Báo cáo khoa học: Crystal structures of bovine odorant-binding protein in complex with odorant molecules doc

Crystal structures of bovine odorant-binding protein in complex with odorant molecules Florence Vincent 1 , Roberto Ramoni 2 , Silvia Spinelli 1 , Stefano Grolli 2 , Mariella Tegoni 1 and Christian Cambillau 1 1 Architecture et Fonction des Macromole ´ cules Biologiques, UMR 6098, CNRS, Marseille, France; 2 Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualita ` e Sicurezza degli Alimenti, Universita ` di Parma, Parma, Italy The structure of bovine odorant-binding protein (bOBP) revealedastrikingfeatureofadimerformedbydomain swapping 2 [Tegoni, M ., Ramoni, R., Bigne tti, E., Spinelli, S. & Cambillau, C. (1996) Nat. Struct. Biol. 3, 863–867; B ian- chet, M.A., Bains, G., Pelosi, P., Pevsner, J., Snyder, S.H., Monaco, H.L. & Amzel, L.M. (1996) Nat. Struct. Biol. 3, 934–939] and t he presence of a n aturally occur ing liga nd [Ramoni, R., Vincent, F., Grolli, S., C onti, V., Malosse, C ., Boyer, F.D., Nagnan-Le Meillour, P., Spinelli, S., Cambil- lau, C. & T egoni, M. (2001) J. Biol. Chem. 276, 7150–7155]. These features led us to investigate the binding of odorant molecules with bOBP in solution and in the crystal. The behavior of odorant m olecules in bOBP resembles that observed with porcine OBP (pOBP), although the latter is monomeric and devoid of ligand when purified. The odorant molecules p resented K d values with bOBP in the m icromolar range. Most of the X-ray structures revealed that odorant molecules interact with a com mon set of residues forming the cavity wall and do no t exhibit spe cific interactions. Depending on the ligand and on the monomer (A or B), a single residue – Phe89 – presents alternate conformation s and might control cross-talking between the subunits. Crystal data o n b oth p OBP a nd bOBP, in contrast w ith binding and s pectroscopic studies on rat OBP in solution, reveal an absence o f significant c onformational changes involving protein loops or backbone. T hus, the role of OBP in signal triggering remains unresolved. 3 Keywords: crystal str ucture; domain swapping; odorant- binding protein; olfaction. Odorant-binding proteins (OBPs), first discovered in the nasal mucus and epithelium o f m ammals at millimolar concentrations [ 4 1,2], w ere identified, b y their sequence, as lipocalins [3], a family of proteins generally involved in the transport of hydrophobic ligands [4]. The hypothesis of their involvement in the olfactory process, perception and transduction of the signal, was derived from their localization and their ability to b ind 2-iso-b utyl-3-metoxy- pyrazine (IBMP), known a s the o dorant with the lo west detection t hreshold i n humans [5]. D ifferent roles for OBPs have previously been proposed [6,7], such as (a) carriers of odorants from the a ir to the o lfactory receptors (ORs) through the aqueous barrier of the mucus [11,12]; (b) scavengers of odorants from the OR after transduction of the olfactory signal [8,9] and/or of odorants present at a high concentration in order to avoid saturation of the OR [10,11]; (c) protectors of the nasal mucosa, which is exposed to airflow and oxidative injuries, by binding cytotoxic and genotoxic molecules, such as alkylic alde- hydes [9,12,13]; or (d) during transduction, to permit recognition of t he OBP–odorant complex by the receptor, as in bacterial chemotaxis [ 14,15]. 5 Indeed, a broad spectrum of activity and a relatively weak affinity for odorants have been found in mammalian OBPs [ 9,12,16– 18], and c lear experimental evidence of the role of OBPs in olfaction and odo rant perception has not yet been produced. Peculiar i n t he case of bovine O BP (bOBP) is the anti-cooperative binding resulting in the stoichiometry of a single molecule of IBMP per dimer of OBP, repeatedly reported in the literature [1,8,9]. We have reported the firs t 3D structure o f bOBP a t 2.0 A ˚ [19]. In fact, the prototype of OBPs was demonstrated to be a Ôspe cialÕ c ase among lipocalins, as it is a dimer with a swapped helix, p ossibly p roviding interdependent properties to the t wo subunits [19]. As p reviously fou nd i n t he case of retinol-binding protein [20] and Major Urinary Protein 6 [21], a natural ligand that co-purified with the protein was observed in the b-barrel cavity o f bOBP [19,22]. The f urther unambiguous identification of this natural ligand as 1-octen-3-ol (OCT), an insect attractant produced by bovine rumination, suggested a role of bOBP in the e cological relationships between bovine and several insect s pecies [23]. Porcine OBP (pOBP) [24] was revealed t o be a monomer and to have a cavity devoid of any ligand, which made it a good candidate for studying t he interaction w ith a broad range o f ligands of bOBP [9,12 ,16,18] o r pOBP [ 16,18]. The structure o f d ifferent complexes of p OBP s howed the presence of the odorants, in a stoichiometr ic molar r atio, Correspondence to M. Tegoni or C. Cambillau, Architecture et Fonction des Macromole ´ cules Biologiques, UMR 6098, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Fax: +33 4 9116 4536, Tel.: +33 4 9116 4501, E-mail: tegoni@afmb.cnrs-mrs.fr or cambillau@afmb.cnrs-mrs.fr Abbreviations: AMA, 1-amino-anthracene; bOBP, bovine odorant- binding protein; BZP, benzophenone; DHM, dihydromyrcenol; IBMP or pyrazine, 2-isobutyl-3-metoxypyrazine; OCT, 1-octen-3-ol; OR, odorant receptor; pOBP, porcine OBP; UND, undecanal. (Received 2 3 June 200 4, accepted 30 July 2004) Eur. J. Biochem. 