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Structural and Membrane Binding Analysis of the Phox Homology Domain of Bem1p

2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês

10.1074/jbc.m702861200

ISSN

1083-351X

Autores

Robert V. Stahelin, Dimitrios Karathanassis, Diana Murray, Roger Williams, Wonhwa Cho,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX domains, we determined the crystal structure of the PX domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the membrane binding properties of the PX domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX domain contains a signature PI-binding site optimized for PtdIns(4)P binding and also harbors basic and hydrophobic residues on the membrane-binding surface. The membrane binding of the Bem1p PX domain is initiated by nonspecific electrostatic interactions between the cationic membrane-binding surface of the domain and anionic membrane surfaces, followed by the membrane penetration of hydrophobic residues. Unlike other PX domains, the Bem1p PX domain has high intrinsic membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial membrane penetration may occur before specific PtdIns(4)P binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-binding Bem1p PX domain provides new insight into the diverse PI specificities and membrane-binding mechanisms of PX domains. Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX domains, we determined the crystal structure of the PX domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the membrane binding properties of the PX domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX domain contains a signature PI-binding site optimized for PtdIns(4)P binding and also harbors basic and hydrophobic residues on the membrane-binding surface. The membrane binding of the Bem1p PX domain is initiated by nonspecific electrostatic interactions between the cationic membrane-binding surface of the domain and anionic membrane surfaces, followed by the membrane penetration of hydrophobic residues. Unlike other PX domains, the Bem1p PX domain has high intrinsic membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial membrane penetration may occur before specific PtdIns(4)P binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-binding Bem1p PX domain provides new insight into the diverse PI specificities and membrane-binding mechanisms of PX domains. Phosphoinositides (PIs), 3The abbreviations used are:PIsphosphoinositidesPtdInsphosphatidylinositolPHpleckstrin homologyFYVEFab1/YOTB/Vac1/EEA1PXPhox homologyENTHepsin N-terminal homologySH3Src homology 3PLDphospholipase DCISKcytokine-independent survival kinasePI3K-C2αphosphoinositide 3-kinase C2αPSphosphatidylserineSPRsurface plasmon resonancePOPC1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholinePOPS1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserinePOPE1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidMADmultiwavelength anomalous dispersionBem1p-PXBem1p PX domainOSBPoxysterol-binding proteinPPIItype II polyproline helix. phosphorylated derivatives of phosphatidylinositol (PtdIns), regulate diverse biological processes such as growth, membrane trafficking, cell survival, and cytoskeletal rearrangement (1Di Paolo G. De Camilli P. Nature. 2006; 443: 651-657Crossref PubMed Scopus (2079) Google Scholar, 2Roth M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (240) Google Scholar). PtdIns is reversibly phosphorylated at the D3, D4, or D5 position to yield seven different PIs, which are both transiently and constitutively present in different cell membranes. Research in the past decade has revealed that a large number of cellular proteins reversibly translocate to these specific subcellular locations to form lipid-protein interactions (3Cho W. Sci. STKE. 2006; 2006: PE7PubMed Google Scholar, 4Teruel M.N. Meyer T. Cell. 2000; 103: 181-184Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). These interactions play a crucial role in cell signaling and membrane trafficking, and the PIs are responsible for regulating not only the localization but also, in many instances, the biological activity of their effector protein (2Roth M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (240) Google Scholar, 5Cremona O. De Camilli P. J. 