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 Å ) 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 ␣-helices and a cooperative thermal unfolding transition of 49°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 ϳ355 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 (ϳ6 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.Self/nonself recognition is a universally important process, encompassing intercellular interactions ranging from vertebrate immune responses to somatic chimera formation in protists, filamentous fungi, sponges, ascidians, and tunicates. Self/ nonself discrimination becomes critical for filamentous fungi during hyphal fusion, which enables the exchange of cytoplasm and nuclei during the assimilative growth phase (1-4). Nonself recognition triggers a postfusion, programmed cell death process known as vegetative incompatibility, which leads to heterokaryon death. The benefits of vegetative incompatibility include prevention of transmission of deleterious cytoplasmic elements (i.e. viruses) as well as restriction of plundering by parasitic genotypes.het genes are known to play a major role in vegetative incompatibility processes that occur in Neurospora crassa and Podospora anserina. het genes exhibit extensive polymorphism and generally encode proteins carrying a HET domain (1-4). Originally, this domain was linked to the het-c2 gene of P. anserina, where it was shown to encode a protein similar in size and with limited sequence homology to mammalian glycolipid transfer...

The crystal structure of meso-tetrasulfonatophenylporphyrin complexed with concanavalin A (ConA) was determined at 1.9 Å resolution.

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.