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. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47DD48V), each containing bound N-nervonoyl-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 sphingosine 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.
Bibliographical noteFunding Information:
This research was supported by Spanish Ministerio de Ciencia e Innovacion (MICINN BFU2010-17711), Russian Foundation for Basic Research (#09-04-00313), USPHS (NIGMS GM45928 and NCI CA121493), the Abby Rockefeller Mauzé Trust, and Maloris and Hormel Foundations. The authors thank Margarita Malakhova for helpful advice, the personnel at ESRF beamline ID 23-1 and ID 14-4 for assistance with X-ray data collection, our colleagues Sandra Delgado, Jevgenia Tamjar, Oscar Millet, and Inma Gómez García from CICbioGUNE for help with fluorescence and DLS measurements, and Helen Pike from the UMN-Hormel Institute for expressing and purifying protein used for transfer activity measurements.