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Cell Biol. 2004; 5: 55-63Crossref PubMed Scopus (92) Google Scholar), and protein kinase C conserved 2 (C2) (23Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 24Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (689) Google Scholar, 25Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 26Cho W. Stahelin R.V. Biochim. Biophys. Acta. 2006; 1761: 838-849Crossref PubMed Scopus (214) Google Scholar) domains. phosphoinositides phosphatidylinositol pleckstrin homology Fab1/YOTB/Vac1/EEA1 Phox homology epsin N-terminal homology Src homology 3 phospholipase D cytokine-independent survival kinase phosphoinositide 3-kinase C2α phosphatidylserine surface plasmon resonance 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid multiwavelength anomalous dispersion Bem1p PX domain oxysterol-binding protein type II polyproline helix. PI metabolism is also crucial to the budding yeast Saccharomyces cerevisiae (27Wera S. Bergsma J.C. Thevelein J.M. FEMS Yeast Res. 2001; 1: 9-13Crossref PubMed Scopus (33) Google Scholar), although the function and regulation of PIs and PI effectors are still less defined. Growth of S. cerevisiae by budding requires polarity establishment to expand the cell wall, and this bud emergence process is tightly regulated and occurs at distinct sites in new cells (28Chant J. Pringle J.R. J. Cell Biol. 1995; 129: 751-765Crossref PubMed Scopus (243) Google Scholar, 29Zahner J.E. Harkins H.A. Pringle J.R. Mol. Cell. Biol. 1996; 16: 1857-1870Crossref PubMed Scopus (194) Google Scholar) following a period of uniform growth during G1. Recent studies have identified a number of key players in the initiation of bud formation: Cdc42p, a small GTPase protein; Cdc24p, a GDP/GTP exchange factor for Cdc42p; and Bem1p, a putative scaffold protein (30Adams A.E. Johnson D.I. Longnecker R.M. Sloat B.F. Pringle J.R. J. Cell Biol. 1990; 111: 131-142Crossref PubMed Scopus (475) Google Scholar, 31Bender A. Pringle J.R. Mol. Cell. Biol. 1991; 11: 1295-1305Crossref PubMed Scopus (351) Google Scholar, 32Hartwell L.H. Exp. Cell Res. 1971; 69: 265-276Crossref PubMed Scopus (475) Google Scholar). Bem1p is a multidomain scaffolding protein that binds Cdc42p with its N-terminal Src homology 3 (SH3) domain (33Bose I. Irazoqui J.E. Moskow J.J. Bardes E.S. Zyla T.R. Lew D.J. J. Biol. Chem. 2001; 276: 7176-7186Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), and this interaction is critical for proper Cdc42p activation (34Irazoqui J.E. Gladfelter A.S. Lew D.J. Nat. Cell Biol. 2003; 5: 1062-1070Crossref PubMed Scopus (204) Google Scholar). Bem1p has been shown to migrate to the plasma membrane during budding and mating, where it can serve as an adaptor for Cdc42p and other proteins (35Ito T. Matsui Y. Ago T. Ota K. Sumimoto H. EMBO J. 2001; 20: 3938-3946Crossref PubMed Scopus (137) Google Scholar, 36Shimada Y. Gulli M.P. Peter M. Nat. Cell Biol. 2000; 2: 117-124Crossref PubMed Scopus (151) Google Scholar). The mechanism behind the plasma membrane translocation of Bem1p is still unknown. Interestingly, Bem1p has been shown to harbor a PX domain that binds PtdIns(4)P (37Ago T. Takeya R. Hiroaki H. Kuribayashi F. Ito T. Kohda D. Sumimoto H. Biochem. Biophys. Res. Commun. 2001; 287: 733-738Crossref PubMed Scopus (92) Google Scholar). PtdIns(4)P has been shown to be localized to both the plasma membrane and secretory machinery in yeast (27Wera S. Bergsma J.C. Thevelein J.M. FEMS Yeast Res. 2001; 1: 9-13Crossref PubMed Scopus (33) Google Scholar). The molecular details of a number of protein-protein interactions have been mapped out for Bem1p (38Vollert C.S. Uetz P. Mol. Cell. Proteomics. 