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• W2891533071 abstract "The glycolipid transfer protein (GLTP) fold defines a superfamily of eukaryotic proteins that selectively transport sphingolipids (SLs) between membranes. However, the mechanisms determining the protein selectivity for specific glycosphingolipids (GSLs) are unclear. Here, we report the crystal structure of the GLTP homology (GLTPH) domain of human 4-phosphate adaptor protein 2 (FAPP2) bound with N-oleoyl-galactosylceramide. Using this domain, FAPP2 transports glucosylceramide from its cis-Golgi synthesis site to the trans-Golgi for conversion into complex GSLs. The FAPP2–GLTPH structure revealed an element, termed the ID loop, that controls specificity in the GLTP family. We found that, in accordance with FAPP2 preference for simple GSLs, the ID loop protrudes from behind the SL headgroup-recognition center to mitigate binding by complex GSLs. Mutational analyses including GLTP and FAPP2 chimeras with swapped ID loops supported the proposed restrictive role of the FAPP2 ID loop in GSL selectivity. Comparative analysis revealed distinctly designed ID loops in each GLTP family member. This analysis also disclosed a conserved H-bond triplet that “clasps” both ID-loop ends together to promote structural autonomy and rigidity. The findings indicated that various ID loops work in concert with conserved recognition centers to create different specificities among family members. We also observed four bulky, conserved hydrophobic residues involved in “sensor-like” interactions with lipid chains in protein hydrophobic pockets and FF motifs in GLTP and FAPP2, well-positioned to provide acyl chain–dependent SL selectivity for the hydrophobic pockets. In summary, our study provides mechanistic insights into sphingolipid recognition by the GLTP fold and uncovers the elements involved in this recognition. The glycolipid transfer protein (GLTP) fold defines a superfamily of eukaryotic proteins that selectively transport sphingolipids (SLs) between membranes. However, the mechanisms determining the protein selectivity for specific glycosphingolipids (GSLs) are unclear. Here, we report the crystal structure of the GLTP homology (GLTPH) domain of human 4-phosphate adaptor protein 2 (FAPP2) bound with N-oleoyl-galactosylceramide. Using this domain, FAPP2 transports glucosylceramide from its cis-Golgi synthesis site to the trans-Golgi for conversion into complex GSLs. The FAPP2–GLTPH structure revealed an element, termed the ID loop, that controls specificity in the GLTP family. We found that, in accordance with FAPP2 preference for simple GSLs, the ID loop protrudes from behind the SL headgroup-recognition center to mitigate binding by complex GSLs. Mutational analyses including GLTP and FAPP2 chimeras with swapped ID loops supported the proposed restrictive role of the FAPP2 ID loop in GSL selectivity. Comparative analysis revealed distinctly designed ID loops in each GLTP family member. This analysis also disclosed a conserved H-bond triplet that “clasps” both ID-loop ends together to promote structural autonomy and rigidity. The findings indicated that various ID loops work in concert with conserved recognition centers to create different specificities among family members. We also observed four bulky, conserved hydrophobic residues involved in “sensor-like” interactions with lipid chains in protein hydrophobic pockets and FF motifs in GLTP and FAPP2, well-positioned to provide acyl chain–dependent SL selectivity for the hydrophobic pockets. In summary, our study provides mechanistic insights into sphingolipid recognition by the GLTP fold and uncovers the elements involved in this recognition. Human 4-phosphate-adaptor-protein-2 (FAPP2) 4The abbreviations used are: FAPP2phosphoinositol 4-phosphate adaptor protein 2GLTPglycolipid transfer proteinGLTPHGLTP homologyGLTP foldthe fold first found in human GLTPSLsphingolipidGSLglycosphingolipidGlcCerglucosylceramideGalCergalactosylceramideLacCerlactosylceramidePOPC1-palmitoyl-2-oleyl phosphatidylcholinePerperylenoylAVanthrylvinylSFsulfatidePDBProtein Data BankLTPlipid-transfer