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Structural basis for glycosphingolipid transfer specificity

Nature volume 430, pages 1048–1053 (2004)Cite this article

Abstract

Lipid transfer proteins are important in membrane vesicle biogenesis and trafficking, signal transduction and immunological presentation processes1,2,3. 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 apoptosis4,5. 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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Figure 1: Overall structures of apo-GLTP and the lactosylceramide–GLTP complex.
Figure 2: Intermolecular interactions in the lactosylceramide–GLTP complex.
Figure 3: Comparison of apo-GLTP and lactosylceramide-bound GLTP.

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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.

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Author notes

  1. Lucy Malinina and Margarita L. Malakhova: These authors contributed equally to this work

Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, 10021, USA

    Lucy Malinina, Alexei Teplov & Dinshaw J. Patel

  2. Hormel Institute, University of Minnesota, Austin, Minnesota, 55912, USA

    Margarita L. Malakhova & Rhoderick E. Brown

Authors
  1. Lucy Malinina
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  2. Margarita L. Malakhova
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Corresponding authors

Correspondence to Rhoderick E. Brown or Dinshaw J. Patel.

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Supplementary information

Supplementary Table S1

X-ray data collection and refinement statistics. (RTF 47 kb)

Supplementary Figure S1

A two-layer topology of the γ-helices. (PDF 106 kb)

Supplementary Figure S2

Structure of the lactosylceramide-GLTP complex. (PDF 1910 kb)

Supplementary Figure S3

Schematic showing the hydrogen bonding between lactosylceramide and protein side chains in the complex. (PDF 98 kb)

Supplementary Figure S4

Glycolipid transfer activities of wtGLTP and representative GLTPs with point mutations. (PDF 220 kb)

Supplementary Figure S5

Superposition of wild type GLTP and the D48V mutant. (PDF 917 kb)

Supplementary Figure S6

Far UV CD spectra of GLTP mutants. (PDF 116 kb)

Supplementary Figure S7

Near UV CD spectra of GLTP mutants. (PDF 118 kb)

Supplementary Figure S8

Electron density map, sequence and secondary structure elements for apo-GLTP. (PDF 2448 kb)

Supplementary Figure S9

Omit electron density map for a ligand. (PDF 733 kb)

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Malinina, L., Malakhova, M., Teplov, A. et al. Structural basis for glycosphingolipid transfer specificity. Nature 430, 1048–1053 (2004). https://doi.org/10.1038/nature02856

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