271, 3832–3842 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04315.x inside the c avity formed by the b-barrel. The orientation of the ligands ins ide the cavity appeared to be opportunistic with no specific target patches f or aromatic o r charged groups and no c orrelation between the number o f contacts and the affinity measured in solution. In particular, n o special characteristic could b e a scribed to the good binders, and upon interaction with all odorants, all residue side- chains lining the cavity kept the conformation observed in the native p rotein. While these results were not in favor of a s pecific carrier role for OBPs [13], other studies performed in solution, although sometimes contradictory, pointed to a contrasting situation 7 . Fluorescence studies in solution with three rat OBPs and 49 d ifferent ligands revealed some preference of each OBP to certain classes of chemical compounds, suggesting a specific filter r ole of OBPs [17]. Recently, biophysical s tudies on a subclass of dimeric rat OBP (OBP- 1F) indicated that one ligand binds per monomer and t hat subtle changes in the side-chains or backbone positions were induced upon binding [25]. A nother group could express a human OR in mammalian c ells an d demonstrate h igh- affinity binding with empty porcine OBP, although binding was further localized in several t issues besides nasal mucosa. Another class of lipocalins – tear lipocalin-1 – was shown to bind with high affinity to a new class of receptor not belonging to the GPCR family as ORs, and to b e further internalized [26]. With a view to collecting hard data based on crystal structures, possibly displaying conformational changes, we chose bOBP as a good candidate for ligand studies – the native structure of bOBP showed some flexibility at Phe89, and its dimer structure seemed favorable for investigating putative cross-talk between both subunits. We report, in this study, the X-ray structures of complexes with five ligands determined at a resolution of 1.7–2.05 A ˚ . These X-ray studies were completed with titration of b OBP by the same odorant m olecules, in competition with 1 -amino-anthracene (AMA). T he odor- ant m olecules were observed inside the two cavities formed by the b-barrels, together with some residual natural ligand (OCT) in three of the complexes. The interactions established with the protein were f ound to be essentially hydrophobic, although few hydrogen bonds were observed with ligands bearing polar groups. The internal cavity exhibits flexibility because Phe89 displays two discrete alternate positions, as in the native protein. These results confirm previous studies on pOBP and define the r ole of bOBP as a ble to recognize efficiently molecules p ertaining to differen t chemical classes. The absence of s ignificant conformational c hange at the protein surface is not in favor of an OR triggering role for bOBP, thus leaving this question still open for new experiments. Experimental procedures Protein and odorants Bovine and porcine OBP were purified from frozen samples of nasal mucosa, as reported previously [23,24]. The purified proteins showed a single band i n S DS/PAGE. AMA was purchased from Fluka. The odorants benzo- phenone (BZP), dihydromyrcenol or 2,6-dimethyl-7-octen- 2-ol (DHM) and undecanal (UND) were purchased fr om Fluka, a nd IBMP and OCT were from Aldrich. Stock solutions (2 m M ) o f t he odorants were prepared in ethanol, then further d iluted in 20 m M Tris/HCl, pH 7.8, 0.5% ( v/v) ethanol (TE buffer). In the binding studies, the odorant solutions in TE buffer were prepared just before each experiment and used only once. Fluorescence-binding assay The fluorescence-binding as say of AMA with bOBP, and the competition between AMA and odorants, were carried out according to a method published previously [27], with minor modifications [23]. T he influ ence of the concentration of ethanol on the chasing process of AMA was tested and found to be negligible up to 1% (v/v), a result in contrast with the behavior of r at OBP r eported by Briand et al .