Structural basis for glycosphingolipid transfer specificity

Abstract

Lipid transfer proteins are important in membrane vesicle biogenesis and trafficking, signal transduction and immunological presentation processes. 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 apoptosis. Here we report crystal structures of apo-GLTP (1.65 A resolution) and lactosylceramide-bound (1.95 A) GLTP, in which the bound glycosphingolipid is sandwiched, after adaptive recognition, within a previously unknown two-layer all-alpha-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 alpha-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.

Glycolipid transfer protein: Clear structure and activity, but enigmatic function

“…Using photo-CIDNP NMR an exposed and flexible Trp residue was clearly seen and identified as Trp142 based on its spectral properties (Mok and Hore, 2004), available structural data (Malinina et al, 2004) and solvent accessibility calculations (Kabsch and Sander, 1983). The photo-CIDNP NMR signals were considerably reduced when GLTP was incubated with vesicles of various compositions.…”

“…Close inspection of the crystal structure data for apo-GLTP and protein containing glycolipid (Malinina et al, 2004) leads to the conclusion that about 20 backbone atoms of amino acid residues in close proximity to the glycolipid change their position in the structure. Since protein flexibility is likely needed for proper accommodation of GlcCer, the influence of GlcCer on protein stability was considered to be of interest.…”

“…GLTP orthologs in fungi and plants were found to be involved in programmed cell death (Brodersen et al, 2002;Mattjus et al, 2003;Saupe et al, 1994) but their function in mammals is largely unknown. The activity of GLTP is preserved after expression in E. coli (Godi et al, 2004;Lin et al, 2000;Malinina et al, 2004;Rao et al, 2004) allowing for detailed structureefunction studies in vitro.…”

“…Crystal structures of GLTP in the absence and presence of bound glycolipid were solved revealing a new folding motif among lipid-binding proteins (Malinina et al, 2004). In combination with mutational studies the structures provided new insights into glycolipid binding (Malakhova et al, 2005).…”

Sphingolipid Homeostasis in the Endoplasmic Reticulum and Beyond

“…These proteins include soluble transporters (e.g., CERT), the S1P GPCRs, and p24, a membrane protein that participates in COPI vesicle formation (Contreras et al 2012). Recent crystallographic studies have illuminated the molecular interactions that enable specific recognition of sphingolipids, revealing hydrophobic binding pockets that can discriminate features such as headgroup structure and acyl chain length (Malinina et al 2004(Malinina et al , 2006Kudo et al 2008;Contreras et al 2012;Hanson et al 2012). Although the above proteins are not known to be homeostatic sensors, such sensors may use similar modes of molecular recognition.…”