Glycosphingolipid (GSL)-enriched rafts are membrane microdomains that putatively function as lateral organizing sites for signaling proteins involved in oncogenesis and as targeting sites for bacteria, their toxins, and envelope viruses. The process by which GSL-enriched domains are formed, maintained, and remodeled are not well defined but are expected to involve specific and highly conserved GSL transfer proteins (GLTPs) that can bind and transfer GSLs between and within cells. Our long-term objectives include elucidating the structure of human GSL-GLTP complexes, determining structure-function relationships of the GLTP liganding site by mutational analysis, and elucidating the gating mechanism most likely used by GLTP to acquire and release GSL ligand. The proposed research is a collaborative effort with the Rhoderick Brown laboratory at the Hormel Institute of the University of Minnesota.
24:1 Galactosylceramide-GLTP Complex
We have also recently solved crystal structures of human GLTP bound to GSLs of diverse acyl chain length, unsaturation, and sugar composition. Structural comparisons show that a highly conserved anchoring of galactosyl- and lactosyl-amide headgroups by the GLTP recognition center. By contrast, acyl chain chemical structure dictates partitioning between sphingosine-in and newly observed sphingosine-out ligand-binding modes. In the sphingosine-out mode, the sphingosine chain is directed outward and enters the hydrophobic tunnel of a partner complex. The structural insights, combined with computed interaction propensity distributions, suggest a concerted sequence of events mediated by GLTP conformational changes during GSL transfer to/from membranes or presentation/transfer to other proteins.
Malinina, L., Malakhova, M. L., Kanack, A. T., Brown, R. E. & Patel, D. J. (2006). The liganding mode of glycolipid transfer protein is controlled by glycolipid acyl structure. PLoS Biology 4, 1996-2011. [PubMed Abstract]
The structural and functional studies on GSL-GLTP complexes will provide a foundation for pursuing the future development of pharmacologic agents that can specifically target GLTP in oncogenic cells displaying aberrant GLTP activity. A second long-term challenge involves the impact of various post-transcriptional modifications on GLTP structure that affect its ability to capture GSLs. A third long-term goal involves structural and mechanistic characterization of complexes that form between GLTP and other important proteins.
18:1 Lactosylceramide-GLTP Complex
We have solved the crystal structure of human apo-GLTP, which forms a previously unknown two-layer all α-helical topology. In addition, the crystal structure of lactosylceramide bound to GLTP has established that the bound GSL is sandwiched, after adaptive recognition, between the α-helical layers of the GLTP. GSL binding specificity is achieved through recognition and anchoring of the sugar-amide headgroup to the GLTP recognition center by hydrogen-bond networks and hydrophobic contacts, and encapsulation of both lipid chains, in a precisely oriented manner within a “molded-to-fit” hydrophobic tunnel. A cleft-like conformational gating mechanism, involving two interhelical loops and one α-helix of GLTP, could enable the GSL chains to enter and leave the tunnel in the membrane-associated state. Mutation and functional analyses of residues in the GSL recognition center and within the hydrophobic tunnel support a framework for understanding how GLTPs, with their structurally conservative and conformationally flexible segments, acquire and release membrane GSLs during lipid transport and presentation processes.
Malinina, L., Malakhova, M. L., Teplov, A., Brown, R. E. & Patel, D. J. (2004). Structural basis for glycosphingolipid transfer specificity. Nature 430, 1048-1053. [PubMed Abstract]