[28]. In brief, the formation of AMA–OBP c omplexes was determined by following an increase of the fluorescence emission at 480 nm, upon excitation at 380 nm, using a PerkinElmer LS 5 0 luminescence spectrometer. The disso- ciation constants of the AMA–OBP complexes were determined from the titration curves using the nonlinear fitting facility, SIGMA PLOT 5.0 (Cambridge Soft Corp., Cambridge, MA, USA). Saturation levels were determined from calibration curves obtained b y incubating increasing concentrations of AMA (0.076–5 l M ) with a fixed, satur- ating concentration of OBP (1 l M ). In the competition curves, the OBP samples (0.5 l M and 1 l M , respectively, for bOBP and pOBP) w ere incubated withafixedamountofAMA(3l M ) and increasing concentrations of odorants (0.39–50 l M ). The chasing of AMA bound to OBP was followed a s a decay of t he emission of the fluorescence intensity at 480 nm, a nd the curves, for each odorant, were analyzed u sing t he SIGMA PLOT 5.0 software for the determination of the apparent dissociation constants (K diss app.). K diss true values were calculated from apparent K diss , using the following formula: K diss true ¼ k diss app:  1=½1 þð1=K diss AMA ½AMAÞ which takes into account the K diss for AMA and the concentration of AMA. Crystallization, data collection and refinement of bovine OBP Except for the bOBP–UND complex, bOBP crystals were obtained by m icrodialysis of a 1 0 mgÆmL )1 protein solu- tion against 28–32% (v/v) ethanol, in 50 m M citrate, pH 5.4, at 4 °C. Crystals of the b OBP–UND co mplex were obtained b y vapour diffusion in t he presence of 18–40% (v/v) ethanol, 20 m M citrate, pH 4.2, 0.05% (v/v) poly(vinylpyrrolidone) 8 . All crystals belong to space group P2 1 ,withcelldimensionsa¼ 55.9 A ˚ ,b¼ 65.5 A ˚ , c ¼ 42.7 A ˚ and b ¼ 98 .8°, and contain one homodimer in the asymmetric u nit. The bOBP–odorant complexes were obtained by soaking the c rystals, overnight a t 4 °C, in synthetic crystallization solutions containing 2 m M odorant. For the chasing of AMA by IBMP, the same procedure as in the solution fluorescence study was followed: a crystal of bOBP was fi rst Ó FEBS 2004 Crystal structures of bOBP–odorant complexes 1 (Eur. J. Biochem. 271) 3833 soaked for 1 h at 4 °C in a reservoir solution containing 2m M AMA and then transferred to a reservoir containing 1m M IBMP and soaked for 4 h . Data collection, scaling and reduction were performed as with native bOBP (Table 1 ). D ata o n m icrodialysis cry stals were collected on a MAR Research 345 image plate (Mar Research, Norderstedt, Germany) placed on a R igaku RU2000 rotating anode with Osmic mirrors (Rigaku Corp., Tokyo, Japan). 9 Data collection was performed at room temperature, as cry ocooling a lways y ielded d ata s ets t hat could not be used in refinement. Only the data set on t he crystal of t he bOBP–UND complex was collected on flash- frozen crystals [12.5% (v/v) methylpentanediol] rm10;11 at 1 00K on DW32 ( LURE ). These data, in contrast with the o ther data sets, could be used and refined. Indexation and integration were performed using DENZO [29], data scaling with SCALA [30], and data reduction with TRUNCATE [30] (Table 1). The refinement of bOBP complexes made use of the previously determined bOBP structure as a starting model (1OBP). The atomic structure of the odorants was built using t he program TURBO [31], w hile topology and force field data were defined i n t he suitable files o f CNS [30] by the automated procedure XDICT (G.L.Kleywegt,Biomedical Centre, Uppsala University, Sweden; http://alpha2.bmc. uu.se/hicup) 12 . The models were then refined using CNS [32] for all bOBP–odorant complexes, e xcept for UND which was refined using REFMAC [33]. Cycles of r efinement were alternated with manual r e-fitting into sigmaA-weighted electron density maps with the graphic program [31]. The final models have R work and R free values of 18.8–22.0% and 22.3–23.9%, respectively (Table 1). The final models have good geometries according t o PROCHECK 13 [34]. The coordi- nates have been deposited with the PDB at RCSB (Table 1). Results Binding of AMA and odorants in solution The dissociation c onstants of the AMA–OBP c omplexes fo r the bovine and porcine OBP isoforms were, respectively, 1 .0 and 1.5 l M and the corresponding maximum saturation levels were 1.7 and 0.85 mol of AMA per mol of OBP. These results were in agreement with values already reported for these proteins when assayed with fluores- cence-binding tests using AMA [23,27,35]. X-ray crystallography experiments and co mpetitive bind- ing tests in solution indicated t hat AMA binds i n the internal cavities of bOBP and c ould be completely d isplaced by the natural ligand, OCT [3]. Therefore, competitive displacement of AMA by different odorants should titrate the same internal binding site of bOBP. The procedure used with O CT for chasing AMA was also used with the five other odorant molecules (Fig. 1). W ith all ligands b ut one (IBMP), t he stoichiometry was found to be close to two molecules of odorant per bOBP dimer. With IBMP, the stoichiometry was found to be close to one molecule of odorant per