2004; 3: 1053-1064Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), but much less is known about its lipid binding properties, in particular the role of its PX domain in the membrane recruitment of Bem1p. The PX domain is a structural module composed of 100-140 amino acids that was first identified in the p40phox and p47phox subunits of NADPH oxidase (39Ponting C.P. Protein Sci. 1996; 5: 2353-2357Crossref PubMed Scopus (266) Google Scholar) and has since been found in a variety of other proteins involved in membrane trafficking (e.g. Mvp1p, Vps5p, Bem1p, Grd19p, and the sorting nexin family of proteins) and cell signaling (e.g. phospholipase D (PLD), PI 3-kinases, cytokine-independent survival kinase (CISK), and five SH3 domains (FISH)). Sequence comparisons of PX domains have shown that they contain several conserved regions, including a proline-rich stretch (PXXP) and a number of basic residues (13Wishart M.J. Taylor G.S. Dixon J.E. Cell. 2001; 105: 817-820Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 14Seet L.F. Hong W. Biochim. Biophys. Acta. 2006; 1761: 878-896Crossref PubMed Scopus (163) Google Scholar). Subsequently, PX domains have been shown to interact with different PIs via conserved basic residues and to target the host proteins to specific subcellular locations (40Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (498) Google Scholar, 41Cheever M.L. Sato T.K. de Beer T. Kutateladze T.G. Emr S.D. Overduin M. Nat. Cell Biol. 2001; 3: 613-618Crossref PubMed Scopus (315) Google Scholar, 42Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (360) Google Scholar, 43Song X. Xu W. Zhang A. Huang G. Liang X. Virbasius J.V. Czech M.P. Zhou G.W. Biochemistry. 2001; 40: 8940-8944Crossref PubMed Scopus (107) Google Scholar, 44Xu Y. Hortsman H. Seet L. Wong S.H. Hong W. Nat. Cell Biol. 2001; 3: 658-666Crossref PubMed Scopus (240) Google Scholar, 45Yu J.W. Lemmon M.A. J. Biol. Chem. 2001; 276: 44179-44184Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). PX domains are similar to the PH domain in that they exhibit broad PI specificity. It was initially reported that PX domains of Vam7p (41Cheever M.L. Sato T.K. de Beer T. Kutateladze T.G. Emr S.D. Overduin M. Nat. Cell Biol. 2001; 3: 613-618Crossref PubMed Scopus (315) Google Scholar), sorting nexin-3 (44Xu Y. Hortsman H. Seet L. Wong S.H. Hong W. Nat. Cell Biol. 2001; 3: 658-666Crossref PubMed Scopus (240) Google Scholar), and p40phox (40Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (498) Google Scholar) specifically interact with PtdIns(3)P in vitro and also target the host proteins to early endosomes in the cell. It was also reported that most of the yeast PX domains bind PtdIns(3)P (45Yu J.W. Lemmon M.A. J. Biol. Chem. 2001; 276: 44179-44184Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), albeit with varying affinities. On the hand other, the PX domain of Class II PI 3-kinase C2α (PI3K-C2α) interacts with PtdIns(4,5)P2 (43Song X. Xu W. Zhang A. Huang G. Liang X. Virbasius J.V. Czech M.P. Zhou G.W. Biochemistry. 2001; 40: 8940-8944Crossref PubMed Scopus (107) Google Scholar, 46Stahelin R.V. Karathanassis D. Bruzik K.S. Waterfield M.D. Bravo J. Williams R.L. Cho W. J. Biol. Chem. 2006; 281: 39396-39406Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), whereas the p47phox PX domain preferentially interacts with PtdIns(3,4)P2 (47Karathanassis D. Stahelin R.V. Bravo J. Perisic O. Pacold C.M. Cho W. Williams R.L. EMBO J. 2002; 21: 5057-5068Crossref PubMed Scopus (259) Google Scholar). Also, the PX domain of the yeast protein PLD1 has specificity for PtdIns(3,4,5)P3 (48Lee J.S. Kim J.H. Jang I.H. Kim H.S. Han J.M. Kazlauskas A. Yagisawa H. Suh P.G. Ryu S.H. J. Cell Sci. 2005; 118: 4405-4413Crossref PubMed Scopus (43) Google Scholar, 49Stahelin R.V. Ananthanarayanan B. Blatner N.R. Singh S. Bruzik K.S. Murray D. Cho W. J. Biol. Chem. 2004; 279: 54918-54926Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), whereas the PX domain of NOXO1 (Nox-organizing protein-1) was reported to bind PtdIns(4)P, PtdIns(5)P, and PtdIns(3,5)P2 (50Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Recent structural and modeling studies of a variety of PX domains have lead to a better understanding of the