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Structural Evidence for Adaptive Ligand Binding of Glycolipid Transfer Protein

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Structural Evidence for Adaptive Ligand Binding of Glycolipid Transfer Protein doi:10.1016/j.jmb.2005.10.031 J. Mol. Biol. (2006) 355, 224–236 Structural Evidence for Adaptive Ligand Binding of Glycolipid Transfer Protein Tomi T. Airenne†, Heidi Kidron†, Yvonne Nymalm, Matts Nylund Gun West, Peter Mattjus and Tiina A. Salminen* Department of Biochemistry Glycolipids participate in many important cellular processes and they are and Pharmacy, A˚ bo Akademi bound and transferred with high specificity by glycolipid transfer protein University, Tykisto¨katu 6A (GLTP). We have solved three different X-ray structures of bovine GLTP at FIN-20520 Turku, Finland 1.4 A˚ , 1.6 A˚ and 1.8 A˚ resolution, all with a bound fatty acid or glycolipid. The 1.4 A˚ structure resembles the recently characterized apo-form of the human GLTP but the other two structures represent an intermediate conformation of the apo-GLTPs and the human lactosylceramide-bound GLTP structure. These novel structures give insight into the mechanism of lipid binding and how GLTP may conformationally adapt to different lipids. Furthermore, based on the structural comparison of the GLTP structures and the three-dimensional models of the related Podospora anserina HET-C2 and Arabidopsis thaliana accelerated cell death protein, ACD11, we give structural explanations for their specific lipid binding properties. q 2005 Elsevier Ltd. All rights reserved. Keywords: crystal structure; homology modeling; conformational change; *Corresponding author cavity; fluorescence Introduction to the diverse roles of glycolipids in the cell, GLTP could potentially function as a modulator or sensor Glycolipid transfer protein (GLTP) is a 24 kDa of glycolipid levels. Most glycosphingolipids are basic cytosolic protein, which has been identified synthesized at the luminal side of Golgi but gluco- from a variety of organisms and cell types.1,2 GLTPs sylceramide, which is the precursor of other glyco- from mammals share high sequence identity sphingolipids and the simplest member of the (w90%) and they have in vitro been shown to glycosphingolipid family, is synthesized on the specifically transfer glycolipids that have glycosyl cytosolic surface of the Golgi membrane6 and units attached to the lipid hydrocarbon backbone another monohexosylceramide, galactosylcera- (either ceramide or diacylglycerol) by b-linkage.3 mide, is synthesized at the cytosolic side of the Glycolipids, and particularly glycosphingolipids, endoplasmic reticulum. It has been suggested that are known to function in many important cellular GLTP could participate in the transfer of glucosyl- processes like development, adhesion and cell–cell ceramide from the cytosolic surface of the Golgi recognition both in eukaryotes and prokaryotes. membrane to the inner leaflet of the plasma The glycosphingolipids can function as cell surface membrane through the cytosol.7,8 markers and modulators of membrane protein GLTP has no sequence homology with other functions, e.g. as binding sites for certain bacteria, lipid-binding proteins found in the animal kingdom toxins and viruses and as stimulators of cell growth, but it shares sequence identity with two lipid- in differentiation and DNA synthesis, and in sorting binding proteins found in fungi and plants; the and trafficking of proteins.4,5 Very little is known HET-C2 protein from Podospora anserina and the about the specific function of GLTP in vivo, but due accelerated cell death protein, ACD11, from Arabi- dopsis thaliana. The inactivation of the het-c gene, which encodes the HET-C2 protein, leads to † T.T.A. & H.K. contributed equally to this work. 9 Abbreviations used: GLTP, glycolipid transfer protein; abnormal ascospore formation in P. anserina, rGLTP, recombinant GLTP; MIR, multiple isomorphous while the inactivation of the acd11 gene in A. thaliana replacement; NEM, N-ethylmaleimide. causes activation of programmed cell death and 10 E-mail address of the corresponding author: expression of defence-related genes. HET-C2 has tiina.salminen@abo.fi been shown to specifically bind glycosphingolipids 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Lipi Binding of Glycolipid Transfer Protein 225 in vitro11 and ACD11 has been proposed to be significance of disulfide bonds for the glycolipid homologous to GLTP, but it does not bind and transfer activity of bovine GLTP was analyzed. transfer glycolipids, instead it has in vitro transfer activity of the single chain lipid sphinghosine.10 We have recently reported the preliminary X-ray Results analysis of GLTP12 and now we present the X-ray structureofbovineGLTPat1.6A˚ resolution X-ray structures of bovine GLTP (intermediate-GLTP) using the multiple isomor- phous replacement (MIR) method. In addition, we Glycolipid transfer proteins recognize a wide have solved two structures with the molecular range of different glycolipids3 and have a high replacement method at 1.4 A˚ (apo-GLTP) and 1.8 A˚ ganglioside transfer activity.14 Mammalian GLTPs resolution (GM3-GLTP) using the MIR structure as share