proteinC1Pceramide-1-phosphateACD11accelerated cell death 11 proteinAUasymmetric unitSPRsurface plasmon resonanceSUMOsmall ubiquitin-like modifier. is a Golgi-associated, two-domain protein consisting of 519 amino acids. The N-terminal pleckstrin homology domain (93 residues) that helps target the Golgi is connected by a 214-residue linker to a C-terminal glycolipid transfer protein homology (GLTPH) domain (212 residues) (1D'Angelo G. Polishchuk E. Di Tullio G. Santoro M. Di Campli A. Godi A. West G. Bielawski J. Chuang C.C. van der Spoel A.C. Platt F.M. Hannun Y.A. Polishchuk R. Mattjus P. De Matteis M.A. Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.Nature. 2007; 449: 62-67Crossref PubMed Scopus (329) Google Scholar, 2Kamlekar R.K. Simanshu D.K. Gao Y.G. Kenoth R. Pike H.M. Prendergast F.G. Malinina L. Molotkovsky J.G. Venyaminov S.Y. Patel D.J. Brown R.E. The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.Biochim. Biophys. Acta. 2013; 1831: 417-427Crossref PubMed Scopus (22) Google Scholar). In vivo, FAPP2 transports glucosylceramides (GlcCer) from the synthesis site in the cis-Golgi to the trans-Golgi/TGN where conversion into more complex glycosphingolipids (GSLs) occurs via sequential sugar addition (1D'Angelo G. Polishchuk E. Di Tullio G. Santoro M. Di Campli A. Godi A. West G. Bielawski J. Chuang C.C. van der Spoel A.C. Platt F.M. Hannun Y.A. Polishchuk R. Mattjus P. De Matteis M.A. Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.Nature. 2007; 449: 62-67Crossref PubMed Scopus (329) Google Scholar, 3D'Angelo G. Uemura T. Chuang C.C. Polishchuk E. Santoro M. Ohvo-Rekila H. Sato T. Di Tullio G. Varriale A. D'Auria S. Daniele T. Capuani F. Johannes L. Mattjus P. Monti M. Pucci P. Williams R.L. Burke J.E. Platt F.M. Harada A. De Matteis M.A. Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi.Nature. 2013; 501: 116-120Crossref PubMed Scopus (115) Google Scholar). When docked with Golgi membranes via the pleckstrin homology domain, the long FAPP2 connecting linker enables the soluble GLTPH domain to move locally within the cytoplasm and approach the Golgi to acquire or release specific sphingolipids (SLs) from/to accessible membrane regions, i.e. act as a lipid-transfer protein (LTP) “on a leash.” phosphoinositol 4-phosphate adaptor protein 2 glycolipid transfer protein GLTP homology the fold first found in human GLTP sphingolipid glycosphingolipid glucosylceramide galactosylceramide lactosylceramide 1-palmitoyl-2-oleyl phosphatidylcholine perylenoyl anthrylvinyl sulfatide Protein Data Bank lipid-transfer protein ceramide-1-phosphate accelerated cell death 11 protein asymmetric unit surface plasmon resonance small ubiquitin-like modifier. Structural homology modeling of the FAPP2–GLTPH domain suggests membership in the GLTP superfamily (1D'Angelo G. Polishchuk E. Di Tullio G. Santoro M. Di Campli A. Godi A. West G. Bielawski J. Chuang C.C. van der Spoel A.C. Platt F.M. Hannun Y.A. Polishchuk R. Mattjus P. De Matteis M.A. Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.Nature. 2007; 449: 62-67Crossref PubMed Scopus (329) Google Scholar, 2Kamlekar R.K. Simanshu D.K. Gao Y.G. Kenoth R. Pike H.M. Prendergast F.G. Malinina L. Molotkovsky J.G. Venyaminov S.Y. Patel D.J. Brown R.E. The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.Biochim. Biophys. Acta. 2013; 1831: 417-427Crossref PubMed Scopus (22) Google Scholar, 4Mattjus P. Glycolipid transfer proteins and membrane interaction.Biochim. Biophys. Acta. 2009; 1788: 267-272Crossref PubMed Scopus (56) Google Scholar), a group of eukaryotic LTPs that selectively transfer SLs between membranes (5Abe A. Yamada K. Sasaki T. A protein purified from pig brain accelerates the inter-membranous translocation of mono- and dihexosylceramides, but not the translocation of phospholipids.Biochem. Biophys. Res. Commun. 1982; 104: 1386-1393Crossref PubMed Scopus (40) Google Scholar, 6Abe A. Sasaki T. Purification and some properties of the glycolipid transfer protein from pig brain.J. Biol. Chem. 1985; 260: 11231-11239Abstract Full Text PDF PubMed Google Scholar, 7Brown R.E. Stephenson F.A. Markello T. Barenholz Y. Thompson T.E. Properties of a specific glycolipid transfer protein from bovine brain.Chem. Phys. Lipids. 