mechanisms of stereospecific PI recognition and membrane binding by PX domains. Earlier structural studies focused on PX domains that interact with PtdIns(3)P. For example, the crystal structure of the p40phox-PtdIns(3)P complex illustrated how the domain achieves the stereospecific recognition of PtdIns(3)P (51Bravo 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 (238) Google Scholar). The structure revealed that basic residues Lys92 and Arg58 specifically form hydrogen bonds with the D1- and D3-phosphates of PtdIns(3)P, respectively. The crystal structure of the CISK PX domain showed that this domain also has all the basic residues necessary for binding the D3-phosphate of PtdIns(3)P (52Xing Y. Liu D. Zhang R. Joachimiak A. Songyang Z. Xu W. J. Biol. Chem. 2004; 279: 30662-30669Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The crystal structures of the free and PtdIns(3)P-bound PX domains of the yeast Grd19p protein showed the lipid-induced local conformational changes in the membrane-binding loop (53Zhou C.Z. de La Sierra-Gallay I.L. Quevillon-Cheruel S. Collinet B. Minard P. Blondeau K. Henckes G. Aufrere 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 (58) Google Scholar). NMR studies of the Vam7p PX domain have also elucidated the origin of its PtdIns(3)P specificity and the membrane-docking mechanism (54Lee S.A. Kovacs J. Stahelin R.V. Cheever M.L. Setty T.G. Burd C. Cho W. Kutateladze T.G. J. Biol. Chem. 2006; 281: 37091-37101Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 55Lu J. Garcia J. Dulubova I. Sudhof T.C. Rizo J. Biochemistry. 2002; 41: 5956-5962Crossref PubMed Scopus (34) Google Scholar). In addition to these studies on PtdIns(3)P-binding PX domains, structural studies on the p47phox (47Karathanassis D. Stahelin R.V. Bravo J. Perisic O. Pacold C.M. Cho W. Williams R.L. EMBO J. 2002; 21: 5057-5068Crossref PubMed Scopus (259) Google Scholar) and PI3K-C2α (46Stahelin R.V. Karathanassis D. Bruzik K.S. Waterfield M.D. Bravo J. Williams R.L. Cho W. J. Biol. Chem. 2006; 281: 39396-39406Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) PX domains that specifically interact with PtdIns(3,4)P2 and PtdIns(4,5)P2, respectively, showed how these PX domains achieve different PI specificities. In particular, the crystal structure of the PX domain of p47phox revealed that this PX domain has a smaller secondary pocket that binds phosphatidic acid or phosphatidylserine (PS) (47Karathanassis D. Stahelin R.V. Bravo J. Perisic O. Pacold C.M. Cho W. Williams R.L. EMBO J. 2002; 21: 5057-5068Crossref PubMed Scopus (259) Google Scholar). A modeling study of the PLD1 PX domain also suggested that it has two binding pockets, a primary site specific for PtdIns(3,4,5)P3 and a second site that interacts nonspecifically with anionic phospholipids (49Stahelin R.V. Ananthanarayanan B. Blatner N.R. Singh S. Bruzik K.S. Murray D. Cho W. J. Biol. Chem. 2004; 279: 54918-54926Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). To date, no structural information is available for the PX domains with specificity for PtdIns(4)P. To gain a better understanding of differential PI recognition and membrane-binding mechanisms of PX domains, we determined the x-ray crystal structure of the Bem1p PX domain, which has been reported to bind PtdIns(4)P (37Ago T. Takeya R. Hiroaki H. Kuribayashi F. Ito T. Kohda D. Sumimoto H. Biochem. Biophys. Res. Commun. 2001; 287: 733-738Crossref PubMed Scopus (92) Google Scholar). We also measured the interaction of this domain and mutations with model membranes containing various PIs by surface plasmon resonance (SPR) and monolayer penetration analyses and calculated the electrostatic potential of the domain in the absence and presence of lipid ligand. The results provide new insight into how the Bem1p PX domain specifically recognizes PtdIns(4)P and how the domain may be targeted to the PtdIns(4)P-containing membranes. Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were from Avanti Polar Lipids (Alabaster, AL). PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 were purchased from Cayman (Ann Arbor, MI). Phospholipid concentrations were determined by phosphate analysis (56Kates M. Amsterdam B.V. Techniques of Lipidology. 2nd Ed. Elsevier Science Publishers, 1986: 114-115Google Scholar). The LiposoFast microextruder and 100-nm polycarbonate filters were from Avestin (Ottawa, Ontario, Canada). Fatty acid-free bovine serum albumin was from Bayer (Kankakee, IL). Restriction endonucleases and other enzymes for molecular biology were from New England Biolabs (Beverly, MA). CHAPS and octyl glucoside were from Sigma and Fisher, respectively. The Pioneer L1 sensor chip was from Biacore (Piscataway, NJ). Structure Determination—For structure determination, DNA encoding the yeast Bem1p PX domain (residues 266-413) was amplified by PCR from yeast genomic DNA and subsequently cloned with a C-terminal His6 affinity tag in the pJL vector. The protein was expressed in the methionine-requiring auxotrophic Escherichia coli strain 834(DE3) and purified by Ni2+ affinity, heparin, and gel filtration chromatography. The protein in gel filtration buffer (20 mm Tris-HCl (pH 7.4 at 25 °C), 100 mm NaCl, and 5 mm dithiothreitol) was concentrated to 5 mg/ml. Crystals were obtained in sitting drops (3 μl of protein plus 3 μl of reservoir solution) that were incubated at 14 °C over a reservoir consisting of 0.2 m NaCl, 0.1 m sodium/potassium phosphate (pH 6.2), 10% polyethylene glycol 8000, and 2 mm dithiothreitol. Crystals were visible after 12 h and grew to full size within 1 week. For diffraction data collection, crystals were cryoprotected by adding Paratone-N to the drop and removing excess mother liquor surrounding the crystal. Loops containing the crystal in Paratone-N with minimal mother liquor were flash-frozen in a nitrogen stream at 100 K. A three-wavelength multiwavelength anomalous dispersion (MAD) data collection was carried out. Table 1 summarizes the data collection statistics. Images were processed with the program MOSFLM (57Leslie A.G.W. Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography. Daresbury Laboratory, Warrington, UK1992Google Scholar) and refined with SCALA (58Collaborative Computational ProjectActa Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19762) Google Scholar). Four selenium sites were located with SOLVE (59Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) and refined with SHARP (60Vonrhein C. Blanc E. Roversi P. Bricogne G. Methods Mol. Biol. 2006; 364: 215-230Google Scholar). After density modification with SOLOMON (61Abrahams J.P. Leslie A.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar) and DM (62Cowtan K. Main P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 487-493Crossref PubMed Scopus (309) Google Scholar), an initial model was automatically built using ARP/wARP (63Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar) and manually adjusted using program O (64Jones T.A. Zou J-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar). The model was refined with REFMAC (65Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13865) Google Scholar). There are two molecules in the asymmetric unit. Residues 266-275 and 411-413 are not ordered in the electron density map. Ramachandran analysis with the program PROCHECK (66Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) showed 92% of residues in the most probable regions and no residues in the disallowed area. The refinement statistics are given in Table 1. A representative section of the experimental and refined electron densities is illustrated in supplemental Fig. 1.TABLE 1Data collection, structure determination, and refinement statisticsPeakaData sets were collected at European Synchrotron Radiation Facility beamline ID14-4 at λ = 0.9793, 0.9796, and 0.9393 Å for peak, inflection, and remote, respectively, using an ADSC detector. The MAD phase refinement was carried out at 1.7-Å resolution. The remote data set was used for the structure refinement. The MAD phasing was carried out using inflection, peak, and remote data sets.InflectionaData sets were collected at