proteins STRUCTURE O FUNCTION O BIOINFORMATICS
STRUCTURE NOTE
Crystal structure of the human odorant binding protein, OBPIIa Andre Schiefner, Regina Freier, Andreas Eichinger, and Arne Skerra* Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl f€ ur Biologische Chemie, Technische Universit€at M€ unchen, 85350 Freising-Weihenstephan, Germany
ABSTRACT Human odorant-binding protein, OBPIIa, is expressed by nasal epithelia to facilitate transport of hydrophobic odorant molecules across the aqueous mucus. Here, we report its crystallographic analysis at 2.6 A˚ resolution. OBPIIa is a monomeric protein that exhibits the classical lipocalin fold with a conserved eight-stranded b-barrel harboring a remarkably large hydrophobic pocket. Basic residues within the four loops that shape the entrance to this ligand-binding site evoke a positive electrostatic potential. Human OBPIIa shows distinct features compared with other mammalian OBPs, including a potentially reactive Cys side chain within its pocket similar to human tear lipocalin. Proteins 2015; 00:000–000. C 2015 Wiley Periodicals, Inc. V
Key words: ligand binding; lipocalin; mammalian odorant binding protein; nasal epithelia; protein crystallization.
INTRODUCTION Odorants are chemically diverse, small hydrophobic volatile molecules that are recognized by olfactory receptors in the nasal epithelium. In order to reach their receptors, these substances first need to cross the hydrophilic barrier of the nasal mucus. Odorant binding proteins (OBPs), which are expressed in the nasal mucosa, are assumed to mediate this transport process by providing hydrophobic ligand pockets. These soluble proteins facilitate the phase transfer for the organic molecules from the hydrophobic gas phase into the aqueous solvent and, conversely, into the hydrophobic binding site of a receptor on the olfactory sensory neuron. In addition, OBPs may act as scavengers to prevent saturation of odorant receptors or to neutralize toxic volatiles. OBPs reversibly bind a variety of hydrophobic compounds with dissociation constants in the micromolar range.1 Several mammalian OBPs have been characterized, including those of cow, pig, rat, mouse, porcupine,
C 2015 WILEY PERIODICALS, INC. V
rabbit, and man. Most of these species express more than one OBP isoform, with up to nine OBP variants reported for porcupine.2 These isoforms can show considerable divergence in their amino acid sequences, which seems to correlate with distinct ligand-binding profiles.3 Nevertheless, all investigated OBPs exhibit a remarkably broad ligand binding activity.
Additional Supporting Information may be found in the online version of this article. Abbreviations: BSA, buried surface area; EDTA, ethylenediamine tetraacetic acid; IMAC, immobilized metal affinity chromatography; OBP, odorant binding protein; RMSD, root mean square deviation; SEC, size exclusion chromatography; Tlc, tear lipocalin Grant sponsor: Helmholtz-Zentrum Berlin. *Correspondence to: Prof. Dr. Arne Skerra, Lehrstuhl f€ ur Biologische Chemie, Technische Universit€at M€ unchen, Emil-Erlenmeyer-Forum 5, 85350 FreisingWeihenstephan, Germany. E-mail: [email protected] Received 16 January 2015; Revised 4 March 2015; Accepted 9 March 2015 Published online 21 March 2015 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.24797
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In humans, two closely related OBP genes have been identified and, based on their high amino acid sequence identity to the rat OBP2 (about 45%), were termed OBPIIa and OBPIIb.4 These genes are preferentially transcribed in different tissues. While OBPIIa is mainly found in nasal epithelia, lachrymal gland, and salivary gland, OBPIIb is primarily expressed in prostate and mammary gland. In addition, several splice variants for the human OBPs have been detected at the transcript level. Both OBPIIa and OBPIIb comprise 170 amino acid residues, including the 15 residues of the N-terminal signal peptide, which is cleaved off during secretion. The mature unglycosylated proteins with 155 residues share 90% identity. Human OBPIIa (hOBPIIa) was isolated from the nasal mucus that covers the olfactory cleft and has been intensely investigated for its ligand-binding activity. hOBPIIa binds a variety of structurally diverse ligands5 such as g-decalactone, a-pinene, and vanillin but also linear and branched aldehydes, branched alcohols, fatty acids as well as retinol and retinoic acid.6 Vertebrate OBPs belong to the lipocalin protein family1 whose members share a characteristic fold with an eight-stranded antiparallel b-barrel that exhibits a central pocket for ligand binding.7 Structures of four different mammalian OBPs, that is, the bovine (bOBP) and porcine (pOBP) protein as well as isoforms 1 and 3 of rat OBP (rOBP) were previously reported.8–12 While pOBP and the two rOBPs exhibit the canonical, monomeric lipocalin fold, bOBP constitutes a unique domainswapped homodimer. Here, we report the crystal structure of the monomeric human OBPIIa at 2.6 A˚ resolution and compare it with the previously determined mammalian OBP structures, revealing distinct features. MATERIALS AND METHODS Description of the expression, purification, X-ray data acquisition, and structure determination methods used in this study is given as Supporting Information. RESULTS AND DISCUSSION Tertiary structure of human OBPIIa
Several recombinant versions of OBPIIa were screened for crystallizability. Only the variant OBPIIa(99S/112N) carrying a short C-terminal His-tag yielded diffraction quality crystals of the space group P41212, diffracting synchroton X-rays to a resolution of 2.6 A˚ (Supporting Information Table S1). The asymmetric unit contained two polypeptide chains, corresponding to a solvent content of 48% and a Matthews coefficient of 2.37 A˚3/Da. Interpretable electron density was observed for residues 8–154 and 2–153 in molecules A and B, respectively, which overall share a similar fold with few local deviations (see below).