dimer, indicating that one of the two AMA molecules could not be chased by IBMP (see the structure section below). The calculated true K diss values were found to ran ge from 0.3 to 3.3 l M (Fig. 1 , Table 2). The values obtained w ith pOBP [13] are also reported for comparison (Table 2). Table 1. D ata collection a nd refinement statistics. AMA, 1-amin o-anthracene; BZP, b en zopheno ne; DHM, d ihydrom yrcenol; IBMP, 2-isobutyl- 3-metoxypyrazine; UND, undecanal. Ligands (upper row) and PDB entry (lower row) UND 1GT4 DHM 1GT3 AMA 1HN2 AMA/IBMP 1GT1 BZP 1GT5 Data collection Temperature (K) 100 300 300 300 300 Resolution limits (A ˚ ) 13–2.06 35–1.7 17–1.8 18.6–1.7 20–2.05 Completeness (%) 96.4 80.3 92.0 88.0 86.4 Redundancy 2.8 3.4 2.8 2.0 2.0 Rsym (all last/shell) 7.6/33 5.2/11.6 5.2/30.1 6.5/26.8 6/24.2 Refinement Resolution limits 9.5–2.1 10–1.8 0–1.8 10–1.71 18–2.08 Number of reflections 16 687 26 271 25 530 28 652 15 517 No. of atoms 2606 2640 2630 2637 2610 R factor /R free (%) 22.1/25.6 20.0/23.9 20.2/22.3 20.3/22.3 18.8/22.8 B factors (molecules A & B) Main chain 27.06/18.86 32.69/27.65 35.11/28.78 36.58/31.52 36.26/29.37 Side-chains Solvent 41.25 50.77 47.83 54.85 46.28 Ligands 68.63/59.9 55.08/48.87 49.07/49.77 50.55/46.92 34.07/34.46 Water molecules 126 184 121 179 97 rmsd Bonds (A ˚ )/angles 0.11/1.64 0.009/1.4 0.013/1.5 0.013/1.4 0.007/1.3 Dihedral angles 26.1 25.8 26.2 25.5 Improper angles 0.70 0.84 0.98 0.68 3834 F. Vincent et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Overall structures of the complexes As described previously, bOBP is a 2 · 159 residue dimer at neutral or basic pH, and a mon omer at pH values below 4.5 [36]. The 2.0 A ˚ resolution structu re of bOBP h as been reported p reviously [19,2 2] and its natural ligand has been identified [23]. Briefly, each monomer is composed of a lipocalin-type n ine-stranded b-bar rel comprising r esidues 15–121 (strands 1 –8) and residues 145–149 (strand 9) f rom the other monomer (Fig. 2). From residue 123 onwards, the topology diverges from the consensus lipocalin fold: the b-barrel is connected by an extended stretch of residues (123–126) to the a-helix protruding out of the b-bar rel and crossing the d imer interface (Fig. 2). A s a consequence, the a-helix of one monomer is placed close t o where the a-helix of the other monomer would be i f b OBP h ad a classical lipocalin fold, in a peculiar arrangement named domain swapping (Fig. 2 ). In the p resent structures, t he two b OBP polypeptidic chains are visible from residues 1–159 and 3–157 for molecules A and B, respectively. The e lectron density map of molecule A is generally better defined than that of molecule B, although its B factors are generally slightly higher (Table 1). When superimposing pairwise the Ca traces of the bOBP dimer for all complexes, the r msd values range b etween 0.06 A ˚ to 0 . 30 A ˚ , v alues w ithin positional errors at this resolution (1.64–1.3 A ˚ , i n t he present study and 1.8 A ˚ for the natural complex OCT–bOBP) [23]. Superposition of monomers A and B for each complex confirms also that the lack o f s ymmetry, owing to a different helix position relative to the b-barrel, is conserved. Complexes with the odorant compounds The internal cavities. An electron density accounting f or the presence of a bound ligand is found in each cavity of monomers A and B of bOBP (Fig. 3). As for the polypeptide chain, the electron d ensity map of the ligand is generally better defined in monomer A than in monomer B. The interpretation of the map is m ore difficult than in pOBP [37], however, because residual binding of the natural ligand ( OCT) occurred with some c omplexes and had to be estimated along th e refinement (Table 3). This is t he case for Fig. 1. 1-Amino-anthracene (AMA) chasing by different odorant molecules. The fluorescence of AMA decreases when chased by the odorant molecules; (A) 1-octen-3-ol, (B) und ecanal, (C) benzophenone, (D) 2-iso-butyl-3-metoxypyrazine and (E) d ihydrom yrcenol. Each fluorescence point on the y-axis s hows the concentration of AMA still b ound per monomer of bovine odorant- binding protein (bOBP) and p o rcine odorant- binding protein (pOBP), relative to t he initial valu e, on a scale of 0–1. These values are plotted as a function of the micromolar concentration of total odorant competing for binding (x-axis). The conc entrations of bOBP and pOBP were, re spectively, 0.5 and 1 l M , w hile AMA w as kept co nstant at 3 l M .(m), bOBP; ( d), pOBP. N on-linear fit was calculated using the nonlinear facility of SIGMA PLOT 5.0 ( Cambridge S oft Corp., Cambridge, M A, USA). Table 2. D issociation constants (K diss in l M ) of the fluorescent probe or of odorant molecules for bovine odorant-binding protein ( bOBP). AMA, 1-amino-anthracene; B ZP, benz ophenon e; DHM, d ihydromyrcen ol; IBMP, 2-isobutyl-3-metoxypyrazine; OCT, 1-octen-3-ol; bOBP, bovine odorant-binding protein; pOBP, porcine odorant-binding protein; UND, undecanal. K diss bOBP K diss pOBP AMA 1 1.5 OCT 1.2 2.7 UND 0.3 2.1 BZP 0.8 3.1 IBMP 3.3 0.3 DHM 0.35 0.1 Ó FEBS 2004 Crystal structures of bOBP–odorant complexes 1 (Eur. J. Biochem. 