1985; 38: 79-93Crossref PubMed Scopus (58) Google Scholar, 8Brown R.E. Jarvis K.L. Hyland K.J. Purification and characterization of glycolipid transfer protein from bovine brain.Biochim. Biophys. Acta. 1990; 1044: 77-83Crossref PubMed Scopus (34) Google Scholar, 9Metz R.J. Radin N.S. Purification and properties of a cerebroside transfer protein.J. Biol. Chem. 1982; 257: 12901-12907Abstract Full Text PDF PubMed Google Scholar, 10Simanshu D.K. Kamlekar R.K. Wijesinghe D.S. Zou X. Zhai X. Mishra S.K. Molotkovsky J.G. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Patel D.J. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.Nature. 2013; 500: 463-467Crossref PubMed Scopus (134) Google Scholar, 11Malinina L. Simanshu D.K. Zhai X. Samygina V.R. Kamlekar R. Kenoth R. Ochoa-Lizarralde B. Malakhova M.L. Molotkovsky J.G. Patel D.J. Brown R.E. Sphingolipid transfer proteins defined by the GLTP-fold.Q Rev. Biophys. 2015; 48: 281-322Crossref PubMed Scopus (24) Google Scholar) and share a common protein fold first established from human GLTP crystal structure (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Structural basis for glycosphingolipid transfer specificity.Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (101) Google Scholar). The GLTP fold is composed of eight α-helices, organized as a curved two-layer “sandwich” that envelopes the SL aliphatic chains within an internal hydrophobic pocket that is accessed via a cleft-like gate, whereas the SL-specific headgroup-binding site includes a surface localized recognition center (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Structural basis for glycosphingolipid transfer specificity.Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (101) Google Scholar). Crystal structures of four other GLTP superfamily members (fungal heterokaryon incompatibility C2 protein, HET-C2, of Podosporaanserina; GLTP-like protein from the thermoacidophilic unicellular red alga, Galdieria sulfuraria; human ceramide-1-phosphate (C1P) transfer protein, CPTP; and accelerated cell death 11 protein (ACD11), an Arabidopsis CPTP homolog) reveal overall similarity with GLTP structure and nearly conserved arrangement of recognition center positions but with distinctions enabling selectivity of SLs with sugar (GSL) versus phosphate (C1P) headgroups (Refs. 10Simanshu D.K. Kamlekar R.K. Wijesinghe D.S. Zou X. Zhai X. Mishra S.K. Molotkovsky J.G. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Patel D.J. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.Nature. 2013; 500: 463-467Crossref PubMed Scopus (134) Google Scholar, 13Kenoth R. Simanshu D.K. Kamlekar R.K. Pike H.M. Molotkovsky J.G. Benson L.M. Bergen 3rd, H.R. Prendergast F.G. Malinina L. Venyaminov S.Y. Patel D.J. Brown R.E. Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold.J. Biol. Chem. 2010; 285: 13066-13078Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 14Simanshu D.K. Zhai X. Munch D. Hofius D. Markham J.E. Bielawski J. Bielawska A. Malinina L. Molotkovsky J.G. Mundy J.W. Patel D.J. Brown R.E. Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels.Cell Rep. 2014; 6: 388-399Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, and PDB code 2I3F), defining two subfamilies, GSL-specific and C1P-specific. Modeling of FAPP2–GLTPH relative to the GLTP structure shows conservation of residues required for sugar headgroup binding (2Kamlekar R.K. Simanshu D.K. Gao Y.G. Kenoth R. Pike H.M. Prendergast F.G. Malinina L. Molotkovsky J.G. Venyaminov S.Y. Patel D.J. Brown R.E. The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.Biochim. Biophys. Acta. 2013; 1831: 417-427Crossref PubMed Scopus (22) Google Scholar). However, FAPP2 prefers simple GSLs. The in vitro transfer of GlcCer and GalCer by FAPP2–GLTPH domain is equally efficient but is reduced by ∼50% with LacCer and is slower yet with complex GSLs, such as ganglioside GM1 that possess five sugars including N-acetylneuraminic acid, or sulfo-GalCer, sulfatide (SF) (2Kamlekar R.K. Simanshu D.K. Gao