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hOBPIIa exists as a monomer in solution. Analytical size exclusion chromatography experiments showed an apparent molecular weight of 18.9 kDa, which is in good agreement with the calculated molecular mass of 18.6 kDa. Analysis with Protein Interfaces, Surfaces and Assemblies (PISA; http://www.ebi.ac.uk/msd-srv/prot_int/ cgi-bin/piserver) confirmed the monomeric state of OBPIIa in the crystal structure. hOBPIIa adopts the classical lipocalin fold comprising a central eight-stranded antiparallel b-barrel (strands A–H) with a C-terminal a-helix that packs against strands FGHA. A ninth short b-strand (I) occurs downstream of the a-helix and runs partially antiparallel to strand A (Fig. 1). Cys59 and Cys151 form a disulfide bridge between bstrand D and the C-terminus, thus restraining its conformational flexibility, as observed for most lipocalins.7,13 However, according to the stereochemical criteria of DSSP (http://swift.cmbi.ru.nl/gv/dssp), hOBPIIa does not exhibit the canonical 310-helix preceding b-strand A that has been described for other members of this protein family.7 The residues at the N-terminus of molecule A adopt a coiled structure and are rather flexible as indicated by the missing electron density for the first seven amino acids. In contrast, the same residues of molecule B adopt a coil followed by a short a-helix [Fig. 1(B)]. On the other hand, two 310-helical segments are found in loop #1 and in the loop connecting the canonical a-helix and strand I for molecule A but not for molecule B. As another peculiarity, b-strands B and I are separated into two shorter segments (B1/B2 and I1/I2) each by a Pro imino acid residue. Strand I1 runs antiparallel to A while strand I2 runs parallel to B1. At the open end of the b-barrel, strands A, G, and H of molecule A are shorter by 1, 2, and 2 residues, respectively, compared with molecule B [according to the conformational definition; cf. Fig. 1(B)]. Superposition of molecules A and B using the 58 conserved Ca positions of the b-barrel,13 corresponding to residues 11–20, 35–41, 46–52, 60–67, 74– 77, 82–89, 94–100, and 109–115 in hOBPIIa, results in a very low RMSD of 0.28 A˚. In contrast, the four loops #1–#4 (residues 21–34, 53–59, 78–81, and 101–108), which connect the antiparallel strands A–H at the open end of the b-barrel in a pairwise manner and thus form the entrance to the ligand pocket, show much larger RMSD values of 3.82, 0.98, 1.06, and 4.88 A˚, respectively. While considerable structural plasticity of these loops has been generally observed across the lipocalin family,13 this significant deviation between the two crystallographically distinct molecules indicates enhanced flexibility of the loop region in human OBP. A search for related three-dimensional structures was performed with PDBeFold (http://www.ebi.ac.uk/msdsrv/ssm). As expected from the overall sequence identity of 43% for the mature polypeptides, human tear lipocalin (Tlc, PDB code 3EYC),14 which in fact had allowed structure solution by molecular replacement (see Supporting Information), exhibits the fold most similar to hOBPIIa (chain A). Tlc shows an overall RMSD of 1.65 A˚
X-Ray Structure of OBPIIa
Figure 1 Crystal structure of human OBPIIa. A: Molecule A in the asymmetric unit: a-helices, b-strands, and loops are colored pale green, light blue, and gray, respectively. The disulfide bond that connects Cys residues 59 and 151 (light orange) and the citrate ion (C: gray; O: red) are depicted as ball-and-sticks. All side chains that interact with the citrate are shown as sticks. The missing side chains of Cys99 and Lys112 inside the cavity were modeled in plausible conformations and depicted as violet sticks. B: Superposition of molecule B (yellow) from the asymmetric unit onto molecule A (light blue). Strands of the central b-barrel are alphabetically labeled. C: Illustration of the OBPIIa ligand pocket. The polypeptide is shown as ribbon (gray) while the pocket surface is colored according to hydrophobicity, from brown (hydrophobic) over white (neutral/peptide backbone) to green (polar). D: Electrostatic surface potential for OBPIIa from 210 kBT/e (red) to 110 kBT/e (blue), with view into the pocket.
