The Phox Domain of Sorting Nexin 5 Lacks Phosphatidylinositol 3-Phosphate (PtdIns(3)P) Specificity and Preferentially Binds to Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2)
2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês
10.1074/jbc.m109.008995
ISSN1083-351X
AutoresLeonardus M. I. Koharudin, William Furey, Hao Liu, Yongjian Liu, Angela M. Gronenborn,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoSubcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 Å resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new α2′-helix and a much longer α3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors. Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 Å resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new α2′-helix and a much longer α3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors. The early work on retromer revealed its role in the trafficking of cargo proteins between endosomes and the trans-Golgi network (TGN), 2The abbreviations used are: TGNtrans-Golgi networkSNXsorting nexinPXphox domainPtdInsphosphatidylinositolPtdIns(0)Pphosphatidylinositol phosphatePtdIns(3)Pphosphatidylinositol 3-phosphatePtdIns(4)Pphosphatidylinositol 4-phosphatePtdIns(5)Pphosphatidylinositol 5-phosphatePtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphatePtdIns(3,4)P2phosphatidylinositol 3,4-bisphosphatePtdIns(3,5)P2phosphatidylinositol 3,5-bisphosphatePtdIns(3,4,5)P3phosphatidylinositol 3,4, 5-trisphosphateMADmultiwavelength anomalous dispersionNOEnuclear Overhauser effectHSQCheteronuclear single quantum correlation.2The abbreviations used are: TGNtrans-Golgi networkSNXsorting nexinPXphox domainPtdInsphosphatidylinositolPtdIns(0)Pphosphatidylinositol phosphatePtdIns(3)Pphosphatidylinositol 3-phosphatePtdIns(4)Pphosphatidylinositol 4-phosphatePtdIns(5)Pphosphatidylinositol 5-phosphatePtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphatePtdIns(3,4)P2phosphatidylinositol 3,4-bisphosphatePtdIns(3,5)P2phosphatidylinositol 3,5-bisphosphatePtdIns(3,4,5)P3phosphatidylinositol 3,4, 5-trisphosphateMADmultiwavelength anomalous dispersionNOEnuclear Overhauser effectHSQCheteronuclear single quantum correlation. although recently, retromer involvement in many other physiological and developmental processes has been uncovered (1.Bonifacino J.S. Rojas R. Nat. Rev. Mol. Cell Biol. 2006; 7: 568-579Crossref PubMed Scopus (494) Google Scholar, 2.Vergés M. Int. Rev. Cell Mol. Biol. 2008; 271: 153-198Crossref PubMed Scopus (17) Google Scholar). The best studied proteins associated with retromer activity are intracellular sorting receptors such as the yeast vacuolar protein-10 (Vps10) and mammalian mannose 6-phosphate receptors (3.Willnow T.E. Petersen C.M. Nykjaer A. Nat. Rev. Neurosci. 2008; 9: 899-909Crossref PubMed Scopus (58) Google Scholar, 4.Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (794) Google Scholar). These receptors sort acid hydrolases, enzymes essential for protein degradation, out of the TGN into the yeast vacuole or the mammalian lysosome. Upon releasing their substrates, these cargos traffic back to the TGN to mediate further rounds of cargo-hydrolase transportation. Similar retrograde trafficking of cargo proteins involving signaling molecules such as Wnt and amyloid precursor protein (APP) are thought to be critical for their secretion and function (5.Eaton S. Dev. Cell. 2008; 14: 4-6Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 6.Mayeux R. Hyslop P.S. Lancet Neurol. 