Lipid transfer proteins are important in membrane vesicle biogenesis and trafficking, signal transduction and immunological presentation processes. The conserved and ubiquitous mammalian glycolipid transfer proteins (GLTPs) serve as potential regulators of cell processes mediated by glycosphingolipids, ranging from differentiation and proliferation to invasive adhesion, neurodegeneration and apoptosis. Here we report crystal structures of apo-GLTP (1.65 Å resolution) and lactosylceramide-bound (1.95 Å) GLTP, in which the bound glycosphingolipid is sandwiched, after adaptive recognition, within a previously unknown two-layer all-α-helical topology. Glycosphingolipid binding specificity is achieved through recognition and anchoring of the sugar-amide headgroup to the GLTP recognition centre by hydrogen bond networks and hydrophobic contacts, and encapsulation of both lipid chains, in a precisely oriented manner within a 'moulded-to-fit' hydrophobic tunnel. A cleft-like conformational gating mechanism, involving two interhelical loops and one α-helix of GLTP, could enable the glycolipid chains to enter and leave the tunnel in the membrane-associated state. Mutation and functional analyses of residues in the glycolipid recognition centre and within the hydrophobic tunnel support a framework for understanding how GLTPs acquire and release glycosphingolipids during lipid intermembrane transfer and presentation processes.
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Acknowledgements We would like to thank C. Shimoda and T. Nakamura for the S. pombe genomic library, T. Carr, H. Okayama and N. Walworth for strains, and T. Matsumoto for introducing X-ray and g-ray irradiation. This study was supported by the COE Grant of the Ministry of Education, Culture, Sports, Science and Technology of Japan. K.N. was a recipient of a predoctoral fellowship from the Japan Society for Promotion of Science.
Acknowledgements We thank the personnel at SBC beamline 19BM of the Advanced Photon Source beamline staff for assistance with data collection from multiwavelength anomalous dispersion; A. Serganov for technical support; X. Lin, T. Chung and H. Pike for their contributions to the cloning and expression of the recombinant human GLTP; X.-M. Li for synthesizing and purifying N-18:1 lactosylceramide; A. J. Windebank for help with DNA sequencing at the Mayo Molecular Biology Core Facility; T. Burghardt for help with recording near-ultraviolet CD spectra; and S. Venyaminov in the Franklyn Prendergast laboratory for recording the far-ultraviolet CD spectra. This research was supported by NIH and the Hormel Foundation. Use of the ANL SBC beamlines at the APS was supported by the US Department of Energy, Office of Energy Research.