high sequence identity; the amino acid an initial model. The apo-GLTP structure reveals a sequences of bovine and porcine GLTP are identical conformation similar to the apo-conformation of and human and bovine GLTP differ only by five human GLTP13 while the other two structures amino acid residues, none of which are involved in represent an intermediate conformation of the ligand binding (Figure 2). In order to investigate the apo-GLTP and lactosylceramide-bound GLTP mechanism of lipid binding to GLTP we have (LacCer-GLTP) (Figure 1).13 The apo-GLTP and the solved three different high-resolution X-ray intermediate-GLTP structures are bound to a fatty structures of bovine GLTP. All the bovine GLTP acid while the GM3-GLTP structure is bound to a structures have a similar fold to the human GLTP;13 double-chain glycolipid, ganglioside GM3. Based a unique two-layer all-a-helical topology with a on the structural analysis of the novel bovine GLTP sugar moiety binding pocket and a channel where structures and the known human GLTP structures hydrophobic ligands can bind (Figures 1 and 4). we suggest a mechanism for lipid recognition and Furthermore, a large positively charged area binding by GLTP. In order to provide structural formed by four lysine residues (Lys87, Lys137, explanations for the specific and distinctive lipid Lys138 and Lys208) is located on the surface of binding of GLTP and the GLTP-related HET-C2 and GLTP in the vicinity of the sugar-binding pocket ACD11 proteins, three-dimensional models of HET- (Figure 3(a)). The intermediate-GLTP (PDB code C2 and ACD11 protein were constructed using the 1TFJ) and GM3-GLTP (PDB code 2BV7) structures GLTP X-ray structures as templates. In addition, the represent a different conformation than either of the Figure 1. A stereo view of X-ray structures of bovine GLTP. The superimposed bovine apo (red), intermediate (grey) and GM3-GLTP (blue) structures are shown as cartoons. Secondary structure elements (a-helices) are numbered according to the apo-GLTP structure. 226 Lipi Binding of Glycolipid Transfer Protein Figure 2. A sequence alignment of 11 GLTP-like sequences. The amino acid numbering follows the bovine GLTP sequence. The a-helices defined by the bovine intermediate-GLTP structure are shown as cylinders. The residues conserved in all sequences are shaded with dark gray, while the residues that are conserved in GLTPs but not in ACD11 are shaded with light gray. Residues of particular interest are indicated in the alignment: lysine residues forming the membrane contact area in bovine GLTP (:), amino acids in the sugar-binding site (C) and amino acids forming the hydrophobic channel (B). The organisms and sequence accession numbers are referred to as follows: Bovine (P17403); Human (Q9NZD2); Mouse (Q9JL62); Drosophila (Drosopila melanogaster, Q9VXV1); P. anserina (Podospora anserina, Q01494); P. involutus (Paxillus involutus, Q5V8K7); FAPP2_Human (Q80W71); FAPP2_Mouse (Q80W71); A. thaliana (Arabidopsis thaliana, Q6NLQ3); C. elegans (Caenorhabditis elegans, Q9BKS2); ACD11 (Arabidopsis thaliana, O64587). Figure 3. Surface properties of GM3-GLTP,HET-C2 and ACD11. The electrostatic potentials of (a) GM3-GLTP,(b) HET-C2 and (c) ACD11 are mapped to the surfaces. The four lysine residues (K87, K137, K138 and K208) forming a locally charged area on the surface of GM3-GLTP are indicated. Only one of these residues is conserved in HET-C2 (K123) and none in ACD11. Lipi Binding of Glycolipid Transfer Protein 227 recently reported human apo or the LacCer-GLTP the hydrophobic cavity. In the bovine GM3-GLTP, structures,13 whereas the apo-GLTP (PDB code the sphingosine chain does not fit into the narrower 1WBE) structure resembles the human apo-GLTP. channel but lies on the surface of the protein. Two of the solved bovine GLTP structures, the apo In all known GLTP structures the acyl chain and intermediate-GLTP, have a bound fatty acid cavity, which is located between the a1, a7 and a8 (Figure 4(a) and (b)), which is likely to be a decanoic helices, is structurally conserved. Small differences acid according to mass spectrometry analysis (data in the acyl chain cavities of human and bovine not shown) and originate from the bacterial GLTP structures are, however, seen probably due to expression of the protein used for crystallization. the crystal packing or different N termini of the Moreover, the intermediate-GLTP structure con- crystallized proteins. All crystallized bovine GLTP tained uninterpretable electron density near the structures reported here contained an N-terminal bound fatty acid within the hydrophobic cavity 6xHis-tag, whereas in the recently reported human (Figure 4(b)). The third bovine GLTP structure, GLTP structures13 the His-tag was proteolytically GM3-GLTP, contains a bound GM3 ligand cleaved off. In the bovine GLTP structures and the (Figure 4(c), the structure of GM3 is shown in human apo-GLTP structure the acyl chain cavity is Supplementary Figure 1), which was introduced open and hence accessible for solvent, while in the during the crystallization as ganglioside micelles human LacCer-GLTP structure the acyl chain cavity and co-crystallized with the protein.
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