271) 3835 the complexes with AMA, AMA/IBMP and DHM. The complexes with BZP and UND do not include residual OCT (Table 3). The positions of the complete sets o f ligands clusters, when s uperimposed, result in a global volume of % 12 · 7 · 3A ˚ 3 (Fig. 4 A). They establish c ontacts w ith 17 residues f orming the cavity walls, among which 11 are hydrophobic and six are polar noncharged (Table 4, Fig. 4B). No contacts are established w ith charged residues as none point inside the cavity. Some r esidues are pre- eminently involved in interactions with the ligands (thresh- old 10 A ˚ 2 ), such as Phe36, Phe40, Phe54 , Phe89, Phe119 , and, above all, Asn103 14 (Table 4). The surface covered by the var ious ligands is rather homogeneous, with an average value o f 1 56 ± 20 A ˚ 2 (Table 4). While electron density maps of the side-chains are w ell defined a nd unique, Phe89 presents two main positions, as reported previously [19,22,35], i.e. in a closed position (Fig. 5A) or an open (Fig. 5 B) position. Phe89 may also be present in a mixture of alternate open and closed positions, leading to different cavity volumes. 15 As se en in Table 5 and in Fig. 5, the volume of the larger cavity is smaller when Phe89 is closed (Fig. 5 A), rather t han open (Fig. 5A), as th e internal volume is split into alternate large and s mall cavities. Indeed, t he cavity volumes with Phe89 in b oth open and closed positions are comparable t o those with Phe89 in the closed conformation. 16 OCT, the natural ligand. Although the structure of the complex of bOBP with OCT has been reported p reviously [23], w e will briefly recall its binding mode here. T he nature of OCT of bOBP was determined by GC/MS. The two enantiomers of OCT (ratio 1 : 1) were fit ted with the most appropriate conformations in the electron d ensity maps within each barrel, and their occupancies were refined in the CNS . The two isomers have very similar orientations, and are quasi superimposed (Fig. 3M,N). The aliphatic chains o f the two isomers are extended and remain close to each other. The hydroxyl groups point to the same direction, although to an area of the cavity walls where no hydrogen bond donor is available. AMA. The structure of the c omplex between bOBP and AMA h as already b een d escribed [23] an d we recall here these results and a more detailed analysis. AMA in complex with bOBP occupies the same place as the natural ligand, i.e. in the internal c avity of t he b-barrel of e ach monomer [19,35]. In cavity A, the initial electron density map for AMA was very clear and a molecule of AMA was fitted readily (Fig. 3A). The AMA molecule accounted for the total e lectron density, and its occupation was m aintained at 100% (Table 3). All residues i n the combining site are well defined in t he electron density map a nd do not exhibit alternate conformations. The side-chain of Phe89 presents Fig. 2. Bovine odorant-binding prote in (bOBP) dimer (secondary s tructure representation) and superposition o f all the ligands (sphere repre- sentation) in both cavities. (A) Mon omer A (green) and m onomer B (blue-grey). (B) The structure is r otated 90° towards t he reader with respect to (A); both monomers are rain- bow-colored. T he figure was produced using PYMOL [48]. 3836 F. Vincent et al.(Eur. J. Biochem. 271) Ó FEBS 2004 only the open conformation among the two observed i n the native protein (Table 5). In contrast, in cavity B t he electron density map around AMA and Phe89 displays complex features. Some residual density appears at several places when only the AMA molecule is taken i nto a ccount for the calculation of t he electron density m ap. These b ulbs of positive difference density disappear when 20% of the natural ligand and 80% of AMA are introduced in the calculations (Fig. 3B, Table 3). Phe89 has two c onforma- tions: the predominant one is the open conformation, found for Phe89 in cavity A; while the other conformation, with a slimmer d ensity, is the closed one, similar to t hat found in the native structure wit h its n atural ligand ( Table 5). The position of AMA in cavity B is v ery similar to that observed in cavity A. BZP. The map of the complex with BZP exhibits a c lear electron density for the ligand i n s ite A associated with low B-factors (Table 1, Fig. 3G). The ligand exhibits a unique conformation, and n o trace of the natural ligand is d etected in the electron density map (Table 3). The two phenyl rings of BZP for m a dihedral angle o f % 40° (Fig. 3 ). The carbonyl g roup of BZP establishes a canonic hydrogen bond (d ¼ 2.8 A ˚ ,angle¼ 126°) with t he hydroxyl group of Thr38. The other contacts are exclusively hydrophobic (Table 4). In cavity