for 139 Ca positions, and of merely 0.99 A˚ for the 58 conserved Ca positions of the b-barrel mentioned above. This hit was followed by the galline extracellular fatty acid binding protein (Ex-FABP; PDB code 3SAO) and the rat epididymal retinoic acid binding protein (ERABP; PDB code 1EPA) with RMSD values of 2.0 A˚ and 1.8 A˚, respectively, for 134 and 132 Ca positions. Ligand pocket of hOBPIIa
Inside the b-barrel, OBPIIa harbors an astonishingly large hydrophobic cavity [Fig. 1(C)], which is in accordance with its binding activity toward a variety of hydrophobic compounds with diverse molecular structures and sizes.5 In contrast, the entrance to the ligand pocket is
lined by polar and mostly positively charged side chains. Depending on the differing conformations of loops #1– #4 in molecules A and B of OBPIIa, the pocket volume varies between 908 and 1316 A˚3 as determined by CASTp (http://sts.bioe.uic.edu/castp). This structural flexibility, together with the intricate shape of the cavity, explains the particularly broad ligand specificity of this lipocalin. Although a pI of 7.9 for mature OBPIIa would suggest an almost uncharged protein under physiological pH conditions, electrostatic calculation with ABPS (http://www. poissonboltzmann.org/docs/calculating) revealed a distinct charge distribution across its protein surface [cf. Fig. 1(D)]. The entire ligand pocket and, especially, the loop region at its entrance exhibit a positive potential. Indeed, all amino acid side chains that point into the cavity are PROTEINS
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Figure 2 Structural comparison of hOBPIIa with other mammalian OBPs. A: Multiple structural superposition between hOBPIIa (chain A, blue), rOBP1 (3FIQ chain A, orange), rOBP3 (3ZQ3 chain B, pink), pOBP (1DZM chain A, green), and bOBP (1GT1 chain segments A1–1211B122–159, brown). Disulfide bridges are indicated as ball-and-sticks. The set of 58 conserved b-barrel residues13 is colored black. B: Structure-based sequence alignment using a cutoff of 3.5 A˚ for matching residues. Lower case letters indicate N- and C-terminal amino acids not resolved in the electron density. The swapped C-terminal sequence segment of bOBP is underlined. Residues in helical and strand conformations according to DSSP are colored light green and light blue, respectively. Consensus secondary structure elements and conserved amino acid residues are labeled above the alignment. In this context, lower case letters indicate conservation in at least four sequences, whereas upper case letters mark full conservation. The 58 structurally conserved b-barrel residues are labeled underneath (=). Cys residues involved in disulfide bridges and unpaired Cys residues are colored yellow and orange, respectively. For the purpose of comparison, the sequence of hOBPIIb has been included in the alignment without highlighting structural elements; here, interspersed lower case letters mark differences from the amino acid sequence of hOBPIIa.
either hydrophobic or positively charged, with the single exception of Asp101 at the mouth of the pocket. Apart from an influence on ligand specificity this charge pattern may also play a role for receptor interactions. The side chains of both Cys99 and Lys112, which were replaced by Ser and Asn, respectively, for the purpose of protein crystallization, point into the ligand cavity. Lys112 is located in strand H and was previously identified as a binding partner for aldehydes.15 Although Cys99 in the neighboring strand G has not been recognized to be important for ligand binding so far, its basic environment may facilitate deprotonation and, thus, allow covalent interaction with aldehydes via thiohemiacetal formation or with odorants carrying thiol groups through a disulfide bridge. The two human isoforms OBPIIa and OBPIIb are expressed in distinct tissues,4 where they may encounter different ligand spectra. However, their overall high sequence similarity mentioned above, with mostly conserved properties of the differing residues (cf. Fig. 2), indicate related ligand profiles. Moreover, only five of the 17 differing amino acids structurally contribute to the binding site. These include positions 75 (Ile/Met), 84 (Phe/Tyr), 103 (Arg/His), 104 (Arg/His), and 108 (Arg/His), of which the first two are located within the pocket and the latter three are situated at its entrance. Unexpectedly, in the crystal structure of hOBPIIa a negatively charged citrate molecule from the crystallization
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buffer was observed bound to molecule A (Fig. 1, Supporting Information Fig. S1). However, citrate-binding activity has not been reported for this lipocalin and is probably not of physiological relevance. Notably, the citrate ion does not bind inside the large hydrophobic pocket but rather at its hydrophilic opening, thereby blocking access to the cavity for other ligands. Nine residues of hOBPIIa are in van der Waals distance to the citrate; among these, residues Arg29, Lys62, and Lys82 form salt bridges to the anion. In addition, citrate participates in three hydrogen bonds, two with residues Arg55 and Tyr78 and one with a water molecule that is trapped inside the ligand pocket (Supporting Information Fig. S1). In contrast, the entrance to the cavity of molecule B is much wider, due to considerably different loop conformations, as explained above [Fig. 1(B)]. Comparison of hOBPIIa with other mammalian OBPs
The crystal structure of hOBPIIa was compared with all available three-dimensional structures of other mammalian OBPs (Fig. 2): bOBP,16 pOBP,10 rOBP1,11 and rOBP3.12 At the amino acid sequence level, these proteins show only distant relation to mature hOBPIIa, with 16, 15, 19, and 20% identity, respectively. To achieve proper superposition of the domain-swapped bOBP dimer with the other monomeric OBPs, its monomers A
X-Ray Structure of OBPIIa
