Abstract
HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 A) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp(66), Asn(70), Lys(73), Trp(109), and His(147)) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at approximately 355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (approximately 6 nm) permitting determination of binding affinities. The unique positioning of Trp(208) at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.
Highlights
HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP)
Structure and Glycolipid Specificity of HET-C2—Limited sequence homology between HET-C2 and porcine glycolipid transfer proteins (GLTPs) was first noted by Saupe et al [5]
To define HET-C2 conformation and localize residues potentially involved in glycolipid binding, the HET-C2 structure was solved by x-ray crystallography
Summary
Expression and Purification of HET-C2—HET-C2 was expressed and purified as detailed for GLTP [22]. Structure Determination—For data collection at cryogenic temperatures, crystals were stabilized in mother liquor supplemented with 20% ethylene glycol. X-ray diffraction data for the methylated HET-C2 crystal was collected on beamline ID24-E at the Advanced Photon Source (Argonne National Laboratory). This data set was processed using the HKL2000 suite [24]. The CDPro program suite [34], a modified version of three methods (SELCON3 [35], CONTIN/LL locally linearized approximation [38] of CONTIN [39], and CDSSTR [40]), was used to calculate HET-C2 secondary structure from far-UV CD spectra. Raw spectral data were transformed into relative molecular masses using the Agilent TOF Protein Confirmation software
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
