Conformational Folding and Stability of the HET-C2 Glycolipid Transfer Protein Fold: Does a Molten Globule-like State Regulate Activity?

Abstract

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP-fold which represents a novel motif for lipid binding/transfer and for reversible interaction with membranes, i.e. peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP-fold. Currently, insights into the folding stability and conformational states that regulate GLTP-fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP-fold, we investigated the effect of changing pH on the fungal HET-C2 GLTP-fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and ten tyrosines in human GLTP). pH-Induced conformational alterations were determined by changes in: i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, anisotropy); ii) circular dichroism over the near-UV and far-UV ranges including thermal stability profiles of the derivatized molar ellipticity at 222 nm; iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid; iv) glycolipid inter-membrane transfer activity monitored by Förster resonance energy transfer. Analyses of our recently solved crystallographic structure of HET-C2 (1.9 Å) enabled identification of side chain electrostatic interactions that contribute to HET-C2 GLTP-fold stability and can be altered by pH change. Side-chain interactions include numerous salt bridges and interchain cation-pi interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP-fold.

Proteomic Analysis of Chinook Salmon (Oncorhynchus tshawytscha) Ovarian Fluid

“…Precisely which molecular events lead to fusion avoidance and death of contacting fungal hyphae is not currently known, but it has been suggested that a conformational change, in the case of HET-C2, may be important to initiate an apoptotic response [66].…”

Sphingolipid transfer proteins defined by the GLTP-fold

“…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.…”

“…heterokaryon incompatibility C2 protein (HET-C2) of Podospora anserina and GLTP in the thermoacidophilic unicellular red alga, Galdieria sulphuraria (PDB: 2I3F) show GLTP-folds with putative lipid recognition centers containing the same essential residues topologically organized in the same way as in mammalian GLTPs complexed with glycolipid (Kenoth et al 2010, 2011; Samygina et al 2011). In the case of HET-C2, functional analyses show more focused glycolipid selectivity than exhibited by human GLTP such as fast transfer of glucosylceramide (GlcCer) or GalCer but almost no transfer of 3-sulfo-GalCer (sulfatide).…”

Arabidopsis Accelerated Cell Death 11, ACD11, Is a Ceramide-1-Phosphate Transfer Protein and Intermediary Regulator of Phytoceramide Levels

“…Yet, ACD11 is unable to transfer glycolipids (Brodersen et al, 2002), consistent with the lack of essential residues needed for glycosphingolipid (GSL) sugar headgroup binding (Petersen et al, 2008). In mammalian GLTPs and HET-C2 fungal GLTP, X-ray structures reveal the molecular details of how glycolipids are recognized and bound by a conserved residue cluster (Asp, Asn, Lys, His, Trp) that form a hydrogen bond network with the GSL sugar-amide region, thus explaining the selectivity and transfer proficiency for various GSLs (Airenne et al, 2006; Kenoth et al, 2011; Kenoth et al, 2010; Malinina et al, 2006; Malinina et al, 2004; Samygina et al, 2011). Currently lacking for ACD11 is establishment of its preferred sphingolipid ligand as well as direct evidence for its functional involvement in the regulation of plant sphingolipid metabolism.…”