Y.G. Kenoth R. Pike H.M. Prendergast F.G. Malinina L. Molotkovsky J.G. Venyaminov S.Y. Patel D.J. Brown R.E. The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.Biochim. Biophys. Acta. 2013; 1831: 417-427Crossref PubMed Scopus (22) Google Scholar). By contrast, human GLTP efficiently transfers both complex and simple GSLs (6Abe A. Sasaki T. Purification and some properties of the glycolipid transfer protein from pig brain.J. Biol. Chem. 1985; 260: 11231-11239Abstract Full Text PDF PubMed Google Scholar, 7Brown R.E. Stephenson F.A. Markello T. Barenholz Y. Thompson T.E. Properties of a specific glycolipid transfer protein from bovine brain.Chem. Phys. Lipids. 1985; 38: 79-93Crossref PubMed Scopus (58) Google Scholar, 9Metz R.J. Radin N.S. Purification and properties of a cerebroside transfer protein.J. Biol. Chem. 1982; 257: 12901-12907Abstract Full Text PDF PubMed Google Scholar), as reviewed in Ref. 11Malinina L. Simanshu D.K. Zhai X. Samygina V.R. Kamlekar R. Kenoth R. Ochoa-Lizarralde B. Malakhova M.L. Molotkovsky J.G. Patel D.J. Brown R.E. Sphingolipid transfer proteins defined by the GLTP-fold.Q Rev. Biophys. 2015; 48: 281-322Crossref PubMed Scopus (24) Google Scholar. Crystal structures of different holo forms of human GLTP provide insights into the structural features responsible for the recognition of GalCer, GlcCer, LacCer, and SF. Also available are two apo structures of other GSL-specific members and different holo forms of two C1P-specific members, providing a potential source for comparative analyses. However, the basis for the focused FAPP2–GLTPH selectivity for simple GSLs has remained a matter of speculation because of the lack of structure determination for FAPP2. To address the issue, we present here the 1.45 Å resolution crystal structure of FAPP2–GLTPH domain (residues 308–519) complexed with the GSL, N-oleoyl-galactosylceramide (18:1-GalCer), and perform comparative analysis that includes other available structures of the GLTP superfamily members. The approach provides insights into the strict preference of FAPP2 for simple GSLs; reveals a unique element, termed the ID-loop, that distinguishes each particular protein of the family; and enables identification of previously unrecognized but important elements of the GLTP fold. After numerous crystallization trials were unsuccessful, we introduced the E377A/E378K double mutation far from functionally important regions (Fig. S1a) to reduce the excessive negative surface charge of FAPP2–GLTPH domain (residues 308–519) (2Kamlekar R.K. Simanshu D.K. Gao Y.G. Kenoth R. Pike H.M. Prendergast F.G. Malinina L. Molotkovsky J.G. Venyaminov S.Y. Patel D.J. Brown R.E. The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.Biochim. Biophys. Acta. 2013; 1831: 417-427Crossref PubMed Scopus (22) Google Scholar). This change did not affect transfer activity (Fig. S1b) but enabled a high-resolution crystal of the GLTPH domain complexed with N-oleoyl-galactosylceramide (18:1-GalCer) to form. Hereafter, “FAPP2–GLTPH” refers to the E377A/E378K mutant. The final model was refined to Rwork/Rfree values of 0.131/0.179 at 1.45 Å resolution. Table 1 summarizes the X-ray data collection and refinement statistics.Table 1X-ray data collection and refinement statistics for human FAPP2–GLTPH·18:1-GalCer structureData collection Space groupP212121 a (Å)66.24 b (Å)74.61 c (Å)93.59 α = β = γ (°)90.0 AU content2 molecules Resolution (Å)58.34–1.45 (1.53–1.45) Rmerge0.047 (0.754) I/σI14.5 (2.0) Completeness (%)99.7 (99.7) Redundancy4.3 (4.3)Refinement Resolution (Å)15.0–1.45 No. of reflections Work78,300 Free4122 Rwork/Rfree0.131/0.179 No. of atoms Protein3454 Lipid102 Water493 B factors (Å2) Protein A/B26.6/29.4 Lipid A/B36.0/34.6 Water45.2 RMSD Bond lengths (Å)0.019 Bond angles (°)1.77PDB code5KDI Open table in a new tab The overall structure of FAPP2–GLTPH with bound 18:1-GalCer (Fig. 1a) resembles GLTP complexed with 18:1 GlcCer (PDB code 3S0K) (15Samygina V.R. Popov A.N. Cabo-Bilbao A. Ochoa-Lizarralde B. Goni-de-Cerio F. Zhai X. Molotkovsky J.G. Patel D.J. Brown R.E. Malinina L. Enhanced selectivity for sulfatide by engineered human glycolipid transfer protein.Structure. 