European Synchrotron Radiation Facility beamline ID14-4 at λ = 0.9793, 0.9796, and 0.9393 Å for peak, inflection, and remote, respectively, using an ADSC detector. The MAD phase refinement was carried out at 1.7-Å resolution. The remote data set was used for the structure refinement. The MAD phasing was carried out using inflection, peak, and remote data sets.RemoteaData sets were collected at European Synchrotron Radiation Facility beamline ID14-4 at λ = 0.9793, 0.9796, and 0.9393 Å for peak, inflection, and remote, respectively, using an ADSC detector. The MAD phase refinement was carried out at 1.7-Å resolution. The remote data set was used for the structure refinement. The MAD phasing was carried out using inflection, peak, and remote data sets.Data collection statistics Resolution (Å) 1.7 1.7 1.5 Completeness (last shell) 88 (51) 88 (51) 99.6 (99.4)Rmerge (last shell)bRmerge = ∑hkl∑i|Ii(hkl) - 〈I(hkl)〉|/∑hkl∑iIi(hkl). 0.049 (0.30) 0.049 (0.29) 0.068 (0.38) Multiplicity (last shell) 3.4 (2.5) 3.4 (2.5) 3.5 (3.6) (last shell) 18.2 (2.5) 17.7 (2.1) 8.9 (1.5) Unit cell (P212121) a = 61.0, b = 71.6, c = 75.3Phasing statisticsaData sets were collected at European Synchrotron Radiation Facility beamline ID14-4 at λ = 0.9793, 0.9796, and 0.9393 Å for peak, inflection, and remote, respectively, using an ADSC detector. The MAD phase refinement was carried out at 1.7-Å resolution. The remote data set was used for the structure refinement. The MAD phasing was carried out using inflection, peak, and remote data sets. Isomorphous phasing powercValues are the ratio of the heavy atom structure factor amplitudes to the lack-of-closure error. 0.5 1.3 Anomalous phasing powercValues are the ratio of the heavy atom structure factor amplitudes to the lack-of-closure error. 1.4 0.9 1.0 Se sites found 4 FOMdFOM, figure of merit. after SHARP 0.32 FOM after SOLOMON 0.78 FOM after DM 0.92Refinement statisticsaData sets were collected at European Synchrotron Radiation Facility beamline ID14-4 at λ = 0.9793, 0.9796, and 0.9393 Å for peak, inflection, and remote, respectively, using an ADSC detector. The MAD phase refinement was carried out at 1.7-Å resolution. The remote data set was used for the structure refinement. The MAD phasing was carried out using inflection, peak, and remote data sets. Resolution range (Å) 51.9-1.5 Number of reflections 50,498 Cutoff (F/σ) None Completeness (%) 99.4 Protein atoms 4339 Average total B factor (Wilson B factor; Å2) 16 (19) Waters 9 RcrysteRcryst and Rfree = ∑||Fo| - |Fc||/∑|Fo|. Rfree was calculated with the percentage of the data shown in parentheses. 0.22 Rfree (% data used)eRcryst and Rfree = ∑||Fo| - |Fc||/∑|Fo|. Rfree was calculated with the percentage of the data shown in parentheses. 0.25 (5.1) r.m.s.d. from idealityfRoot mean square deviations are for bond angles and lengths with regard to the parameters of Engh and Huber (89).Bonds (Å) 0.012Angles 1.2°Dihedrals4.9°a Data sets were collected at European Synchrotron Radiation Facility beamline ID14-4 at λ = 0.9793, 0.9796, and 0.9393 Å for peak, inflection, and remote, respectively, using an ADSC detector. The MAD phase refinement was carried out at 1.7-Å resolution. The remote data set was used for the structure refinement. The MAD phasing was carried out using inflection, peak, and remote data sets.b Rmerge = ∑hkl∑i|Ii(hkl) - 〈I(hkl)〉|/∑hkl∑iIi(hkl).c Values are the ratio of the heavy atom structure factor amplitudes to the lack-of-closure error.d FOM, figure of merit.e Rcryst and Rfree = ∑||Fo| - |Fc||/∑|Fo|. Rfree was calculated with the percentage of the data shown in parentheses.f Root mean square deviations are for bond angles and lengths with regard to the parameters of Engh and Huber (89Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2545) Google Scholar). Open table in a new tab Mutagenesis and Protein Expression—Mutagenesis of the Bem1p PX domain (Bem1p-PX) was performed using the overlap extension PCR method (67Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6

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