2008; 7: 2-3Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Retrograde transportation is highly regulated by the heteropentameric retromer complex that consists of a sorting nexin (SNX) dimer (e.g. Vps5 and Vps17 in yeast) and a Vps26/29/35 trimer (7.Bonifacino J.S. Hurley J.H. Curr. Opin. Cell Biol. 2008; 20: 427-436Crossref PubMed Scopus (384) Google Scholar). In mammals, the binding of the SNX dimer to specific phosphatidylinositol (PtdIns) determines its subcellular membrane association and governs the recruitment of the Vps trimer to endosomal compartments. Mammalian orthologs of the trimer have been biochemically characterized, and their interaction and function in cargo protein trafficking is well established (8.Seaman M.N. Trends Cell Biol. 2005; 15: 68-75Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). More recently, crystal structures of three Vps proteins in the trimer suggested how this trimer interacts with the SNX dimer and cargo proteins as well as with curved membranes (9.Shi H. Rojas R. Bonifacino J.S. Hurley J.H. Nat. Struct. Mol. Biol. 2006; 13: 540-548Crossref PubMed Scopus (139) Google Scholar, 10.Wang D. Guo M. Liang Z. Fan J. Zhu Z. Zang J. Zhu Z. Li X. Teng M. Niu L. Dong Y. Liu P. J. Biol. Chem. 2005; 280: 22962-22967Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 11.Collins B.M. Skinner C.F. Watson P.J. Seaman M.N. Owen D.J. Nat. Struct. Mol. Biol. 2005; 12: 594-602Crossref PubMed Scopus (115) Google Scholar, 12.Hierro A. Rojas A.L. Rojas R. Murthy N. Effantin G. Kajava A.V. Steven A.C. Bonifacino J.S. Hurley J.H. Nature. 2007; 449: 1063-1067Crossref PubMed Scopus (219) Google Scholar). In the SNX dimer, SNX1 and SNX2 are thought to be interchangeable Vps5 orthologs (13.Carlton J. Bujny M. Peter B.J. Oorschot V.M. Rutherford A. Mellor H. Klumperman J. McMahon H.T. Cullen P.J. Curr. Biol. 2004; 14: 1791-1800Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 14.Rojas R. Kametaka S. Haft C.R. Bonifacino J.S. Mol. Cell. Biol. 2007; 27: 1112-1124Crossref PubMed Scopus (182) Google Scholar). The NMR structure of SNX1 revealed details of PI(3)P specific binding, thereby explaining its role in endosomal trafficking (15.Zhong Q. Watson M.J. Lazar C.S. Hounslow A.M. Waltho J.P. Gill G.N. Mol. Biol. Cell. 2005; 16: 2049-2057Crossref PubMed Scopus (35) Google Scholar). The identity for SNX5 as a potential functional mammalian ortholog of Vps17, however, was not revealed until recently. trans-Golgi network sorting nexin phox domain phosphatidylinositol phosphatidylinositol phosphate phosphatidylinositol 3-phosphate phosphatidylinositol 4-phosphate phosphatidylinositol 5-phosphate phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 3,4-bisphosphate phosphatidylinositol 3,5-bisphosphate phosphatidylinositol 3,4, 5-trisphosphate multiwavelength anomalous dispersion nuclear Overhauser effect heteronuclear single quantum correlation. trans-Golgi network sorting nexin phox domain phosphatidylinositol phosphatidylinositol phosphate phosphatidylinositol 3-phosphate phosphatidylinositol 4-phosphate phosphatidylinositol 5-phosphate phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 3,4-bisphosphate phosphatidylinositol 3,5-bisphosphate phosphatidylinositol 3,4, 5-trisphosphate multiwavelength anomalous dispersion nuclear Overhauser effect heteronuclear single quantum correlation. Although initially identified as a Fanconi anemia complementation group A (FANCA)-binding protein (16.Otsuki T. Kajigaya S. Ozawa K. Liu J.M. Biochem. Biophys. Res. Commun. 1999; 265: 630-635Crossref PubMed Scopus (56) Google Scholar), SNX5 was later shown to play an important role in membrane trafficking (17.Liu H. Liu Z.Q. Chen C.X. Magill S. Jiang Y. Liu Y.J. Biochem. Biophys. Res. Commun. 2006; 342: 537-546Crossref PubMed Scopus (36) Google Scholar, 18.Kerr M.C. Lindsay M.R. Luetterforst R. Hamilton N. Simpson F. Parton R.G. Gleeson P.A. Teasdale R.D. J. Cell Sci. 2006; 119: 3967-3980Crossref PubMed Scopus (113) Google Scholar, 19.Lim J.P. Wang J.T. Kerr M.C. Teasdale R.D. Gleeson P.A. BMC Cell Biol. 2008; 9: 58Crossref PubMed Scopus (42) Google Scholar). SNX5 contains a PX domain (SNX5-PX) that is the signature feature in defining the SNX family, composed of 30 members at present (20.Xu Y. Seet L.F. Hanson B. Hong W. Biochem. 