2011; 19: 1644-1654Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) with respect to lipid position and binding mode. The recognition center (Fig. 1b) displays a similar network of interactions as GLTP for anchoring the polar region of GalCer (16Malinina L. Malakhova M.L. Kanack A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. The liganding of glycolipid transfer protein is controlled by glycolipid acyl structure.PLos Biol. 2006; 4: e362Crossref PubMed Scopus (51) Google Scholar) with Trp407 serving as a stacking plate for the GSL sugar, whereas Asp360, Asn364, Lys367, and His445 form hydrogen bonds with the galactose-amide moiety. In addition, the C-terminal Val519 makes tight van der Waals contacts with two hydroxyls and the ceramide C1 atom, whereas residue Glu403 contacts with the sugar C6′ atom (Fig. 1b). Both nonpolar aliphatic chains are encapsulated by GLTPH via the so-called “sphingosine-in” binding mode (Fig. 1a) previously observed for human GLTP bound by GSLs containing an N-oleoyl-acyl chain (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Structural basis for glycosphingolipid transfer specificity.Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (101) Google Scholar, 15Samygina V.R. Popov A.N. Cabo-Bilbao A. Ochoa-Lizarralde B. Goni-de-Cerio F. Zhai X. Molotkovsky J.G. Patel D.J. Brown R.E. Malinina L. Enhanced selectivity for sulfatide by engineered human glycolipid transfer protein.Structure. 2011; 19: 1644-1654Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Nonpolar residues such as Phe, Leu, and Val line the FAPP2 hydrophobic pocket, creating an appropriate environment for encapsulating the ceramide chains (Fig. 1c). Comparison of the two molecules of GLTPH·18:1-GalCer complexes that occupy the asymmetric unit (AU) reveals moderate lateral mobility of α6-helix, noticeable flexibility of loop L7/8 (Fig. 1d), and different positions for the sphingosine chain of bound 18:1-GalCer in each molecule (Fig. 1e). Thus, high similarity exists with GLTP (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Structural basis for glycosphingolipid transfer specificity.Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (101) Google Scholar, 15Samygina V.R. Popov A.N. Cabo-Bilbao A. Ochoa-Lizarralde B. Goni-de-Cerio F. Zhai X. Molotkovsky J.G. Patel D.J. Brown R.E. Malinina L. Enhanced selectivity for sulfatide by engineered human glycolipid transfer protein.Structure. 2011; 19: 1644-1654Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 16Malinina L. Malakhova M.L. Kanack A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. The liganding of glycolipid transfer protein is controlled by glycolipid acyl structure.PLos Biol. 2006; 4: e362Crossref PubMed Scopus (51) Google Scholar). However, compared with GLTP, FAPP2 allows different sphingosine chain positioning (Fig. 1e) within the same binding mode and for the same GSL molecule. Fig. 2a shows the structure of FAPP2–GLTPH domain bound with 18:1-GalCer in superposition with other three members of the GSL-specific subfamily with known X-ray structures. Superimposition that includes C1P-specific members of the GLTP superfamily with known 3D structures is shown in Fig. S1a. Differences between ACD11 versus CPTP and CPTP versus FAPP2–GLTPH are highlighted in Fig. S1 (c and d). Poststructural sequence alignment of all six proteins (Fig. 2b) indicates conserved/semiconserved residues of the GLTP fold. Key recognition center residues (Fig. 2b, blue letters) identify five conserved positions in the GSL-specific subfamily and five conserved positions in the C1P-specific subfamily, four of which coincide in both subfamilies. Together they define the basic six-point pattern for the recognition center in the GLTP fold, with spatial templates DNKxWH and DKxRRH (where x indicates a nonconserved residue) characterizing the GSL-specific and C1P-specific families, respectively (Figs. 1b and 2b). The conserved recognition center templates for each subfamily suggest that specificity variations within a subfamily are controlled by other elements. Global comparisons (Fig. 2a and Fig. S1a) identify variable elements of the GLTP-fold including the “nonalignable” (Fig. 2b) loop L3/4 that differs among each member of the GLTP family. Also evident are three previously unrecognized but conserved interaction sites (Fig. 2a, labeled in orange as 1, 2, and 3) that are presumably important for GLTP-fold function. The differing structural features of the L3/4 loop in FAPP2–GLTPH and other subfamily members are shown in Fig. 2a, whereas the variability of L3/4-loop length and sequence for each GLTP superfamily member can be appreciated in Fig. 2b (red box). Significant differences in L3/4-loop conformations in the GSL-specific subfamily and in the GLTP superfamily are highlighted in stereo mode in Fig. 3 (a and b, respectively). Because each L3/4 loop is unique, we refer to it as the “ID loop,” i.e. individual or identification loop. What makes the ID loop special compared with other helix–helix connectors is that each loop end interacts with each other to generate a pinched-together “clasp-like” structure (Fig. 2a, linkage 1). In FAPP2–GLTPH, this clasp is secured by an H-bond triplet (Fig. 4a, shaded area) formed by the side-chain carboxyl of Glu389, located in the C-terminal region of α3-helix, and two consecutive main-chain NH-groups (401 and 402) plus a side-chain hydroxyl (Thr402), grouped at the N terminus of α4-helix. Glu389 provides three acceptor vacancies for the triplet, whereas the partners from α4-helix provide three H-bond donors. The same H-bond triplet pattern, involving the same residues at the same locations, connects the ends of helices 3 and 4 in GLTP (Fig. 4b, shaded area) and is strictly conserved in other GSL-specific GLTP superfamily members (Fig. 4c). Moreover, as illustrated in Fig. 4d, the interaction that “locks” the ID loop persists in the C1P-specific subfamily, although Glu is replaced by Asp in ACD11 and Thr is replaced by Cys in CPTP. Regardless, the fastener for the ID loop remains intact, albeit with minor modifications (Fig. 4d). Thus, the ID-loop clasp is a conserved feature of the GLTP fold that endows the ID loop with relative structural autonomy and rigidity. Examination of the ID-loop structural details in FAPP2–GLTPH shows that the “loop body” is stabilized via a network of tight intraloop interactions involving main-chain hydrogen bonding and van der Waals contacts of side chains (Fig. 4a). The interactions span the entire loop and rigidify its conformation. In GLTP, the ID loop also maintains conformational rigidity by a system of intraloop interactions encompassing the entire loop body (Fig. 4b). Further support for the ID-loop rigidity arises from the very similar backbone conformations observed by superposition of different molecular structures available for GLTP (20 molecules; Fig. 4e), ACD11 (12 molecules; Fig. 4f), and CPTP (15 molecules; Fig. 4g). Although each protein backbone displays some conformational mobility (Fig. 4, e–g), the variations are less than those observed for most loops (e.g. see Fig. 7a). Even though the ID loops are longer, on average, in C1P-specific members ACD11 and CPTP than those in GSL-specific members (Fig. 2b), the similar conformations found in 12 different ACD11 molecules and 15 different CPTP molecules strongly support the relative rigidity of these ID loops (Fig. 4, f and g). We conclude that ID-loop conformations differ among superfamily members, but each ID loop is relatively rigid. Taken together, these data support the importance of the ID loop in the GLTP fold. Previous functional analyses show very different transfer activity by FAPP2–GLTPH for simple versus complex GSLs (2Kamlekar R.K. Simanshu D.K. Gao Y.G. Kenoth R. Pike H.M. Prendergast F.G. Malinina L. Molotkovsky J.G. Venyaminov S.Y. Patel D.J. Brown R.E. The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.Biochim. Biophys. Acta. 2013; 1831: 417-427Crossref PubMed Scopus (22) Google Scholar). Compared with GalCer and GlcCer, LacCer transfer efficiency declines by ∼50%, whereas transfer of SF and ganglioside GM1 is quite low. By contrast, GLTP efficiently transfers both simple and complex GSLs, including GalCer, GlcCer, LacCer, gangliosides, and sulfatides (17Brown R.E. Mattjus P. Glycolipid transfer proteins.Biochim. Biophys. Acta. 2007; 1771: 746-760Crossref PubMed Scopus (70) Google Scholar, 18Mattjus P. Specificity of the mammalian glycolipid transfer proteins.Chem. Phys. Lipids. 