and B were split between residues 121 and 122 and the combined polypeptide segments 1–121 from chain A and 122–159 from chain B were treated as one entity. Structural sequence alignment with SALIGN (http:// modbase.compbio.ucsf.edu/salign) indicated that conserved residues occur only at four equivalent positions among these five OBPs [Fig. 2(B)]. Two of these residues form the lipocalin signature motif “GXW” within bstrand A.7 In further 22 structurally equivalent positions the same amino acid occurs in four of the five OBPs. Contrasting with the crystal structures of mammalian OBPs reported previously, which show high mutual similarity, hOBPIIa exhibits significant deviations in its loop regions, both at the open end (#1–#4) and at the closed end of the b-barrel. In particular, the segment comprising loop #1, the adjoining b-strand B1, and loop #3 are bent away from the b-barrel axis. The conformational difference of strand B1 also causes a displacement of the C-terminal coiled segment (beyond strand I), which adopts a conformation in OBPIIa that differs from all other OBP structures. Except for bOBP, which lacks any Cys residues, all other OBPs exhibit the typical disulfide bridge that links bstrand D and the C-terminus. A second disulfide bridge is found in rOBP1, while unpaired Cys residues only occur in hOBPIIa and rOBP3. However, only in hOBPIIa the free Cys side-chain points into the ligand pocket, where it may be involved in ligand binding. Interestingly, this unpaired Cys is structurally conserved in human Tlc.14 A sequence search through the UniProt database (http://www.uniprot.org) revealed several presumed OBP sequences in primates. These were directly followed by a putative bovine OBP2b sequence (UniProt ID: F1MK50) that shows 57 and 61% identical residues, respectively, with hOBPIIa and hOBPIIb. Remarkably, in contrast to the above mentioned domain-swapped bOBP structure, this up to now uncharacterized bovine protein appears to carry the canonical disulfide bridge between b-strand D and the C-terminus and, thus, should also be a monomeric lipocalin. Moreover, bOBP2b exhibits a Cys residue equivalent to Cys99 in both human orthologs. On the other hand, while the amino acid sequence of rOBP2 also shares high homology with hOBPIIa (45% sequence identity), a porcine OBP isoform with comparable sequence similarity to the human OBPs (versus the merely 15% identity of pOBP mentioned above) is unknown to date. Interestingly, the most similar sequence to hOBPIIa in pigs is that of porcine tear lipocalin, with 39% sequence identity, a number close to the 43% sequence identity for human Tlc already mentioned above. Again, both the disulfide bridge and the free Cys residue within the ligand pocket are conserved in this case. Taken together, elucidation of the crystal structure for hOBPIIa has unraveled previously unknown features of this abundant protein, which provide a basis for future studies on ligand- and receptor-binding functions as well as its broader biological role. Coordinates and structure
factors of the refined hOBPIIa X-ray structure have been deposited in the Protein Data Bank (PDB; www.rcsb.org) under accession code 4RUN.
AUTHORS CONTRIBUTION A. Sc., A. E., and A. Sk. designed the research. A. Sc., A. E., and R. F. performed the experiments and data analysis. A. Sc. and A. Sk. wrote the article. REFERENCES 1. Tegoni M, Pelosi P, Vincent F, Spinelli S, Campanacci V, Grolli S, Ramoni R, Cambillau C. Mammalian odorant binding proteins. Biochim Biophys Acta 2000;1482:229–240. 2. Ganni M, Garibotti M, Scaloni A, Pucci P, Pelosi P. Microheterogeneity of odorant-binding proteins in the porcupine revealed by Nterminal sequencing and mass spectrometry. Comp Biochem Physiol B Biochem Mol Biol 1997;117:287–291. 3. L€ obel D, Jacob M, V€ olkner M, Breer H. Odorants of different chemical classes interact with distinct odorant binding protein subtypes. Chem Senses 2002;27:39–44. 4. Lacazette E, Gachon AM, Pitiot G. A novel human odorant-binding protein gene family resulting from genomic duplicons at 9q34: differential expression in the oral and genital spheres. Hum Mol Genet 2000;9:289–301. 5. Briand L, Eloit C, Nespoulous C, Bezirard V, Huet JC, Henry C, Blon F, Trotier D, Pernollet JC. Evidence of an odorant-binding protein in the human olfactory mucus: location, structural characterization, and odorant-binding properties. Biochemistry 2002;41: 7241–7252. 6. Breustedt DA, Sch€ onfeld DL, Skerra A. Comparative ligand-binding analysis of ten human lipocalins. Biochim Biophys Acta 2006;1764: 161–173. 7. Flower DR. The lipocalin protein family: structure and function. Biochem J 1996;318:1–14. 8. Tegoni M, Ramoni R, Bignetti E, Spinelli S, Cambillau C. Domain swapping creates a third putative combining site in bovine odorant binding protein dimer. Nat Struct Biol 1996;3:863–867. 9. Bianchet MA, Bains G, Pelosi P, Pevsner J, Snyder SH, Monaco HL, Amzel LM. The three-dimensional structure of bovine odorant binding protein and its mechanism of odor recognition. Nat Struct Biol 1996;3:934–939. 10. Vincent F, Spinelli S, Ramoni R, Grolli S, Pelosi P, Cambillau C, Tegoni M. Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes. J Mol Biol 2000;300:127–139. 11. White SA, Briand L, Scott DJ, Borysik AJ. Structure of rat odorantbinding protein OBP1 at 1.6 A˚ resolution. Acta Crystallogr D Biol Crystallogr 2009;65:403–410. 12. Portman KL, Long J, Carr S, Briand L, Winzor DJ, Searle MS, Scott DJ. Enthalpy/entropy compensation effects from cavity desolvation underpin broad ligand binding selectivity for rat odorant binding protein 3. Biochemistry 2014;53:2371–2379. 13. Skerra A. Lipocalins as a scaffold. Biochim Biophys Acta 2000;1482: 337–350. 14. Breustedt DA, Chatwell L, Skerra A. A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity. Acta Crystallogr D Biol Crystallogr 2009;65:1118–1125. 15. Tcatchoff L, Nespoulous C, Pernollet JC, Briand L. A single lysyl residue defines the binding specificity of a human odorant-binding protein for aldehydes. FEBS Lett 2006;580:2102–2108. 16. Vincent F, Ramoni R, Spinelli S, Grolli S, Tegoni M, Cambillau C. Crystal structures of bovine odorant-binding protein in complex with odorant molecules. Eur J Biochem 2004;271:3832–3842.
