The Crystal Structure of D7r4, a Salivary Biogenic Amine-binding Protein from the Malaria Mosquito Anopheles gambiae
2007; Elsevier BV; Volume: 282; Issue: 50 Linguagem: Inglês
10.1074/jbc.m706410200
ISSN1083-351X
AutoresBen J. Mans, Eric Calvo, José M. C. Ribeiro, John F. Andersen,
Tópico(s)Insect and Pesticide Research
ResumoThe D7-related (D7r) proteins of the malaria vector Anopheles gambiae have been shown to bind the biogenic amines serotonin, norepinephrine, and histamine with high affinity. One member of the group (D7r1 or hamadarin) has also been shown to have an anticoagulant/antikinin activity. To understand the mechanistic details of its antihemostatic/anti-inflammatory effects, we have determined the crystal structure of one member of this group, D7r4, along with the structures of ligand complexes with serotonin, tryptamine, histamine, and norepinephrine. The D7 fold consists of an arrangement of eight α-helices stabilized by three disulfide bonds. The structure is similar to those of the arthropod odorant-binding proteins, a relationship that had been predicted based on sequence comparisons. Although odorant-binding proteins commonly have six α-helices, D7r4 has eight, resulting in significantly different positioning and structure of the ligand binding pocket. The pocket itself is lined by hydrophobic side chains along with polar and charged groups oriented to form hydrogen bonds with the aliphatic amino group and with groups on the aromatic portions of the ligands. These structures, along with accompanying mutagenesis studies, have allowed us to identify critical residues for biogenic amine binding and to predict which members of the large D7 protein family found in blood-feeding nematocerous Diptera will function as biogenic amine-binding proteins. The D7-related (D7r) proteins of the malaria vector Anopheles gambiae have been shown to bind the biogenic amines serotonin, norepinephrine, and histamine with high affinity. One member of the group (D7r1 or hamadarin) has also been shown to have an anticoagulant/antikinin activity. To understand the mechanistic details of its antihemostatic/anti-inflammatory effects, we have determined the crystal structure of one member of this group, D7r4, along with the structures of ligand complexes with serotonin, tryptamine, histamine, and norepinephrine. The D7 fold consists of an arrangement of eight α-helices stabilized by three disulfide bonds. The structure is similar to those of the arthropod odorant-binding proteins, a relationship that had been predicted based on sequence comparisons. Although odorant-binding proteins commonly have six α-helices, D7r4 has eight, resulting in significantly different positioning and structure of the ligand binding pocket. The pocket itself is lined by hydrophobic side chains along with polar and charged groups oriented to form hydrogen bonds with the aliphatic amino group and with groups on the aromatic portions of the ligands. These structures, along with accompanying mutagenesis studies, have allowed us to identify critical residues for biogenic amine binding and to predict which members of the large D7 protein family found in blood-feeding nematocerous Diptera will function as biogenic amine-binding proteins. Anopheles gambiae is the major African vector of the malaria parasite Plasmodium falciparum that infects 300-500 million people and causes 1-2 million deaths annually. Malaria transmission occurs during ingestion of a blood meal by the female mosquito when the sporozoite stage of the parasite is passed to the host in the insect saliva. Blood feeding is facilitated by a salivary mixture of biologically active peptides and small molecules that target the processes of blood clotting, vasodilation, inflammation, tissue remodeling, and immunity (1Ribeiro J.M.C. Infect. Agents Dis. 1995; 4: 143-152PubMed Google Scholar). Inhibition of antihemostatic defenses by these components is essential for the efficient ingestion of blood and, consequently, for egg production and possibly parasite transmission.Saliva from female A. gambiae contains a plethora of potent antihemostatic proteins, including the thrombin inhibitor anophelin (2Valenzuela J.G. Francischetti I.M. Ribeiro J.M. Biochemistry. 