AC2 6 LYS A 273 GLY A 275 LYS A 344 HIS A 356 SITE 2AC2 6SO4 A501 HOHA541 10 20 30 40 50 MDEGGGGEGG SVPEDLSLEE REELLDIRRR KKELIDDIER LKYEIAEVMT 60 70 80 90 100 EIDNLTSVEE SKTTQRNKQI AMGRKKFNMD PKKGIQFLIE NDLLQSSPED 110 120 130 140 150 VAQFLYKGEG LNKTVIGDYL GERDDFNIKV LQAFVELHEF ADLNLVQALR 160 170 180 190 200 QFLWSFRLPG EAQKIDRMME AFASRYCLCN PGVFQSTDTC YVLSFAIIML 210 220 230 240 250 NTSLHNHNVR DKPTAERFIT MNRGINEGGD LPEELLRNLY ESIKNEPFKI 260 270 280 290 300 PEDDGNDLTH TFFNPDREGW LLKLGGRVKT WKRRWFILTD NCLYYFEYTT 310 320 330 340 350 DKEPRGIIPL ENLSIREVED PRKPNCFELY NPSHKGQVIK ACKTEADGRV 360 370 380 390 399 VEGNHVVYRI SAPSPEEKEE WMKSIKASIS RDPFYDMLAT RKRRIANKK 1FGZ, 1FGY, 1U2B 4IP (261-387,1-399), (13-434) ), 1FHW,1FHX(264,A265,B266-391,1- 399),2R09,2R0D(63-399,1-399) MSE SELENOMETHIONINE 2(C5 H11 N O2 SE) 4IP INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE C6 H16 O18 P4 HELIX 1 1SERA364ASPA3821 19 SHEET 1 A9SERA314 VALA318 0 SHEET 2 A9CYSA326 TYRA330-1 O CYSA326 N VALA318 SHEET 3 A9VALA357 SERA361-1 N TYRA358 O LEUA329 SHEET 4 A9ARGA267 LEUA274-1 O LEUA272 N SERA361 SHEET 5 A9TRPA281 THRA289-1 O LYSA282 N LYSA273 SHEET 6 A9CYSA292 PHEA296-1 O CYSA292 N THRA289 SHEET 7 A9GLYA306 PROA309-1 O GLYA306 N TYRA295 SHEET 8 A9CYSA342 THRA344-1 O CYSA342 N ILEA307 SHEET 9 A9VALA350 GLUA352-1 O VALA351 N LYSA343 LINK C TRPA371 N MSEA372 1555 1555 1.33 LINK C MSEA372 N LYSA373 1555 1555 1.33 LINK C ASPA386 N MSEA387 1555 1555 1.34 SITE 1 AC1 6 SO4 A 202 LYS A 273 LYS A 282 ARG A 284 SITE 2AC1 6TYR A295 ARGA305 SITE 1AC2 7HOH A 21 SO4A201 LYSA 273 GLY A275 SITE 2 AC2 7 GLY A 276 LYS A 343 HIS A 355 1FHW,1FHX(264,A265,B266-391,1-399), I5P INOSITOL-(1,3,4,5,6)-PENTAKISPHOSPHATE 2(C6 H17 O21 P5) HELIX 1 1SERA364ASPA3821 19 HELIX 2 2SERB364ARGB3811 18 HELIX 3 3ARGB381ALAB3891 9 SHEET 1 A9SERA314 GLUA317 0

29 SHEET 2 A9CYSA326 TYRA330-1 N GLUA328 O ARGA316 SHEET 3 A9VALA357 SERA361-1 O TYRA358 N LEUA329 SHEET 4 A9ARGA267 LEUA274-1 N LEUA272 O SERA361 SHEET 5 A9TRPA281 THRA289-1 O LYSA282 N LYSA273 SHEET 6 A9CYSA292 PHEA296-1 O CYSA292 N THRA289 SHEET 7 A9GLYA306 PROA309-1 O GLYA306 N TYRA295 SHEET 8 A9CYSA342 THRA344-1 O CYSA342 N ILEA307 SHEET 9 A9VALA350 GLUA352-1 N VALA351 O LYSA343 SHEET 1 B9SERB314 GLUB317 0 SHEET 2 B9CYSB326 TYRB330-1 N GLUB328 O ARGB316 SHEET 3 B9VALB357 SERB361-1 N TYRB358 O LEUB329 SHEET 4 B9ARGB267 LEUB274-1 O LEUB272 N SERB361 SHEET 5 B9TRPB281 THRB289-1 O LYSB282 N LYSB273 SHEET 6 B9CYSB292 PHEB296-1 O CYSB292 N THRB289 SHEET 7 B9GLYB306 PROB309-1 O GLYB306 N TYRB295 SHEET 8 B9CYSB342 THRB344-1 N CYSB342 O ILEB307 SHEET 9 B9VALB350 GLUB352-1 N VALB351 O LYSB343 SSBOND 1CYSA 342 CYSB 342 1555 1555 2.03 SITE 1AC1 4ASN A312 ASNA331 HOHB 31 LYS B340 SITE 1AC215HOHA 12 HOHA 46 HOHA 48 HOHA 50 SITE 2 AC2 15 HOH A 121 HOH A 156 LYS A 273 GLY A 276 SITE 3 AC2 15 ARG A 277 LYS A 282 ARG A 284 TYR A 295 SITE 4 AC2 15 ARG A 305 LYS A 343 HIS A 355 SITE 1 AC3 14 HOH B 22 HOH B 139 HOH B 140 HOH B 141 SITE 2 AC3 14 LYS B 273 GLY B 275 GLY B 276 ARG B 277 SITE 3 AC3 14 VAL B 278 ARG B 284 TYR B 295 ARG B 305 SITE 4 AC3 14 LYS B 343 HIS B 355 1FAO,1FB8 (148-273,1-280), 10 20 30 40 50 MGRAELLEGK MSTQDPSDLW SRSDGEAELL QDLGWYHGNL TRHAAEALLL 60 70 80 90 100 SNGCDGSYLL RDSNETTGLY SLSVRAKDSV KHFHVEYTGY SFKFGFNEFS 110 120 130 140 150 SLKDFVKHFA NQPLIGSETG TLMVLKHPYP RKVEEPSIYE SVRVHTAMQT 160 170 180 190 200 GRTEDDLVPT APSLGTKEGY LTKQGGLVKT WKTRWFTLHR NELKYFKDQM 210 220 230 240 250 SPEPIRILDL TECSAVQFDY SQERVNCFCL VFPFRTFYLC AKTGVEADEW 260 270 280 IKILRWKLSQ IRKQLNQGEG TIRSRSFIFK 4FES 0T9 (3S,8S,9S,10R,13S,14S,17R)-3-HYDROXY-10,13-DIMETHYL-17-[(2S,6S)-

30