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STRUCTURE NOTE
Crystal structure of the human odorant binding protein, OBPIIa Andre Schiefner, Regina Freier, Andreas Eichinger, and Arne Skerra* Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl f€ ur Biologische Chemie, Technische Universit€at M€ unchen, 85350 Freising-Weihenstephan, Germany
ABSTRACT Human odorant-binding protein, OBPIIa, is expressed by nasal epithelia to facilitate transport of hydrophobic odorant molecules across the aqueous mucus. Here, we report its crystallographic analysis at 2.6 A˚ resolution. OBPIIa is a monomeric protein that exhibits the classical lipocalin fold with a conserved eight-stranded b-barrel harboring a remarkably large hydrophobic pocket. Basic residues within the four loops that shape the entrance to this ligand-binding site evoke a positive electrostatic potential. Human OBPIIa shows distinct features compared with other mammalian OBPs, including a potentially reactive Cys side chain within its pocket similar to human tear lipocalin. Proteins 2015; 00:000–000. C 2015 Wiley Periodicals, Inc. V
Key words: ligand binding; lipocalin; mammalian odorant binding protein; nasal epithelia; protein crystallization.
INTRODUCTION Odorants are chemically diverse, small hydrophobic volatile molecules that are recognized by olfactory receptors in the nasal epithelium. In order to reach their receptors, these substances first need to cross the hydrophilic barrier of the nasal mucus. Odorant binding proteins (OBPs), which are expressed in the nasal mucosa, are assumed to mediate this transport process by providing hydrophobic ligand pockets. These soluble proteins facilitate the phase transfer for the organic molecules from the hydrophobic gas phase into the aqueous solvent and, conversely, into the hydrophobic binding site of a receptor on the olfactory sensory neuron. In addition, OBPs may act as scavengers to prevent saturation of odorant receptors or to neutralize toxic volatiles. OBPs reversibly bind a variety of hydrophobic compounds with dissociation constants in the micromolar range.1 Several mammalian OBPs have been characterized, including those of cow, pig, rat, mouse, porcupine,
C 2015 WILEY PERIODICALS, INC. V
rabbit, and man. Most of these species express more than one OBP isoform, with up to nine OBP variants reported for porcupine.2 These isoforms can show considerable divergence in their amino acid sequences, which seems to correlate with distinct ligand-binding profiles.3 Nevertheless, all investigated OBPs exhibit a remarkably broad ligand binding activity.
Additional Supporting Information may be found in the online version of this article. Abbreviations: BSA, buried surface area; EDTA, ethylenediamine tetraacetic acid; IMAC, immobilized metal affinity chromatography; OBP, odorant binding protein; RMSD, root mean square deviation; SEC, size exclusion chromatography; Tlc, tear lipocalin Grant sponsor: Helmholtz-Zentrum Berlin. *Correspondence to: Prof. Dr. Arne Skerra, Lehrstuhl f€ ur Biologische Chemie, Technische Universit€at M€ unchen, Emil-Erlenmeyer-Forum 5, 85350 FreisingWeihenstephan, Germany. E-mail: [email protected] Received 16 January 2015; Revised 4 March 2015; Accepted 9 March 2015 Published online 21 March 2015 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.24797
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In humans, two closely related OBP genes have been identified and, based on their high amino acid sequence identity to the rat OBP2 (about 45%), were termed OBPIIa and OBPIIb.4 These genes are preferentially transcribed in different tissues. While OBPIIa is mainly found in nasal epithelia, lachrymal gland, and salivary gland, OBPIIb is primarily expressed in prostate and mammary gland. In addition, several splice variants for the human OBPs have been detected at the transcript level. Both OBPIIa and OBPIIb comprise 170 amino acid residues, including the 15 residues of the N-terminal signal peptide, which is cleaved off during secretion. The mature unglycosylated proteins with 155 residues share 90% identity. Human OBPIIa (hOBPIIa) was isolated from the nasal mucus that covers the olfactory cleft and has been intensely investigated for its ligand-binding activity. hOBPIIa binds a variety of structurally diverse ligands5 such as g-decalactone, a-pinene, and vanillin but also linear and branched aldehydes, branched alcohols, fatty acids as well as retinol and retinoic acid.6 Vertebrate OBPs belong to the lipocalin protein family1 whose members share a characteristic fold with an eight-stranded antiparallel b-barrel that exhibits a central pocket for ligand binding.7 Structures of four different mammalian OBPs, that is, the bovine (bOBP) and porcine (pOBP) protein as well as isoforms 1 and 3 of rat OBP (rOBP) were previously reported.8–12 While pOBP and the two rOBPs exhibit the canonical, monomeric lipocalin fold, bOBP constitutes a unique domainswapped homodimer. Here, we report the crystal structure of the monomeric human OBPIIa at 2.6 A˚ resolution and compare it with the previously determined mammalian OBP structures, revealing distinct features. MATERIALS AND METHODS Description of the expression, purification, X-ray data acquisition, and structure determination methods used in this study is given as Supporting Information. RESULTS AND DISCUSSION Tertiary structure of human OBPIIa
Several recombinant versions of OBPIIa were screened for crystallizability. Only the variant OBPIIa(99S/112N) carrying a short C-terminal His-tag yielded diffraction quality crystals of the space group P41212, diffracting synchroton X-rays to a resolution of 2.6 A˚ (Supporting Information Table S1). The asymmetric unit contained two polypeptide chains, corresponding to a solvent content of 48% and a Matthews coefficient of 2.37 A˚3/Da. Interpretable electron density was observed for residues 8–154 and 2–153 in molecules A and B, respectively, which overall share a similar fold with few local deviations (see below).