1999; 38: 11209-11215Crossref PubMed Scopus (83) Google Scholar), the platelet aggregation inhibitor apyrase (3Arca B. Lombardo F. Capurro M. della Torre A. Spanos L. Dimopoulos G. Louis C. James A.A. Coluzzi M. Parassitologia. 1999; 41: 483-487PubMed Google Scholar, 4Francischetti I.M. Valenzuela J.G. Pham V.M. Garfield M.K. Ribeiro J.M. J. Exp. Biol. 2002; 205: 2429-2451Crossref PubMed Google Scholar), and catechol oxidase-peroxidase, which serves as a vasodilator (5Ribeiro J.M. Nussenzveig R.H. Tortorella G. J. Med. Entomol. 1994; 31: 747-753Crossref PubMed Scopus (51) Google Scholar, 6Ribeiro J.M. Valenzuela J.G. J. Exp. Biol. 1999; 202: 809-816Crossref PubMed Google Scholar). Among the most abundant salivary molecules are the D7-related (D7r) proteins (7Calvo E. deBianchi A.G. James A.A. Marinotti O. Insect Biochem. Mol. Biol. 2002; 32: 1419-1427Crossref PubMed Scopus (12) Google Scholar, 8Calvo E. Mans B.J. Andersen J.F. Ribeiro J.M. J. Biol. Chem. 2006; 281: 1935-1942Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 9James A.A. Blackmer K. Marinotti O. Ghosn C.R. Racioppi J.V. Mol. Biochem. Parasitol. 1991; 44: 245-253Crossref PubMed Scopus (127) Google Scholar), a group of five polypeptides (D7r1-D7r5) that interfere with various aspects of host physiology. D7r1 (also known as hamadarin) was initially shown to have an anticoagulant/antikinin activity resulting from strong binding interactions with high molecular weight kininogen and factor XIIa, two components of the contact activation system of coagulation (8Calvo E. Mans B.J. Andersen J.F. Ribeiro J.M. J. Biol. Chem. 2006; 281: 1935-1942Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 10Isawa H. Orito Y. Iwanaga S. Jingushi N. Morita A. Chinzei Y. Yuda M. Insect Biochem. Mol. Biol. 2007; 37: 466-477Crossref PubMed Scopus (31) Google Scholar). More recently, D7r1-D7r4 were found to bind the biogenic amines serotonin, norepinephrine, and histamine, thereby reducing the concentrations of these effectors at the feeding site (8Calvo E. Mans B.J. Andersen J.F. Ribeiro J.M. J. Biol. Chem. 2006; 281: 1935-1942Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar).The process of blood clotting, the maintenance of vascular tone, and inflammatory responses are all regulated to some extent by biogenic amines. The widespread distribution of biogenic amine-binding lipocalin proteins in the saliva of tick and triatomine bug species suggests that inhibition of these processes is essential for efficient blood feeding (11Andersen J.F. Francischetti I.M. Valenzuela J.G. Schuck P. Ribeiro J.M. J. Biol. Chem. 2003; 278: 4611-4617Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 12Paesen G.C. Adams P.L. Harlos K. Nuttall P.A. Stuart D.I. Mol. Cell. 1999; 3: 661-671Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 13Ribeiro J.M. Walker F.A. J. Exp. Med. 1994; 180: 2251-2257Crossref PubMed Scopus (134) Google Scholar, 14Sangamnatdej S. Paesen G.C. Slovak M. Nuttall P.A. Insect Mol. Biol. 2002; 11: 79-86Crossref PubMed Scopus (132) Google Scholar). Interestingly, D7r5 does not bind biogenic amines but is also poorly expressed, suggesting that it may be nonfunctional and on an evolutionary path toward silencing (8Calvo E. Mans B.J. Andersen J.F. Ribeiro J.M. J. Biol. Chem. 2006; 281: 1935-1942Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar).Binding studies using recombinant D7r proteins have revealed significantly different ligand selectivities for the different forms, a possible driving force in the maintenance of this cluster of similar genes in the A. gambiae genome (8Calvo E. Mans B.J. Andersen J.F. Ribeiro J.M. J. Biol. Chem. 2006; 281: 1935-1942Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). All of the proteins, with the exception of D7r5, bind serotonin with high affinity and histamine with somewhat lower affinity. D7r2 and D7r3 bind epinephrine and norepinephrine with much higher affinity than D7r1 and D7r4 suggesting that these two members of the group may be