Structural insight into the glycosylphosphatidylinositol transamidase subunits PIG-K and PIG-S from yeast.
The addition of glycosylphosphatidylinositol (GPI) anchors to eukaryotic proteins in the lumen of the endoplasmic reticulum is catalyzed by the transamidase complex, composed of at least five subunits (PIG-K, PIG-S, PIG-T, PIG-U and GPAA1). Here PIG-K(24-337) and PIG-S(38-467) from yeast, including the residues 24-337 and 38-467 of the entire 411 and 534 residue protein, respectively, was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that yPIG-K(24-377) comprises 52% α-helix and 12% β-sheet, whereas yPIG-S(38-467) involves 58% α-helix and 18% β-sheet. The radius of gyration (R(g)) and the maximum size (D(max)) of both proteins have been analyzed by small angle X-ray scattering (SAXS) and determined to be 2.64±0.3 and 10.3±0.1 nm (yPIG-K(24-377)) as well as 3.06±0.02 nm (R(g)) and 16.9±0.4 nm (D(max)) in the case of yPIG-S(38-467), respectively. Using an ab initio approach, the first low-resolution solution structures of both proteins were restored. yPIG-K(24-377) is an elongated particle consisting of an egg-like portion and a small globular segment linked together by an 1.9 nm long stalk. yPIG-S(38-467) forms an elongated molecule in solution with a larger domain of 10.1 nm in length, a diameter of 9.1 nm and a smaller domain of 6.7 nm in length and 3.4 nm in width. The two domains of yPIG-S(38-467) are tilted relative to each other. Finally, the arrangements of PIG-K and PIG-S inside the ensemble of the transamidase complex are discussed.
Bioinformatics Institute, (BII), Agency for Science, Technology and Research (A∗STAR), Singapore, Singapore.
Yew Kwang
YK
Toh
Neelagandan
N
Kamariah
Sebastian
S
Maurer-Stroh
Manfred
M
Roessle
Frank
F
Eisenhaber
Sharmila
S
Adhikari
Birgit
B
Eisenhaber
Gerhard
G
Grüber
D019881:Aminoacyltransferases / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D019295:Computational Biology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015003:Yeasts / Q000201:enzymology*
D017261:Glycosylphosphatidylinositols; D019881:Aminoacyltransferases; C023175:transamidases