Ceramide-1-phosphate Binding Specificty
Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2-α (cPLA2-α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signaling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear.
Nonvesicular Trafficking by a Ceramide-1-Phosphate Transfer Protein that Regulates Eicosanoids
Here, in collaboration with the Rhoderick Brown (Hormel Institute), Charles Chalfont (Virginia Commonwealth University) and Edward Hindcliffe (Hormel Institute) laboratories, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2-α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.
Simanshu, D. K., Kamlekar, R. K., Wijesinghe, D. S., Zou, X., Zhai, X., Mishra, S. K., Molotkovsky, J. G., Malinina, L., Hincliffe, E. H., Chalfant, C. E., Brown, R. E. & Patel, D. J. (2013). Nonvesicular trafficking by ceramide-1-phosphate transfer protein regulates eicosanoid production. Nature 500, 463-467.
Glycosphingolipid Binding Specificty
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.
Enhanced Selectivity for Sulfatide by Engineered Human Glycolipid Transfer Protein
Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Here in a project championed by the Lucy Malinina laboratory (CIC bioGUNE, Bilbao, Spain), we report structure-guided engineering of the lipid-binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D, D48V), each containing bound N-nervonyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of ‘portal entrance’ residues, A47 and D48, reversibly regulate sphingosene access to the hydrophobic pocket via a mechanism that could involve homodimerization. ‘Door opening’ conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of ‘engineered GLTPs’ with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.
Samygina, V., 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. (2011). A designer human glycolipid transfer protein with enhanced transfer selectivity for sulfatide. Structure 19, 1625-1634.
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]
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]