B, the position of BZP is also well defined and unique (Fig. 3 H, Table 3). The h ydrogen bond with Thr38 is longer than in subunit A (3.4 A ˚ )andits geometry less optimum. Phe89 is observed in both t he open and the closed positions in subunit A, a nd only in the closed position in subunit B (Table 5). DHM. The m ap of the DHM–bOBP c omplex exhibits intricate features. First, Phe89 displays alternate conforma- tions in both monomers, as in the case of O CT (Tab le 5). This is not surprising as both OCT and D HM exhibit a Fig. 3. View of differe nt ligands and the elec- tron density observed in c avities A and B o f bovine o dorant-binding protein (bOBP), respectively. (A,B) 1-Octen-3-ol (OCT) (bo th enantiomers) co-purified with b O BP. (C,D) 1-Amino-anthrace ne (AMA) and AMA and OCT. (E ,F) Benzophenone ( BZP). (G,H) D i- hydromyrcenol (DHM) and OCT. (I,J) AMA plus 2-iso-butyl-3-metoxypyrazine (IBMP), and AMA plus IBMP plus OCT. (K ,L) Undecanal (UND). Electron density maps were produced using TURBO [31] a nd contoured at the 1 r level. Li gands are r ep- resented in stick mode; color code: carbon ¼ yellow, oxygen ¼ red, nitrogen ¼ blue. Table 3. E stimates in percentage occupancy of the ligands and of the endogenous ligand [1-octen-3-ol (OCT)] in the cavity of bOBP m onomers A and B. AMA, 1-amino- anthracene ; BZP, benzoph enone; DHM, dihydromyrcenol; IBMP, 2-isobut yl-3-metoxypyrazine ; UND, unde - canal. Molecule A Molecule B OCT A OCT B AMA 100 80 0 20 AMA/IBMP 60 IBMP/ 40 AMA 50 IBMP/ 40 AMA 010 BZP 100 100 0 0 DHM 70 70 30 30 UND 100 100 0 0 Ó FEBS 2004 Crystal structures of bOBP–odorant complexes 1 (Eur. J. Biochem. 271) 3837 similar elongated shape. Furthermore, the electron density map could not be accounted fo r using only DHM, but the introduction of 70% DHM and 30% remaining OCT almost suppresses residual e lectron density m ap (Fig. 3I,J). The hydroxyl group of DHM exhibits a hydr ogen bond with the Asn103 Nd2 atom (3.0 A ˚ ). Fig. 4. Ribbon representation and molecular surface o f cavity A of bovine odo rant-binding protein ( bOBP). All ligands are shown super- imposed. (A) The ligands are r epresent ed (sticks, indivi dual colors) i nside the s labbed volume of the c avity. Left and right views a re rotated by 9 0°. ( B) Stereo view of the super- position of all ligands (sphere representation, individual color s) in cavity A of bOBP (transparent molecular surface). The side- chains (stick rep resentation, color code a s in Fig. 3) of the r esidues in the c avity interacting with the ligan ds are shown. T he figure was produced using PYMOL [48]. Table 4 . Inte raction surfac es (A ˚ 2 ) of the ligands with residues of c avities in monomers A an d B. These values were calculated by the surface value of each re sidue when t he cavity is empty f rom the surface value wh en the cavity is filled by the ligand (using the program TURBO )[31].AMA,1-amino- anthracene; BZP, b enz ophenone; DHM, dihydromyrcenol; IBMP, 2-is obutyl-3-metoxypyrazine; O CT, 1-octen-3-ol; UND, undecanal. OCT AMA AMA/IBMP BZP DHM UND ABABABABABAB Ile22 8 9 7 9 7 3 8 9 8 8 9 6 Phe36 18 21 18 19 18 13 18 20 18 19 17 17 Thr38 8 9 9 9 7 5 8 8 9 9 8 10 Phe40 11 9 13 10 12 12 11 10 12 11 13 6 Phe54 10 11 17 11 21 12 14 12 11 10 17 14 Phe56 8 11 8 9 8 10 8 8 8 9 8 9 Val69 7 6 6 6 6 5 6 5 7 7 8 6 Ala81 4 5 4 4 5 5 4 6 4 5 5 8 Tyr83 10 9 9 8 8 8 9 7 10 8 8 9 Asn87 7 8 6 5 7 5 5 4 6 5 6 6 Phe89 13 17 15 15 14 11 15 15 18 14 15 26 Ala101 3 3 10 3 13 4 4 4 4 3 11 3 Asn103 17 17 24 15 23 18 18 21 16 20 24 11 Leu115 8 8 8 7 9 6 8 7 7 7 7 8 Thr116 2 3 2 3 2 3 2 3 3 3 3 3 Gly117 2 3 3 3 3 3 3 3 3 3 2 3 Phe119 9 9 14 11 15 11 11 10 10 10 15 9 Total 145 154 173 147 178 134 152 152 154 151 168 154 3838 F. Vincent et al.(Eur. J. Biochem. 271) Ó FEBS 2004 IBMP. A c omplex with IBMP has b een submitted to X-ray analysis, aiming to u nderstand the peculiar behavior of IBMP in solution (Introduction and Experimental pro- cedures). With this aim, the procedure used t o obtain the complex reproduced exactly the fluorescence experiment, i.e. IBMP c hasing AMA bound to bOBP (see the Experimental procedures). In the s tructure, t he electron density m ap at both sites differed from that of AMA in the bOBP–AMA complex, and the size of the density indicates that some AMA is still bound (Fig. 3E,F). IBMP h as been modeled in t he electron density map of cavity A a nd B, and AMA h as been introduced. After refinement with CNS , including occupancy refinement for IBMP and AMA, the electon density map could b e modeled with 40% AMA and 60% IBMP, in cavity A (Fig. 3E, Table 3). In cavity B, some of the n atural lig and had also to be introduced, leading to o ccupation values of 42% AMA, 42% IBMP and 16% OCT ( Fig. 3F, T able 3). Although the occu pancy values may d iffer i n different experiments and should be interpreted w ith caution, these results confirm that in t he crystal, as in solution, only