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hOBPIIa exists as a monomer in solution. Analytical size exclusion chromatography experiments showed an apparent molecular weight of 18.9 kDa, which is in good agreement with the calculated molecular mass of 18.6 kDa. Analysis with Protein Interfaces, Surfaces and Assemblies (PISA; http://www.ebi.ac.uk/msd-srv/prot_int/ cgi-bin/piserver) confirmed the monomeric state of OBPIIa in the crystal structure. hOBPIIa adopts the classical lipocalin fold comprising a central eight-stranded antiparallel b-barrel (strands A–H) with a C-terminal a-helix that packs against strands FGHA. A ninth short b-strand (I) occurs downstream of the a-helix and runs partially antiparallel to strand A (Fig. 1). Cys59 and Cys151 form a disulfide bridge between bstrand D and the C-terminus, thus restraining its conformational flexibility, as observed for most lipocalins.7,13 However, according to the stereochemical criteria of DSSP (http://swift.cmbi.ru.nl/gv/dssp), hOBPIIa does not exhibit the canonical 310-helix preceding b-strand A that has been described for other members of this protein family.7 The residues at the N-terminus of molecule A adopt a coiled structure and are rather flexible as indicated by the missing electron density for the first seven amino acids. In contrast, the same residues of molecule B adopt a coil followed by a short a-helix [Fig. 1(B)]. On the other hand, two 310-helical segments are found in loop #1 and in the loop connecting the canonical a-helix and strand I for molecule A but not for molecule B. As another peculiarity, b-strands B and I are separated into two shorter segments (B1/B2 and I1/I2) each by a Pro imino acid residue. Strand I1 runs antiparallel to A while strand I2 runs parallel to B1. At the open end of the b-barrel, strands A, G, and H of molecule A are shorter by 1, 2, and 2 residues, respectively, compared with molecule B [according to the conformational definition; cf. Fig. 1(B)]. Superposition of molecules A and B using the 58 conserved Ca positions of the b-barrel,13 corresponding to residues 11–20, 35–41, 46–52, 60–67, 74– 77, 82–89, 94–100, and 109–115 in hOBPIIa, results in a very low RMSD of 0.28 A˚. In contrast, the four loops #1–#4 (residues 21–34, 53–59, 78–81, and 101–108), which connect the antiparallel strands A–H at the open end of the b-barrel in a pairwise manner and thus form the entrance to the ligand pocket, show much larger RMSD values of 3.82, 0.98, 1.06, and 4.88 A˚, respectively. While considerable structural plasticity of these loops has been generally observed across the lipocalin family,13 this significant deviation between the two crystallographically distinct molecules indicates enhanced flexibility of the loop region in human OBP. A search for related three-dimensional structures was performed with PDBeFold (http://www.ebi.ac.uk/msdsrv/ssm). As expected from the overall sequence identity of 43% for the mature polypeptides, human tear lipocalin (Tlc, PDB code 3EYC),14 which in fact had allowed structure solution by molecular replacement (see Supporting Information), exhibits the fold most similar to hOBPIIa (chain A). Tlc shows an overall RMSD of 1.65 A˚
X-Ray Structure of OBPIIa
Figure 1 Crystal structure of human OBPIIa. A: Molecule A in the asymmetric unit: a-helices, b-strands, and loops are colored pale green, light blue, and gray, respectively. The disulfide bond that connects Cys residues 59 and 151 (light orange) and the citrate ion (C: gray; O: red) are depicted as ball-and-sticks. All side chains that interact with the citrate are shown as sticks. The missing side chains of Cys99 and Lys112 inside the cavity were modeled in plausible conformations and depicted as violet sticks. B: Superposition of molecule B (yellow) from the asymmetric unit onto molecule A (light blue). Strands of the central b-barrel are alphabetically labeled. C: Illustration of the OBPIIa ligand pocket. The polypeptide is shown as ribbon (gray) while the pocket surface is colored according to hydrophobicity, from brown (hydrophobic) over white (neutral/peptide backbone) to green (polar). D: Electrostatic surface potential for OBPIIa from 210 kBT/e (red) to 110 kBT/e (blue), with view into the pocket.
for 139 Ca positions, and of merely 0.99 A˚ for the 58 conserved Ca positions of the b-barrel mentioned above. This hit was followed by the galline extracellular fatty acid binding protein (Ex-FABP; PDB code 3SAO) and the rat epididymal retinoic acid binding protein (ERABP; PDB code 1EPA) with RMSD values of 2.0 A˚ and 1.8 A˚, respectively, for 134 and 132 Ca positions. Ligand pocket of hOBPIIa
Inside the b-barrel, OBPIIa harbors an astonishingly large hydrophobic cavity [Fig. 1(C)], which is in accordance with its binding activity toward a variety of hydrophobic compounds with diverse molecular structures and sizes.5 In contrast, the entrance to the ligand pocket is
lined by polar and mostly positively charged side chains. Depending on the differing conformations of loops #1– #4 in molecules A and B of OBPIIa, the pocket volume varies between 908 and 1316 A˚3 as determined by CASTp (http://sts.bioe.uic.edu/castp). This structural flexibility, together with the intricate shape of the cavity, explains the particularly broad ligand specificity of this lipocalin. Although a pI of 7.9 for mature OBPIIa would suggest an almost uncharged protein under physiological pH conditions, electrostatic calculation with ABPS (http://www. poissonboltzmann.org/docs/calculating) revealed a distinct charge distribution across its protein surface [cf. Fig. 1(D)]. The entire ligand pocket and, especially, the loop region at its entrance exhibit a positive potential. Indeed, all amino acid side chains that point into the cavity are PROTEINS
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A. Schiefner et al.