2016; 194: 72-78Crossref PubMed Scopus (13) Google Scholar). The ID-loop location behind the bound GSL headgroup (Figs. 2a and 5, a and d) suggests a potential regulatory role (negative or positive) in binding GSLs containing more than one sugar. Compared with GLTP, the main chain of the FAPP2 ID loop (especially Arg398 and Asn399) protrudes toward the GSL (Fig. 4h), where it can potentially interfere with complex headgroup binding, thereby limiting functional selectivity of FAPP2 to simple GSLs. To test the idea, we modeled the FAPP2·LacCer and FAPP2·SF complexes by superposition of the current crystal structure with GLTP·18:1-LacCer (PDB code 1SX6) and GLTP·3-O-SF (PDB code 3RZN and 4H2Z). In Fig. 5 (b, c, e, and f), the protein molecule is represented by FAPP2–GLTPH domain from the current crystal structure, but GalCer is replaced by LacCer or SF from the superimposed GLTP complexes. Because the ID loop is rigid, such models allow one to assess whether the loop and the GSL headgroup will collide in the potential complexes. For reference, the original structure of FAPP2–GLTPH·GalCer is shown in Fig. 5 (a and d). The protruding region of FAPP2 ID loop (R398 and N399 in Fig. 5, a–c) resembles a wall behind the GSL-sugars. The nearby Lys367 residue of α2-helix forms an H-bond with bound galactose (Fig. 5d). Accordingly, Lys367 projects inward in similar fashion to Lys55 in GLTP·GalCer complexes. The bound galactose is too far from the ID loop to be affected by nearby “wall” components, Asn399 and Arg398 (Fig. 5, a and d). However, in the modeled FAPP2–GLTPH·LacCer complex (Fig. 5, b and e), the second sugar ring of lactose moderately clashes against the wall. When GSLs have more than two headgroup sugars (e.g. gangliosides, globosides), serious interference by this wall effect is expected to significantly diminish GSL binding. This finding is consistent with functional data showing low transfer activity of FAPP2–GLTPH for ganglioside GM1 compared with GLTP (2Kamlekar R.K. Simanshu D.K. Gao Y.G. Kenoth R. Pike H.M. Prendergast F.G. Malinina L. Molotkovsky J.G. Venyaminov S.Y. Patel D.J. Brown R.E. The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.Biochim. Biophys. Acta. 2013; 1831: 417-427Crossref PubMed Scopus (22) Google Scholar). Because the recognition center residues target mostly the initial sugar, binding of more complex heads is expected to involve additional nearby favorable interactions. Otherwise, the binding efficiency would decrease because of the increased size and complexity of the headgroup. Based on the favorable GM1 transfer by human GLTP (13Kenoth R. Simanshu D.K. Kamlekar R.K. Pike H.M. Molotkovsky J.G. Benson L.M. Bergen 3rd, H.R. Prendergast F.G. Malinina L. Venyaminov S.Y. Patel D.J. Brown R.E. Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold.J. Biol. Chem. 2010; 285: 13066-13078Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), the complex double subloop architecture of the GLTP ID loop (Fig. 3a) appears likely to produce no local obstructions for the GM1 headgroup while promoting some additional interactions to tether the distal sugars to the protein surface. In the modeled FAPP2–GLTPH·SF complex (Fig. 5, c and f), the proximity of the protruding ID loop to the potential 3-O-sulfo-group position (Fig. 5c) results in conformational" @default.
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• W2891533071 date "2018-10-01" @default.
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• W2891533071 title "Structural analyses of 4-phosphate adaptor protein 2 yield mechanistic insights into sphingolipid recognition by the glycolipid transfer protein family" @default.
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• W2891533071 doi "https://doi.org/10.1074/jbc.ra117.000733" @default.
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