one IBMP molecule binds per bOBP dimer. In subunit A, Phe89 is found in the open conformation, while the closed one is found in subunit B (Table 5). UND. The UND molecule is better defined in subunit A where all atoms are present in the electron density map (Fig. 3 K). In subunit B , the last atoms of t he alkyl chain (C9-C11) are poorly defined (Fig. 3L). In both s ubunits, the occupancy of UND is 100% (Table 3). UND adopts a n extended conformation up to carbon 5 a nd then bends to form a U-shaped s tructure (Fig. 3K,L). The aldehyde function does not establish any hydrogen bond with the residues o f the cavity. The complex with UND is the only one where Phe89 does not assume alternate positions in either subunits: P he89 is in the open position in subunit A and in the closed position in subunit B (Table 5). In relation to the position o f P he89, t he position o f t he U-shaped part of UN D adopts opposite d irections in su bunits A and B (Fig. 3 K,L). Furthermore, the position adopted by UND in subunit A would not be compatible with the closed position of Phe89 in subunit B. Communication of the internal cavity with the bulk solvent The cavity of bOBP has no direct access to the solvent, which i s also t he case for other lipocalins, such as p OBP [24] or aphrodisin [3]. A unique side-chain shields the cavity from the solvent, however. Combined with t he open position of P he89, the rotation of Tyr83 by 120° (Fig. 6 Aa) opens a communication path to the internal cavity through which ligands might find access ( Fig. 6B). This rotation is not hindered by neighboring residues, and should therefore require a small amount of energy. This suggests strongly that Tyr83 may be the ÔdoorÕ of the cavity, and might trigger the access of t he natural substrate (Fig. 6 ). Furthermore, no crystal packing contacts involve this part of the bOBP surface, which faces water crystal channels. T his i s indeed the r eason why soaking odorant molecules in the crystals was successful. It is worthy of mention that in pOBP, Tyr83 is well situated to perform a similar r ole, and that the corresponding residue (Tyr76) in aphrodisin has a lready been proposed as being the cavity door [23]. Discussion The average of the K diss values for bOBP is comparable (1.2 l M ) t o that f ound for p OBP (1.6 l M ) (Table 2). The bOBP natural ligand, OCT, presents a slightly better affinity for bOBP than for pOBP. Amazingly, IBMP, taken historically as the r eference compound for binding studies with OBPs [1,2], is the poorest ligand of bOBP and an excellent ligand of pOBP; the affinity for bOBP i s 10 times smaller than for pOBP. There is no evident correlation between the bulkiness, the flexibility or the chemical functions borne by the different compounds and t heir affinity with either OBP. It should b e noted that the r atio of the K diss values for b OBP are grouped w ithin a factor of 10, meaning that the differences in energy involved are rather low. This range o f values i s s imilar to that observed for rat OBP-F1 (Nespoulos) and for rat OBPs 1–3, for e ach o f the proteins analyzed individually [17]. As mention ed above, the bOBP cavity is mostly hydro- phobic. Besides BZP, the ligands make little use of the five semipolar residues in the cavity to establish hydrogen Fig. 5. Rep resentation of the molecular surfaces of the cavities in monomer A. Left, bovine odoran t-binding protein-1/amino-anthracene (bOBP–AMA) complex. The Phe89 side-chain prese nts only the open position (yellow) and the cavity is unique ( pink). Right, b ovine odor- ant-binding prote in/benz yl-benzo ate ( bOBP–BZ P) c omplex, w ith t he Phe89 s ide-ch ain in alternate positions: th e open position is yellow, the closed p osition is orange. Note t hat the closed position of Phe89 sep- arates the c avity (as seen in the l eft view) into two (light and dark b lue). The figure w as produced u sing GRASP [49]. See also Table 5. Table 5. P osition o f Phe89 in the c avity of monomer A and B , in each complex. The positions can be open, closed or alternate (alt). The corresponding volumes (in A ˚ 3 ) o f the cavities are presented. AMA, 1-amino-anthracene; B ZP, benz ophenon e; DHM, d ihydromyrcen ol; IBMP, 2-isobutyl-3-metoxypyrazine; OCT, 1-octe n-3-ol; UND, undecanal. OCT AMA AMA/IBMP BZP DHM UND Phe89 A Alt Open Open Alt Alt Open B Alt Alt Alt Closed Alt Closed Volumes of the cavities A 396 491 504 396 400 443 B 377 378 401 375 377 377 Ó FEBS 2004 Crystal structures of bOBP–odorant complexes 1 (Eur. J. Biochem. 