specifically adapted for local catecholamine removal around a feeding site.Proteins showing sequence similarity to the D7r proteins occur in other blood-feeding Diptera, including mosquito species in the genera Anopheles (15Calvo E. Andersen J. Francischetti I.M. deL Capurro M. deBianchi A.G. James A.A. Ribeiro J.M. Marinotti O. Insect Mol. Biol. 2004; 13: 73-88Crossref PubMed Scopus (88) Google Scholar, 16Calvo E. Dao A. Pham V.M. Ribeiro J.M. Insect Biochem. Mol. Biol. 2007; 37: 164-175Crossref PubMed Scopus (82) Google Scholar), Aedes (17Valenzuela J.G. Pham V.M. Garfield M.K. Francischetti I.M. Ribeiro J.M. Insect Biochem. Mol. Biol. 2002; 32: 1101-1122Crossref PubMed Scopus (156) Google Scholar), and Culex (18Ribeiro J.M. Charlab R. Pham V.M. Garfield M. Valenzuela J.G. Insect Biochem. Mol. Biol. 2004; 34: 543-563Crossref PubMed Scopus (125) Google Scholar), sand flies in the genera Lutzomyia and Phlebotomus (19Anderson J.M. Oliveira F. Kamhawi S. Mans B.J. Reynoso D. Seitz A.E. Lawyer P. Garfield M. Pham M. Valenzuela J.G. BMC Genomics. 2006; 7: 52Crossref PubMed Scopus (127) Google Scholar) and Culicoides sp. (20Campbell C.L. Vandyke K.A. Letchworth G.J. Drolet B.S. Hanekamp T. Wilson W.C. Insect Mol. Biol. 2005; 14: 121-136Crossref PubMed Scopus (74) Google Scholar). Some of these, including "long form" D7, the first described member of the group from the yellow fever mosquito Aedes aegypti, are larger proteins that contain two domains, each being comparable with that of the entire D7r protein (9James A.A. Blackmer K. Marinotti O. Ghosn C.R. Racioppi J.V. Mol. Biochem. Parasitol. 1991; 44: 245-253Crossref PubMed Scopus (127) Google Scholar). Sequence comparisons have suggested a distant relationship between the D7/D7r proteins and the arthropod odorant (or pheromone)-binding protein (OBP) 2The abbreviation used is: OBP, odorant-binding protein. 2The abbreviation used is: OBP, odorant-binding protein. family (8Calvo E. Mans B.J. Andersen J.F. Ribeiro J.M. J. Biol. Chem. 2006; 281: 1935-1942Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar).In this study, we describe the three-dimensional structure of D7r4 along with the structures of a number of ligand complexes. Details of the structures reveal how the D7r proteins act to inhibit hemostatic and inflammatory processes by binding a diverse set of ligands with high affinity. The study also confirms the relationship of the D7r proteins with the OBPs, suggesting a possible origin for the group.EXPERIMENTAL PROCEDURESMaterials—Crystallization reagents were obtained from Hampton Research. The selenomethionine media kit (SelenoMet) was obtained from Molecular Dimensions Ltd. Serotonin, tryptamine, and histamine were obtained from Sigma as hydrochloride salts. l-Norepinephrine bitartrate salt was also obtained from Sigma.Preparation of Protein and Crystallization—The D7r4 cDNA, cloned into the expression vector pET 17b, was used to produce recombinant protein in Escherichia coli as described previously. Inclusion bodies were prepared 3 h after induction, solubilized, and refolded as described by Calvo et al. (8Calvo E. Mans B.J. Andersen J.F. Ribeiro J.M. J. Biol. Chem. 2006; 281: 1935-1942Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The refolded proteins were purified by a combination of gel filtration and cation exchange chromatography. Selenomethionine-containing protein was produced in E. coli using SelenoMet medium (Molecular Dimensions Ltd.) according to the manufacturer's instructions. The protein was refolded and purified in a manner similar to the wild type protein. Crystals were obtained using the hanging drop-vapor diffusion method with 20% polyethylene glycol 6000, 100 mm Tris-HCl, pH 8.0, as a precipitant. Prior to data collection, crystals were flash-frozen after a short soak in 30% PEG 6000, 100 mm Tris-HCl, pH 8.0, containing 10% glycerol. Co-crystals were grown by adding serotonin or tryptamine to the precipitant solution at a concentration of 1 mm. Ligands were soaked into crystals by placing a crystal into a 5-μl drop of cryoprotectant solution containing a 20-100 μm ligand concentration and allowing it to soak for several minutes before flash freezing.Data Collection and Structure Determination—Data were collected at beamlines 19-ID and 19-BM at the Structural Biology Center, APS, Argonne National Laboratory. Diffraction data for the selenomethionine derivative was collected at two wavelengths near the selenium edge and integrated and scaled using HKL 3000 (21Minor W. Cymborowski M. Otwinowski Z. Chruszcz M. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2006; 62: 859-866Crossref PubMed Scopus (1514) Google Scholar). Initial phases were also obtained using the HKL 3000 package which combines SHELXD-E (22Schneider T.R. Sheldrick G.M. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1574) Google Scholar, 23Sheldrick G.M. Z. Kristallogr. 2002; 217: 644-650Crossref Scopus (360) Google Scholar), MLPHARE (24Otwinowski Z. Wolf W. Evans P.R. Leslie A.G.W. Proceedings of the CCP4 Study Weekend. Daresbury Laboratory, Warrington, UK1991: 60-68Google Scholar), and DM (25Cowtan K. Main P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 487-493Crossref PubMed Scopus (309) Google Scholar) for the location of selenium sites, solvent flattening, phasing, and density modification. Some of the helical segments of the model were built using RESOLVE (26Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (432) Google Scholar), whereas the remainder of the protein backbone structure and the side chains were built manually using a native D7r4 data set. Numerous cycles of rebuilding and refinement were performed with Coot (27Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22799) Google Scholar) and REFMAC (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar) to obtain the final structure of the unliganded protein. Various manipulations of reflection and coordinate data during the course of structure determination were made using the CCP4 package (29P4 CC Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar). Model quality was checked using the MOLPROBITY web server (30Lovell S.C. Davis I.W. Arendall W.B. II I de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3790) Google Scholar). The structural figures presented here were produced with PyMOL (DeLano Scientific).In the histamine and norepinephrine complexes, where the crystals were soaked with ligand-containing cryoprotectant solutions, a change in unit cell dimensions was observed that resulted in two molecules of D7r4 in the asymmetric unit rather than one. The space group of both crystals was P43 and transformation from the small to the larger unit cell can be described as follows: a′= a + b, b′=-a + b, c′= c.The two molecules in the asymmetric unit of the large unit cell were positioned by molecular replacement using PHASER (31McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1596) Google Scholar), and the structures were rebuilt and refined as described above. The crystallographic and phasing data for all structures are given in Table 1.TABLE 1Data collection, phasing, and refinement statistics for D7r4 and ligand complexesCrystalSeMet-inf (SeMet-hr)aData collection at two wavelengths on a single crystal, with values for the second data set being in parentheses. SeMet indicates selenomethionine.UnligandedSerotoninTryptamineHistamineNorepinephrineResolution (Å)30.0-2.5062.6/2.0050.0-2.0063.1-2.2088.3/2.0087.1/2.32Beamline19-BM19-ID19-ID19-ID19-BM19-BMWavelength (Å)0.978907 (0.971077)0.97915330.97926010.979078010.9791150.978743Completeness99.7/97.9 (99.7/98.8)98.1/85.899.6/98.599.3/92.899.5/99.799.8/99.7Average redundancy (total/high resolution shell)4.9/4.3 (3.0/2.9)4.4/3.47.9/7.47.9/8.04.3/4.06.3/6.3Rmerge (total/high resolution shell)7.1/54.6 (3.9/25.9)3.9/30.37.0/40.95.6/18.97.6/44.78.2/36.7I/sigI (total/high resolution shell)6.6/1.7 (7.8/2.5)17.8/3.316.2/9.224.9/16.87.6/2.77.1/4.2Observed reflections56,172 (34,578)48,90678,48568,16299,11992,717Unique reflections11,400 (11,430)10,6579,9768,19421,61913,953Space groupP43P43P43P43P43P43Unit cell dimensions (Å)a62.652 (62.733)62.65163.35863.10188.39287.807b62.652 (62.733)62.65163.35863.10188.39287.807c43.125 (43.157)42.77342.61742.59643.37343.488α, β, γ (°)909090909090Phasing statisticsNo. of selenium sites4FOM (RESOLVE)0.87r.m.s.br.m.s. indicates root mean square. deviationsbond lengths (Å)0.0090.0080.0120.0070.010bond angles (°)1.060.9651.350.8521.15Ramachandran plot (favored/allowed)93.9/10093.9/10093.8/10092.4/10093.1/100Mean B value for all atoms44.126.927.427.542.7Rcryst/Rfree21.9/23.719.3/23.817.2/24.818.4/21.818.3/25.0a Data collection at two wavelengths on a single crystal, with values for the second data set being in parentheses. SeMet indicates selenomethionine.b r.m.s. indicates root mean square. Open table in a new tab Site-directed Mutagenesis and Binding Studies—Site-directed mutagenesis was performed using a PCR-based procedure and verified by DNA sequencing. Protein expression and purification were performed using the same methods as for wild type D7r4. Binding studies were performed by isothermal titration calorimetry using a Microcal VP-ITC microcalorimeter at a temperature of 30 °C. Protein and ligand solutions were prepared in 20 mm Tris-HCl, pH 7.4, 0.15 m NaCl. The protein solution was added to the microcalorimeter sample cell, and ligand solutions were injected in 5-10-μl aliquots. The measured heats were converted to enthalpies (per injection) and analyzed using the Microcal-Origin software package.RESULTS AND DISCUSSIONStructure of D7r4—The structure of D7r4 without ligands was determined by multiple anomalous dispersion methods using a selenomethionine derivative of the protein. Additionally, the structures of ligand complexes containing serotonin, tryptamine, norepinephrine, and histamine were determined using molecular replacement or difference Fourier methods. The serotonin and tryptamine complexes were obtained by co-crystallization of protein and ligand, whereas the norepinephine and histamine complexes were obtained by soaking crystals with ligand-containing solutions. In all cases, interpretable electron density was observed for the ligand (Fig. 2B).The D7r protein fold consists of an arrangement of eight helices stabilized by three disulfide bonds (Fig. 1). The helices surround a small, centrally located ligand binding pocket. A narrow channel bounded by helices B, G, and H leads to the binding pocket and is the most likely ligand entry path. In both the ligand-free and ligand-bound structures, the loops connecting helices A-B and B-C are more mobile than the rest of the structure, exhibiting larger temperature factors and poor electron density for the side chains. This suggests that flexibility in these areas of the protein may play a role in ligand entry.FIGURE 1Comparison of the D7r4 structure with the published structures of the pheromone-binding protein of the silkmoth, B. mori (Protein Data Bank code 1DQE (35Sandler B.H. Nikonova L. Leal W.S. Clardy J. Chem. Biol. 2000; 7: 143-151Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar)), and the hemolymph protein THP12 (Protein Data Bank code 1C3Y (32Rothemund S. Liou Y.C. Davies P.L. Krause E. Sonnichsen F.D. Structure (Lond.). 1999; 7: 1325-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar)) from the beetle T. molitor shown for comparison. Disulfide bonds in each structure are shown in yellow. Helical segments are labeled as in the text. A, D7r4 (red), with serotonin (green) shown in the binding pocket. Disulfide bond linking helices A and C is partially obscured by the ligand. B, pheromone-binding protein (blue) with the ligand bombykol (green) shown in the binding pocket. C, structure of the hemolymph protein THP12 (cyan) without ligands.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Despite being only 12-15% identical in amino acid sequence (by alignment with ClustalW), D7r4 is similar in structure to the arthropod OBPs, a group of all-helical proteins that putatively function in shuttling odorant molecules from the surface of hair-like antennal olfactory sensillae, through the sensillar lymph to olfactory receptors at the neural membrane. The OBP group also contains THP12, a protein of unknown function found in the hemolymph of the beetle Tenebrio molitor (32Rothemund S. Liou Y.C. Davies P.L. Krause E. Sonnichsen F.D. Structure (Lond.). 