Figure 2 Structural comparison of hOBPIIa with other mammalian OBPs. A: Multiple structural superposition between hOBPIIa (chain A, blue), rOBP1 (3FIQ chain A, orange), rOBP3 (3ZQ3 chain B, pink), pOBP (1DZM chain A, green), and bOBP (1GT1 chain segments A1–1211B122–159, brown). Disulfide bridges are indicated as ball-and-sticks. The set of 58 conserved b-barrel residues13 is colored black. B: Structure-based sequence alignment using a cutoff of 3.5 A˚ for matching residues. Lower case letters indicate N- and C-terminal amino acids not resolved in the electron density. The swapped C-terminal sequence segment of bOBP is underlined. Residues in helical and strand conformations according to DSSP are colored light green and light blue, respectively. Consensus secondary structure elements and conserved amino acid residues are labeled above the alignment. In this context, lower case letters indicate conservation in at least four sequences, whereas upper case letters mark full conservation. The 58 structurally conserved b-barrel residues are labeled underneath (=). Cys residues involved in disulfide bridges and unpaired Cys residues are colored yellow and orange, respectively. For the purpose of comparison, the sequence of hOBPIIb has been included in the alignment without highlighting structural elements; here, interspersed lower case letters mark differences from the amino acid sequence of hOBPIIa.
either hydrophobic or positively charged, with the single exception of Asp101 at the mouth of the pocket. Apart from an influence on ligand specificity this charge pattern may also play a role for receptor interactions. The side chains of both Cys99 and Lys112, which were replaced by Ser and Asn, respectively, for the purpose of protein crystallization, point into the ligand cavity. Lys112 is located in strand H and was previously identified as a binding partner for aldehydes.15 Although Cys99 in the neighboring strand G has not been recognized to be important for ligand binding so far, its basic environment may facilitate deprotonation and, thus, allow covalent interaction with aldehydes via thiohemiacetal formation or with odorants carrying thiol groups through a disulfide bridge. The two human isoforms OBPIIa and OBPIIb are expressed in distinct tissues,4 where they may encounter different ligand spectra. However, their overall high sequence similarity mentioned above, with mostly conserved properties of the differing residues (cf. Fig. 2), indicate related ligand profiles. Moreover, only five of the 17 differing amino acids structurally contribute to the binding site. These include positions 75 (Ile/Met), 84 (Phe/Tyr), 103 (Arg/His), 104 (Arg/His), and 108 (Arg/His), of which the first two are located within the pocket and the latter three are situated at its entrance. Unexpectedly, in the crystal structure of hOBPIIa a negatively charged citrate molecule from the crystallization
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buffer was observed bound to molecule A (Fig. 1, Supporting Information Fig. S1). However, citrate-binding activity has not been reported for this lipocalin and is probably not of physiological relevance. Notably, the citrate ion does not bind inside the large hydrophobic pocket but rather at its hydrophilic opening, thereby blocking access to the cavity for other ligands. Nine residues of hOBPIIa are in van der Waals distance to the citrate; among these, residues Arg29, Lys62, and Lys82 form salt bridges to the anion. In addition, citrate participates in three hydrogen bonds, two with residues Arg55 and Tyr78 and one with a water molecule that is trapped inside the ligand pocket (Supporting Information Fig. S1). In contrast, the entrance to the cavity of molecule B is much wider, due to considerably different loop conformations, as explained above [Fig. 1(B)]. Comparison of hOBPIIa with other mammalian OBPs
The crystal structure of hOBPIIa was compared with all available three-dimensional structures of other mammalian OBPs (Fig. 2): bOBP,16 pOBP,10 rOBP1,11 and rOBP3.12 At the amino acid sequence level, these proteins show only distant relation to mature hOBPIIa, with 16, 15, 19, and 20% identity, respectively. To achieve proper superposition of the domain-swapped bOBP dimer with the other monomeric OBPs, its monomers A
X-Ray Structure of OBPIIa
and B were split between residues 121 and 122 and the combined polypeptide segments 1–121 from chain A and 122–159 from chain B were treated as one entity. Structural sequence alignment with SALIGN (http:// modbase.compbio.ucsf.edu/salign) indicated that conserved residues occur only at four equivalent positions among these five OBPs [Fig. 2(B)]. Two of these residues form the lipocalin signature motif “GXW” within bstrand A.7 In further 22 structurally equivalent positions the same amino acid occurs in four of the five OBPs. Contrasting with the crystal structures of mammalian OBPs reported previously, which show high mutual similarity, hOBPIIa exhibits significant deviations in its loop regions, both at the open end (#1–#4) and at the closed end of the b-barrel. In particular, the segment comprising loop #1, the adjoining b-strand B1, and loop #3 are bent away from the b-barrel axis. The conformational difference of strand B1 also causes a displacement of the C-terminal coiled