271) 3839 bonds. Consequently, 80% of the contact surface involves hydrophobic residues (Table 4). Bulky molecules, like AMA and BZP, exhibit very close positions in both subunits. However, e ven in these two clear-cut cases, the side-chain of Phe89 exhibits d ifferent orien tations in subunits A and B. Sligh t differences have also been observed in the position of the OCT molecules [ 35] a nd ar e obser ved here for the molecules of DHM. Only the UND m olecule exhibits a t otally opposite position in subunits A a nd B, clearly associated with the orientation of Phe89. The position o f Phe89 can be s omewhat r ationalized in terms of subunit. In subunit A , t he open position i s f avored, w ith three complexes exhibiting it, w hile three others present a mixture of closed a nd open positions; n o closed-only position is observed. In subunit B, two closed, four alternate and no open-only positions are observed (Table 5). All the conformational changes observed, however, are internal to bOBP, and do not affect the protein surface. It cannot be excluded, however, that the different conformations of Phe89 in each subunit result in a cross-talk of the monomers, triggere d b y s ubtle differences in b ackbone/ side-chain dynamics and controlling the access of a second molecule after t he binding of the first one. This mechanism might be more general in dime ric OBPs, as rat O BP-F1 has been shown t o d isplay an anti-cooperative mechanism [25]. Our results do not report a specificity of interaction between bOBP and the limited number of odorant molecules studied here. H owever, considering the drastic d ifferences between the m olecules chosen, a fine discriminating role of bOBP seems to be excluded. The situation concerning a putative role of OBPs in OR triggering, upon odorant binding, is far from clear. The high-affinity binding of empty pOBP to a human OR in mammalian cells does not favor t he role of OBP as ligand carrier [38]. Furthermore, the activity of the ORs in response to odorant molecules alone has been reported [39]. If a model s uch as bacterial chemotaxis was applicable to mammalian O BPs, it would b e expected that they would undergo a significant conformational change upon com- plexation. Such a behavior has been demonstrated for serum retinol-binding protein, which, upon a conforma- tional change o f a loop, can i nteract w ith a second protein, transthyretin, and prevent retinol excretion [40,41]. With bOBP, the only conformational change observed i n the crystal structure is internal to the m olecule. S imilarly, n o conformational changes have been detected by the struc- tural comparison between unliganded and liganded pOBP, nor were detected by IR spectroscopy [27]. However, solution binding or spectroscopic studies on rat OBPs [17,25] have demonstrated a certain specificity of rOBPs for wide substrate c lasses as well a s some conformational change upon receptor binding. In insects, antennal O BPs, pheromone-binding proteins (PBPs) and Chemosensory P roteins (CSPs) 17; areableto perform considerable conformational changes [42] and c ould be candidates for receptor t riggering, as suggested recently from electro-sensillar r ecording [ 43]. Fully sequenced insect genomes have shown the presence of multiple copies of putative OBP/PBP ORFs: 38 m embers in D rosophila mel- anogaster [44] and 29 members in Anopheles gambiae [45]. Amazingly, the number of ORs expressed in Drosophila is % 40 (on 6 2 candidates) [46,47], not disallowing the possible existence of f unctional O BP–OR c ouples. I n m ammals, t he number of O Rs is in the order of several hundred, while the number o f OBPs is at best a handful, not sufficient, by far, to suggest a specific functional interaction between a given OR with an OBP. Acknowledgements We thank D r Virna Conti f or valuable t echnical assistance. References 1. Bignetti, E., Cavaggioni, A., P elosi, P., Persaud, K.C., Sorbi, R.T. & T irindelli, R. (1985) P urification and characterisation o f an odorant-binding p rote in f rom c ow nasal t issue. Eur. J. Biochem. 149, 2 27–231. 2. Pevsner, J., Trifiletti, R.R., S trittmatter, S.M. & S nyder, S.H. (1985) Isolation and characterization of an olfactory receptor Fig. 6. Solvent-accessible surface o f b ovine o dora nt-bind ing pr otein ( bOBP ). (A) T he ribbo n r epre sentatio n (pu rple) a nd mole cular s urface we re calculated without taking i nto a ccount Tyr83, l eaving the c avity ope n. 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W The PYMOL Molecular Graphics System (http:// www.pymol.org) DeLano Scientific LLC, San Carlos, CA, USA 49 Nicholls, A., Sharp, K & Honig, B Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons Proteins: Struct Funct Genet 11, 281–296 . Crystal structures of bovine odorant- binding protein in complex with odorant molecules Florence Vincent 1 , Roberto Ramoni 2 , Silvia Spinelli 1 , Stefano Grolli 2 ,. changes involving protein loops or backbone. T hus, the role of OBP in signal triggering remains unresolved. 3 Keywords: crystal str ucture; domain swapping; odorant- binding protein; olfaction. Odorant- binding. ihydromyrcen ol; IBMP, 2-isobutyl-3-metoxypyrazine; OCT, 1-octen-3-ol; bOBP, bovine odorant- binding protein; pOBP, porcine odorant- binding protein; UND, undecanal. K diss bOBP K diss pOBP AMA