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Our previous biochemical study suggested a specific interaction between SNX5 and SNX1 through which the two SNXs mutually influence each other's effect in endosomal trafficking of epidermal growth factor receptor upon epidermal growth factor stimulation (17.Liu H. Liu Z.Q. Chen C.X. Magill S. Jiang Y. Liu Y.J. Biochem. Biophys. Res. Commun. 2006; 342: 537-546Crossref PubMed Scopus (36) Google Scholar). In support of this observation are several recent reports that indicate a critical role of SNX5 and the closely related SNX6, beyond that of SNX1 and SNX2, on retrograde sorting of mannose 6-phosphate receptor (24.Wassmer T. Attar N. Bujny M.V. Oakley J. Traer C.J. Cullen P.J. J. Cell Sci. 2007; 120: 45-54Crossref PubMed Scopus (187) Google Scholar, 27.Hara S. Kiyokawa E. Iemura S. Natsume T. Wassmer T. Cullen P.J. Hiai H. Matsuda M. Mol. Biol. Cell. 2008; 19: 3823-3835Crossref PubMed Scopus (26) Google Scholar). Therefore, SNX5 and SNX6 may be functionally interchangeable orthologs of Vps17 in mammalian cells (7.Bonifacino J.S. Hurley J.H. Curr. Opin. Cell Biol. 2008; 20: 427-436Crossref PubMed Scopus (384) Google Scholar, 24.Wassmer T. Attar N. Bujny M.V. Oakley J. Traer C.J. Cullen P.J. J. Cell Sci. 2007; 120: 45-54Crossref PubMed Scopus (187) Google Scholar). Furthermore, in contrast to some reports (18.Kerr M.C. Lindsay M.R. Luetterforst R. Hamilton N. Simpson F. Parton R.G. Gleeson P.A. Teasdale R.D. J. Cell Sci. 2006; 119: 3967-3980Crossref PubMed Scopus (113) Google Scholar, 30.Merino-Trigo A. Kerr M.C. Houghton F. Lindberg A. Mitchell C. Teasdale R.D. Gleeson P.A. J. Cell Sci. 2004; 117: 6413-6424Crossref PubMed Scopus (54) Google Scholar), SNX5 partially localizes to late endosomes and the TGN, exhibiting very low binding affinity for PtdIns(3)P (17.Liu H. Liu Z.Q. Chen C.X. Magill S. Jiang Y. Liu Y.J. Biochem. Biophys. Res. 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There are about a dozen structurally characterized PX domains from the SNX family or other PX domain-containing proteins currently deposited in the Protein Data Bank (PDB) data base. Their structures all share common core features, a three-stranded β-sheet that is abutted by three α-helices and an irregular strand containing the PXXP region. Analyses of the representative p47-PX and SNX3-PX domain structures suggested that PtdIns(3)P binding involves two conserved Arg residues at positions equivalent to Arg58 and Arg105 in p40-PX (36.Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Because equivalent Arg residues are found in the PX domains of most SNX family members, it is generally assumed that all SNX proteins interact with the PtdIns(3)P-enriched elements of the early endocytic compartments. The amino acid sequences of the PX domains of both SNX5 and SNX6, however, lack the two conserved Arg residues that are involved in PtdIns(3)P binding as well as comprising a ∼30-residue insertion immediately after the PXXP motif (Fig. 1A). In addition, the PXXP motif is extended into a double PXXP motif with the sequence PXXPXXP. These unique sequence features set SNX5/6 apart from the other SNX family members. In the p40-PX domain and yeast SNX3, the two conserved Arg residues, the loop between the PXXP motif, and the α3-helix are involved in forming the binding pocket for the phosphate groups of PtdIns(3)P (36.Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 37.Zhou C.Z. de La Sierra-Gallay I.L. Quevillon-Cheruel S. Collinet B. Minard P. Blondeau K. Henckes G. Aufrère R. Leulliot N. Graille M. Sorel I. Savarin P. de la Torre F. Poupon A. Janin J. van Tilbeurgh H. J. Biol. Chem. 2003; 278: 50371-50376Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Therefore, changes in length and sequence in this region in SNX5/6-PX are expected to have profound impact on the specific structure and conformation required for PtdIns recognition. To elucidate how its unique sequence features influence the function of SNX5 in retromer-mediated retrograde membrane trafficking, we structurally investigated the SNX5-PX domain by NMR spectroscopy and x-ray crystallography. Using direct NMR titrations, we established the PtdIns binding specificity of SNX5-PX. The high resolution (1.5 Å) crystal structure of the domain revealed its distinct features when compared with previously known family members. Our results demonstrate that the SNX5-PX domain is indeed unique, both with respect to its structure as well as with respect to ligand binding. These findings have important implications for the function of SNX5 in the subcellular membrane trafficking and retrograde sorting. The SNX5-PX domain (residues 1–180) derived from the rat SNX5 gene was expressed in Escherichia coli BL21(DE3) (Stratagene) as a N-terminal His-tagged fusion protein using the vector pET-15B (Novagen). After purification on a Ni2+ column (GE Healthcare), fractions were dialyzed into 50 mm Tris-HCl, 100 mm NaCl, 5 mm CaCl2, pH 7.5, and the His tag was cleaved off the protein with thrombin at room temperature overnight. In addition to the canonical thrombin site after the His tag, a secondary cleavage site was discovered between residue Arg19 and Ser20. Complete cleavage at this site was allowed to occur (confirmed by mass spectrometry; data not shown). The cleaved protein was separated from the His tag and N-terminal peptide on a Superdex-75 gel filtration column in 20 mm Tris·HCl, 100 mm NaCl, 0.02% NaN3, pH 8.0. Purified protein fractions were collected and concentrated using Centriprep devices (Millipore). For crystallization, the protein solution in gel filtration buffer was concentrated to 8 mg/ml. The Se-Met derivative and the 2.19 Å crystals were obtained using an optimized initial crystallization condition of 8 μl of protein versus 1 μl of reservoir solution (0.2 m (NH4)2SO4, 0.1 m sodium cacodylate trihydrate, pH 6.5, 30% polyethylene glycol 8000) at 4 °C by sitting drop vapor diffusion. The 1.47 Å crystal was obtained using an optimized second crystallization condition (0.2 m NH4CH3COO, 0.1 m sodium cacodylate trihydrate, pH 6.5, 30% polyethylene glycol 4000). The x-ray diffraction data for the Se-Met derivative crystals obtained from the first crystallization conditions were collected at the Southeast Regional Collaborative Access Team(SERCAT) facility sector 22-ID beam line of the Advance Photon Source at the Argonne National Laboratory, Chicago, IL. The MAD data at wavelengths corresponding to the edge, peak, and remote point of the anomalous scattering plots for selenium (0.9795, 0.9793, and 0.9718, respectively) were processed using the d*TREK software (38.Pflugrath J.W. Acta Crystallogr D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1417) Google Scholar). The crystal belongs to a primitive orthorhombic space group P212121 with one molecule per asymmetric unit. The selenium atom sites and MAD phases after solvent flattening were automatically determined using the program BnP (39.Weeks C.M. Blessing R.H. Miller R. Mungee R. Potter S.A. Rappleye J. Smith G.D. Xu H. Furey W. Zeitschrift für Kristallographie. 2002; 217: 686-693Crossref Scopus (54) Google Scholar). Initial model building with additional iterative density improvement was automatically carried out using the RESOLVE program (40.Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (433) Google Scholar). The generated, incomplete initial model was further refined through cycles of rebuilding and refinement using the program Coot (41.Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23204) Google Scholar) and RefMac (42.Nishida N. Sumikawa H. Sakakura M. Shimba N. Takahashi H. Terasawa H. Suzuki E. Shimada I. Nat. Struct. Biol. 2003; 10: 53-58Crossref PubMed Scopus (68) Google Scholar). The MAD structure refined at 2.56 Å resolution was used as a molecular replacement model to extend the resolution of the data collected on the home source (Rigaku FR-E generator with a Saturn 944 CCD detector and high flux VariMax optics) for the second, similar crystal form to 2.19 Å. This model was refined to working and free R-factors of 23.6 and 28.4%, respectively, using the RefMac program (42.Nishida N. Sumikawa H. Sakakura M. Shimba N. Takahashi H. Terasawa H. Suzuki E. Shimada I. Nat. Struct. Biol. 2003; 10: 53-58Crossref PubMed Scopus (68) Google Scholar) in the CCP4 package (43.Collaborative Computational Project, Number 4 Acta Crystallogr D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19745) Google Scholar). For a crystal obtained using the second condition, a diffraction data set was collected on the home source (Rigaku FR-E generator with a Saturn 944 CCD detector and high flux VariMax optics). The data were processed with the d*TREK software (38.Pflugrath J.W. Acta Crystallogr D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1417) Google Scholar). The crystal diffracted to 1.47 Å and belonged to a primitive monoclinic space group P21, with one molecule per asymmetric unit. The 2.19 Å orthorhombic structure was used as a molecular replacement probe for solving the structure in the monoclinic crystal form. The model was refined to working and free R-factors of 18.4 and 23.4%, respectively, using the RefMac program (42.Nishida N. Sumikawa H. Sakakura M. Shimba N. Takahashi H. Terasawa H. Suzuki E. Shimada I. Nat. Struct. Biol. 2003; 10: 53-58Crossref PubMed Scopus (68) Google Scholar) in the CCP4 package (43.Collaborative Computational Project, Number 4 Acta Crystallogr D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19745) Google Scholar). The final model has 98.4 and 1.60% of all residues in the favored and allowed regions of the Ramachandran plot, respectively, and contains no residues in the disallowed region as evaluated by PROCHECK (44.Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The electron density of the final 1.47 Å resolution map clearly reveals two conformations for 10 side chains (Asn26, Ile33, Ser40, Thr78, Asp84, Ile90, Gln107, Glu131, Ser141, and Ser142). Refinement statistics for all models are provided in supplemental Table S1. The atomic coordinates of SNX5-PX have been deposited in the RCSB Protein Data Bank under accession codes 3HPB and 3HPC for the 2.19 Å orthorhombic and the 1.47 Å monoclinic crystals, respectively. All structure figures were generated with the atomic coordinates of the monoclinic crystal using the program Chimera (45.Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Comput Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (27876) Google Scholar). NMR spectra were recorded at 25 °C on Bruker AVANCE800, AVANCE700, and AVANCE600 spectrometers equipped with 5-mm triple-resonance, three-axis gradient probes or z axis gradient cryoprobes. Spectra for backbone resonance assignments were recorded on a 13C/15N-labeled sample in 20 mm Tris buffer, 100 mm NaCl, 0.02% NaN3, pH 7.4. The maximum protein concentration attainable was ∼0.5 mm due to limited protein solubility. Three-dimensional HNCACB, CBCA(CO)NH, and 1H-15N NOE spectroscopy HSQC (mixing time 120 ms) experiments (46.Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (791) Google Scholar, 47.Sattler M. Maurer M. Schleucher J. Griesinger C. J. Biomol. NMR. 1995; 5: 97-102Crossref PubMed Scopus (78) Google Scholar) were used and allowed for ∼99% complete backbone atom chemical shift assignments. All spectra were processed with NMRPipe (48.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11520) Google Scholar) and analyzed using NMRView (49.Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2676) Google Scholar). Binding of soluble di-C8 PtdIns (Echelon Biosciences) was investigated at 25 °C by NMR titration experiments using 15N-labeled protein in 20 mm Tris buffer, 100 mm NaCl, 0.02% NaN3, pH 7.4. Increasing amounts of PtdIns from a stock solution in NMR buffer were added to the protein solution, and 1H-15N HSQC spectra were recorded for each addition up to a final molar ratio of PtdIns:protein of 20:1. Titration isotherms were plotted for four resonances that exhibited sizable and saturable shifts and no peak overlap or broadening. Dissociation constants were calculated by non-linear best fitting the data for four 1HN titration curves simultaneously using KaleidaGraph (Synergy Software, Reading, PA). Structural assessment of SNX5-PX was carried out in solution by NMR spectroscopy for rat SNX5-PX, which exhibits ∼99% amino acid sequence identity with the human homolog. The protein sequence comprises Met1–Lys180, His-tagged at the N terminus. However, mass spectrometric analysis of the protein after thrombin cleavage and removal of the His tag revealed that an additional cleavage had occurred between residues Arg19 and Ser20. The initial NMR analysis prior to cleavage showed that the first 19 residues of SNX5-PX were highly flexible and essentially unstructured. Therefore, susceptibility to proteolysis is not surprising. The final protein after cleavage and purification that was used for structural work by NMR and crystallography contains residues Ser20–Lys180. The 1H-15N HSQC spectrum of the protein (Fig. 2) exhibits well dispersed and narrow resonances, indicative of a well folded, monomeric structure. Complete backbone assignments were achieved using three-dimensional HNCACB, CBCA(CO)NH, and 1H-15N NOE spectroscopy HSQC spectra. For the C-terminal residues (Leu172 to Arg175), two sets of resonances are observed, most likely caused by nonspecific cleavage of the protein at the C terminus. This was confirmed by mass spectrometry. The x-ray structure of the SNX5-PX domain was determined at 1.47 Å resolution. Clear electron density was observed for residues Val21–Val174. Ser20 and the last six C-terminal residues exhibit high B-factors and were excluded from the model as they could not be accurately traced. The overall architecture of SNX5-PX comprises a three-stranded antiparallel β-sheet abutted by three α-helices. In addition, a one-turn 310-helix that connects helices 4 and 3 and a polyproline region that leads into a long, protruding helical hairpin are present (Fig. 3A). The three β-strands are formed by residues Leu31–Glu41, Lys44–Thr53, and Glu62–Arg67, respectively, with β-strand 1 containing a β-bulge between residues Asp34 and Pro36. The four α-helices comprise residues His69–Glu81 (α1), Asp100–Ser115 (α2′), Lys118–Leu133 (α3′), Ala134–Ser152 (α3), and Arg160–Glu167 (α4). Helices α2′ and α3′ are novel features in SNX5-PX that are absent in other known PX domain structures. The α2′-helix follows after the polyproline (PXXPXXP) region that comprises residues Leu88 to Phe99. The short 310-helix is formed by residues Val155–Lys158. The rest of the structure consists of loops and turns connecting the regular secondary structure elements. At the N terminus, residues Val21–Asp28 form a long, irregular strand and exhibit random coil ϕ and ϕ backbone angles. Interestingly, however, good electron density is observed for these residues because they engage in intermolecular interactions and hydrogen bonding with residues located in the β1-strand of the adjacent molecule (Fig. 3B). The first two PX domain structures that were determined were the NMR structure of p47-PX (50.Hiroaki H. Ago T. Ito T. Sumimoto H. Kohda D. Nat. Struct. Biol. 2001; 8: 526-530Crossref PubMed Scopus (149) Google Scholar) and the x-ray structure of the p40-PX/PtdIns
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