1999; 7: 1325-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Arthropod OBPs and THP12 are hexahelical proteins, lacking the C-terminal helices G and H of D7r4 (Fig. 1 and Fig. 2A).The Bombyx mori pheromone-binding protein (BmPBP) and other olfactory OBPs contain three disulfide bonds connecting helix A with C, helix E with F, and helix C with F. The former two bonds are conserved in D7r4, linking Cys-6 with Cys-38 and Cys-77 with Cys-96 (Fig. 1). A third disulfide bond in D7r4 is unique to the group and links Cys-19 of helix B with Cys-144 of helix H (Fig. 1). THP12 retains the conserved disulfide bonds connecting helix A with C and helix E with F, but it is missing the bond linking helix C with F that is seen in BmPBP (Fig. 1).As a rule, members of the OBP family are either truncated at the end of helix F or have a nonhelical C-terminal portion. A functionally important exception is BmPBP that forms an additional helix in a pH-dependent conformational change. The ligand-bound protein found at pH 6.5 exhibits an extended conformation at the C terminus, which forms a seventh helix when the pH is lowered to 4.5 (33Horst R. Damberger F. Luginbuhl P. Guntert P. Peng G. Nikonova L. Leal W.S. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14374-14379Crossref PubMed Scopus (237) Google Scholar, 34Lee D. Damberger F.F. Peng G. Horst R. Guntert P. Nikonova L. Leal W.S. Wuthrich K. FEBS Lett. 2002; 531: 314-318Crossref PubMed Scopus (86) Google Scholar, 35Sandler B.H. Nikonova L. Leal W.S. Clardy J. Chem. Biol. 2000; 7: 143-151Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The C-terminal helix inserts into the pheromone binding pocket and presumably displaces the pheromone ligand (33Horst R. Damberger F. Luginbuhl P. Guntert P. Peng G. Nikonova L. Leal W.S. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14374-14379Crossref PubMed Scopus (237) Google Scholar). The pH dependence of this process is potentially important because the pH at the receptor membrane surface, where ligand release occurs, is apparently lower than the pH of 6.5 measured for the bulk sensillar lymph (33Horst R. Damberger F. Luginbuhl P. Guntert P. Peng G. Nikonova L. Leal W.S. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14374-14379Crossref PubMed Scopus (237) Google Scholar).Helix A of the OBPs is longer than that of D7r4 and contributes significantly to the structure of the binding pocket (Fig. 1B). THP12 also has an extended N terminus (Fig. 1), but it forms a disordered coil giving rise to an open binding groove, rather than the closed ligand-binding pocket of the OBPs (32Rothemund S. Liou Y.C. Davies P.L. Krause E. Sonnichsen F.D. Structure (Lond.). 1999; 7: 1325-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). D7r4 has helices A-F arranged in a similar manner to THP12, but the binding pocket is closed by the additional C-terminal helical segments G and H (Fig. 1A and Fig. 2A). These differences significantly change the position and structure of the binding pocket in D7r4 relative to the sensory OBPs. In the latter, helices D-F make up a large part of the pocket structure, whereas in D7r4 they are largely displaced by helices G and H and, with the exception of Tyr-94, do not contribute to the structure of the pocket.Details of the Ligand Binding Pocket and Ligand Complex Structures—D7r proteins bind a variety of structurally distinct biogenic amines with high affinity, yet employ only a single binding site. This broad ligand specificity is accomplished via a generally hydrophobic binding pocket with polar or charged side chains placed at various locations where they can form hydrogen bonds with ligand functional groups in different arrangements (Fig. 4). The apparent entry path to the binding pocket is surrounded by the anionic side chains of Asp-111, Glu-114, and Asp-139, which act to stabilize the aliphatic amino group of the bound ligand (Fig. 4). The interior of the pocket is lined by aromatic and hydrophobic residues, including Tyr-24 and Phe-110, which contact the aromatic rings and aliphatic side chain of biogenic amine ligands (Fig. 3). At the closed end of the pocket, opposite the ligand entry channel, Glu-7 and His-35 are positioned to form hydrogen bonds with hydroxyl groups found on the aromatic portions of serotonin and norepinephrine (Figs. 3 and 5).FIGURE 4Stereoview showing the hydrogen bonding interactions in the serotonin (A) and tryptamine (B) complexes. Hydrogen bonds are indicated as red dashed lines; water molecules are red spheres; protein residues are colored in green, and ligands are shown in cyan.