Glycosphingolipids (GSLs) are ubiquitous components of eukaryotic plasma membranes and mediate numerous biological functions, from differentiation and proliferation to invasive adhesion, neurodegeneration, and apoptosis [1
]. The importance of GSLs and their metabolites in processes such as tissue development is illustrated by the lethal effect of disruption of GSL biosynthesis during embryonic development [3
]. Whereas the bulk of GSLs reside in the outer leaflet of the plasma membrane where their sugar headgroups project into the extracellular environment, increasing evidence suggests that GSLs also localize to membranes of intracellular organelles (e.g., nucleus and mitochondria) [4
]. Vesicular trafficking plays a dominant role in distributing GSLs intracellularly after synthesis in the Golgi. However, a nonvesicular pathway also exists, possibly involving glycolipid transfer proteins (GLTPs) [7
Based on their ability to selectively accelerate the intermembrane transfer of glycolipids, GLTPs were discovered in the cytosolic extracts of bovine spleen and porcine brain, and later in a wide variety of tissues [9
]. The 23–24-kDa GLTPs display absolute specificity for glycolipids [12
], are highly conserved among mammals [13
], and include plant and fungal orthologs that have been implicated in programmed cell death responses [14
]. Recently, we determined the first x-ray structure of human GLTP in both the GSL-free and lactosylceramide (LacCer)-bound forms [16
]. In addition to displaying a novel architecture that defines GLTP as the founding member of a new protein superfamily [17
] (see http://supfam.org/SUPERFAMILY/cgi-bin/scop.cgi?sunid=110004
; accessed 12 September 2006) with a novel protein fold for membrane interaction and for lipid binding/transfer [19
], the structure of the 18:1 LacCer-GLTP complex revealed the basis for GSL binding specificity by GLTP. The liganding site consists of a sugar headgroup recognition center that anchors the ceramide-linked sugar to the protein surface and a hydrophobic tunnel that accommodates the hydrocarbon chains of ceramide. Comparative structural analyses, including crystallographic B-factor distributions of apo-GLTP and the LacCer-GLTP complex, suggest that liganding of the glycolipid most likely occurs via an adaptive recognition process [16
]. A cleft-like gating mechanism, involving conformational changes to two interhelical loops and one α helix, appears to facilitate entry and exit of the lipid chains in the membrane-associated state when the GSL headgroup is attached to the sugar headgroup recognition center.
An important feature of the GLTP hydrophobic tunnel is its ability to expand to accommodate the GSL ceramide moiety. In the case of the 18:1 LacCer-GLTP complex, the 18-carbon oleoyl and sphingosine chains of LacCer reside side by side within the hydrophobic channel [16
]. However, naturally occurring mammalian glycolipids typically have acyl chains with lengths ranging from 16 to 26 carbons with occasional monounsaturation. To test the conformational properties and accommodation limits of the GLTP hydrophobic tunnel, we synthesized glycolipids (LacCer or galactosylceramide [GalCer]) containing short acyl chains (e.g., octanoyl and dodecanoyl), medium unsaturated acyl chains (e.g., linoleoyl), as well as long, physiologically relevant unsaturated acyl chains (e.g., nervonyl), and we structurally characterized their complexes with GLTP. To elucidate conformational changes caused by the sugar headgroup binding in the GLTP, we also structurally characterized the complex between GLTP and the model compound n
-glycoside, which, unlike GSLs, has a single, very short hydrocarbon chain lacking an amide linkage.
The present structural studies provide definitive evidence for a novel GSL-GLTP complexation mode characterized by a difference in the accommodation of the ceramide lipid chains but not the sugar headgroups. Chief among the differences is bending of the sphingosine chain, causing outward projection from the hydrophobic tunnel. Comparative analysis of all GSL-GLTP structures reveals that the hydrophobic tunnel consists of two functionally distinct compartments, enabling GLTP to accommodate GSL acyl chains of different lengths and conformational restrictions. Equally importantly, our findings suggest a concerted sequence of events during GSL liganding, in which the sphingosine chain is the last part of the glycolipid to enter GLTP and the first part to leave GLTP during interaction with membranes. Interaction propensity distribution computations indicate a potential GLTP interface for interactions with other proteins and/or membranes, which coincide with a region of the proposed GLTP gate mediating GSL binding and release.