Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold Journal Article
| Authors: | Kenoth, R.; Simanshu, D. K.; Kamlekar, R. K.; Pike, H. M.; Molotkovsky, J. G.; Benson, L. M.; Bergen, H. R. 3rd; Prendergast, F. G.; Malinina, L.; Venyaminov, S. Y.; Patel, D. J.; Brown, R. E. |
| Article Title: | Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold |
| 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 Å) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp66, Asn70, Lys73, Trp109, and His147) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by α-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-UVCDwas unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at ∼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 (∼6 nm) permitting determination of binding affinities. The unique positioning of Trp208 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. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; protein expression; carrier protein; unclassified drug; nonhuman; mass spectrometry; proteins; carboxy terminal sequence; fluorescence; protein binding; protein purification; nucleotide sequence; carrier proteins; cell membrane; temperature; protein structure, tertiary; binding affinities; binding energy; amino acids; protein folding; protein secondary structure; sequence homology; crystallization; aspartic acid; fungi; lysine; fungal protein; asparagine; histidine; structural homology, protein; tryptophan; sugar (sucrose); x ray diffraction; emission wavelength; glyco lipids; glycolipid transfer proteins; tryptophan fluorescence; glycolipid; glycolipid transfer protein; glycolipids; glycosphingolipids; fungal proteins; circular dichroism; blue-shifted; c terminus; conformational change; electrospray injections; emission intensity; evolutionary adaptation; filamentous fungi; optical activity; secondary structures; structural determination; surface localization; thermal unfolding transitions; trp-fluorescence; hall effect devices; radio telescopes; membranes; heterokaryon incompatibility c2 protein; alpha helix; electrospray mass spectrometry; podospora anserina; ultraviolet spectroscopy; x-ray diffraction |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 285 |
| Issue: | 17 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2010-04-23 |
| Start Page: | 13066 |
| End Page: | 13078 |
| Language: | English |
| DOI: | 10.1074/jbc.M109.093203 |
| PUBMED: | 20164530 |
| PROVIDER: | scopus |
| PMCID: | PMC2857133 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 1" - "Export Date: 20 April 2011" - "CODEN: JBCHA" - "Source: Scopus" |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work