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Stereoview of the detailed structure of the ligand binding pocket of D7r4 (white) with a bound molecule of serotonin (red) and electron density (Fo - Fc) contoured at 1σ covering the ligand. The figure highlights the role of Arg-22, Glu-114, Tyr-94, and Phe-110 in orienting the ligand in the binding pocket.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Stereoview showing the hydrogen bonding interactions in the norepinephrine (A) and histamine (B) complexes. Hydrogen bonds are indicated as red dashed lines; water molecules are red spheres; protein residues are colored in green, and ligands are shown in cyan.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the serotonin complex, the ligand is oriented with its indole nucleus directed toward the closed end of the pocket and the aliphatic amino group toward the apparent access channel. The 5-hydroxyl group forms hydrogen bonds with the side chains of Glu-7 and His-35 (Fig. 3 and Fig. 4A). The side chain of Arg-22 extends from the protein backbone parallel to the plane of the indole ring system and crosses over it (Fig. 3). It is bent 90° at C-δ allowing the guanidino moiety to contact the edge of the ring as well as its face. The side chain of Tyr-24 forms a third side of the box-like pocket holding the ligand (Fig. 3). An apparent salt bridge between Arg-22 and Glu-114 positions the latter to form a hydrogen bond with the amino group of serotonin (Fig. 3). Also interacting with the amino group are Asp-111, which forms a hydrogen bond via its carboxylate, and Asp-139, which stabilizes a water molecule that forms a hydrogen bond with the amino group (Fig. 4A). The indole nitrogen of serotonin forms a long hydrogen bond (3.07 Å) with the phenolic hydroxyl of Tyr-94, the only residue from helix D that impinges on the binding pocket (Fig. 4A).Not surprisingly, tryptamine and serotonin occupy nearly identical positions in the binding pocket despite the absence of a 5-hydroxyl group in tryptamine (Fig. 4B). All of the hydrogen bonding interactions with the aliphatic amino group seen in the serotonin complex are maintained in the tryptamine complex, as is the long hydrogen bond between the Tyr-94 hydroxyl and the indole nitrogen of the ligand (Fig. 4B). The near equivalence of the serotonin and tryptamine binding modes allowed us to estimate the energetic contributions of hydrogen bonding interactions with the 5-hydroxyl group of serotonin using isothermal titration calorimetry. The 4.7 kcal/mol difference in the binding enthalpy (ΔΔH) seen between the two ligands is apparently because of the loss of hydrogen bonding interactions with Glu-8 and His-35 in the D7r4-tryptamine complex (Table 2). The more favorable entropy change exhibited in the tryptamine binding reaction is probably due to the more hydrophobic nature of this compound.TABLE 2Thermodynamic parameters for binding of serotonin (top) and tryptamine (bottom) with D7r4 and various binding pocket mutantsMutantLigandΔHaValues are given as kcal/mol.ΔΔHaValues are given as kcal/mol.,bΔΔH indicates ΔHmutant − ΔHwild type.TΔSaValues are given as kcal/mol.KdcValues are given as nanomolar.ΔGaValues are given as kcal/mol.ΔΔGaValues are given as kcal/mol.,dΔΔG indicates ΔGmutant − ΔGwild type.Wild typeSerotonin−21.1−9.22.4−11.9Tryptamine−16.4−6.353−10.1E7LSerotonin−14.86.3−4.984−9.82.1Tryptamine−10.06.4−1.7980−8.31.8H35LSerotonin−30.6−9.5−16.50.1−14.0−2.1Tryptamine−28.3−11.9−15.60.6−12.7−2.6E7L/H35LSerotonin−15.95.2−7.0360−8.93.0Trypt
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