segment (beyond strand I), which adopts a conformation in OBPIIa that differs from all other OBP structures. Except for bOBP, which lacks any Cys residues, all other OBPs exhibit the typical disulfide bridge that links bstrand D and the C-terminus. A second disulfide bridge is found in rOBP1, while unpaired Cys residues only occur in hOBPIIa and rOBP3. However, only in hOBPIIa the free Cys side-chain points into the ligand pocket, where it may be involved in ligand binding. Interestingly, this unpaired Cys is structurally conserved in human Tlc.14 A sequence search through the UniProt database (http://www.uniprot.org) revealed several presumed OBP sequences in primates. These were directly followed by a putative bovine OBP2b sequence (UniProt ID: F1MK50) that shows 57 and 61% identical residues, respectively, with hOBPIIa and hOBPIIb. Remarkably, in contrast to the above mentioned domain-swapped bOBP structure, this up to now uncharacterized bovine protein appears to carry the canonical disulfide bridge between b-strand D and the C-terminus and, thus, should also be a monomeric lipocalin. Moreover, bOBP2b exhibits a Cys residue equivalent to Cys99 in both human orthologs. On the other hand, while the amino acid sequence of rOBP2 also shares high homology with hOBPIIa (45% sequence identity), a porcine OBP isoform with comparable sequence similarity to the human OBPs (versus the merely 15% identity of pOBP mentioned above) is unknown to date. Interestingly, the most similar sequence to hOBPIIa in pigs is that of porcine tear lipocalin, with 39% sequence identity, a number close to the 43% sequence identity for human Tlc already mentioned above. Again, both the disulfide bridge and the free Cys residue within the ligand pocket are conserved in this case. Taken together, elucidation of the crystal structure for hOBPIIa has unraveled previously unknown features of this abundant protein, which provide a basis for future studies on ligand- and receptor-binding functions as well as its broader biological role. Coordinates and structure
factors of the refined hOBPIIa X-ray structure have been deposited in the Protein Data Bank (PDB; www.rcsb.org) under accession code 4RUN.
AUTHORS CONTRIBUTION A. Sc., A. E., and A. Sk. designed the research. A. Sc., A. E., and R. F. performed the experiments and data analysis. A. Sc. and A. Sk. wrote the article. REFERENCES 1. Tegoni M, Pelosi P, Vincent F, Spinelli S, Campanacci V, Grolli S, Ramoni R, Cambillau C. Mammalian odorant binding proteins. Biochim Biophys Acta 2000;1482:229–240. 2. Ganni M, Garibotti M, Scaloni A, Pucci P, Pelosi P. Microheterogeneity of odorant-binding proteins in the porcupine revealed by Nterminal sequencing and mass spectrometry. Comp Biochem Physiol B Biochem Mol Biol 1997;117:287–291. 3. L€ obel D, Jacob M, V€ olkner M, Breer H. Odorants of different chemical classes interact with distinct odorant binding protein subtypes. Chem Senses 2002;27:39–44. 4. Lacazette E, Gachon AM, Pitiot G. A novel human odorant-binding protein gene family resulting from genomic duplicons at 9q34: differential expression in the oral and genital spheres. Hum Mol Genet 2000;9:289–301. 5. Briand L, Eloit C, Nespoulous C, Bezirard V, Huet JC, Henry C, Blon F, Trotier D, Pernollet JC. Evidence of an odorant-binding protein in the human olfactory mucus: location, structural characterization, and odorant-binding properties. Biochemistry 2002;41: 7241–7252. 6. Breustedt DA, Sch€ onfeld DL, Skerra A. Comparative ligand-binding analysis of ten human lipocalins. Biochim Biophys Acta 2006;1764: 161–173. 7. Flower DR. The lipocalin protein family: structure and function. Biochem J 1996;318:1–14. 8. Tegoni M, Ramoni R, Bignetti E, Spinelli S, Cambillau C. Domain swapping creates a third putative combining site in bovine odorant binding protein dimer. Nat Struct Biol 1996;3:863–867. 9. Bianchet MA, Bains G, Pelosi P, Pevsner J, Snyder SH, Monaco HL, Amzel LM. The three-dimensional structure of bovine odorant binding protein and its mechanism of odor recognition. Nat Struct Biol 1996;3:934–939. 10. Vincent F, Spinelli S, Ramoni R, Grolli S, Pelosi P, Cambillau C, Tegoni M. Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes. J Mol Biol 2000;300:127–139. 11. White SA, Briand L, Scott DJ, Borysik AJ. Structure of rat odorantbinding protein OBP1 at 1.6 A˚ resolution. Acta Crystallogr D Biol Crystallogr 2009;65:403–410. 12. Portman KL, Long J, Carr S, Briand L, Winzor DJ, Searle MS, Scott DJ. Enthalpy/entropy compensation effects from cavity desolvation underpin broad ligand binding selectivity for rat odorant binding protein 3. Biochemistry 2014;53:2371–2379. 13. Skerra A. Lipocalins as a scaffold. Biochim Biophys Acta 2000;1482: 337–350. 14. Breustedt DA, Chatwell L, Skerra A. A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity. Acta Crystallogr D Biol Crystallogr 2009;65:1118–1125. 15. Tcatchoff L, Nespoulous C, Pernollet JC, Briand L. A single lysyl residue defines the binding specificity of a human odorant-binding protein for aldehydes. FEBS Lett 2006;580:2102–2108. 16. Vincent F, Ramoni R, Spinelli S, Grolli S, Tegoni M, Cambillau C. Crystal structures of bovine odorant-binding protein in complex with odorant molecules. Eur J Biochem 2004;271:3832–3842.
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