Human GLTP: Three Distinct Functions for the Three Tryptophans in a Novel Peripheral Amphitropic Fold

Abstract

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.

Sphingolipid transfer proteins defined by the GLTP-fold

“…The findings were supported by Trp point mutational studies of GLTP (Kamlekar et al 2010; West et al 2006; Zhai et al 2009). Analogous Trp functionality occurs in various lectins during interaction with carbohydrate (Diehl et al 2010; Laughrey et al 2008; Saraboji et al 2012; Sujatha & Balaji, 2004; Sujatha et al 2004; Weis & Drickamer, 1996).…”

“…What is clear is that the localized changes to the glycolipid recognition center in apo-HET-C2 have marginal impact on protein temperature stability. At neutral pH, the unfolding transition temperature mid-point for HET-C2 is ~49 °C (Kenoth et al 2011), which is only 4–5 °C lower than that of human GLTP (Kamlekar et al 2010). The two Cys residues, i.e.…”

“…It is noteworthy that the dimeric contact interface is highly conserved in different crystal polymorphs and complexes, is generated by different glycolipid species and encompasses the glycolipid-binding site while overlapping the putative membrane-docking region of GLTP (Kamlekar et al 2010; Malinina et al 2006). Initially, the homodimerization was viewed primarily as a crystal-related indicator of the membrane interaction region that encompasses the glycolipid-binding site in GLTP (Malinina et al 2006).…”

Phosphatidylserine Stimulates Ceramide 1-Phosphate (C1P) Intermembrane Transfer by C1P Transfer Proteins

“…Spontaneous C1P transbilayer migration is expected to be highly restricted due to the unfavorable energetics of moving the phosphate polar headgroup through the nonpolar hydrocarbon matrix. The interactions of GLTP homologs with membranes occur in a nonperturbing and weakly penetrating manner that leaves the sphingolipid pool in the inner leaflet of the vesicle bilayer undisturbed and inaccessible to protein (14,37,44,55). However, a condition known to influence the initial sphingolipid transbilayer distributions and expected to impact the C1P pool size in the bilayer outer leaflet is the lipid composition during formation of high curvature, small unilamellar vesicles (e.g.…”

GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanolamine

“…6× His-S-tag cleavage yielded native GLTP, avoiding subtle changes to protein conformation and glycolipid binding induced by Nterminal fusion tags ( 13 ). Near-UV CD confi rmed native global folding ( 15,16,19 ). Protein purity and concentration were determined by SDS-PAGE and bicinchoninic acid assay ( 31 ).…”