Structural Studies of Sgt2, a Component of the GET Pathway that Mediates the
Sorting of Tail-anchored Proteins to the ER Membrane.
Nuri Sung, Sukyeong Lee, Amadeo B. Biter, and Francis T.F. Tsai
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College
of Medicine, One Baylor Plaza, Houston, TX 77030
Sgt2 (small glutamine-rich tetratricopeptide repeat (TPR)-containing protein 2) is a 38-kDa
protein and a major component of the GET (Guided Entry of Tail-anchored Proteins)
complex that mediates the sorting of tail-anchored proteins to the ER membrane (1, 2). At
the molecular level, Sgt2 consists of an N-terminal dimerization domain, a TPR domain, and
a glutamine/methionine-rich C-terminal domain. It was recently shown that Sgt2 binds to
members of distinct ATP-dependent heat-shock protein families (3), notably Hsp70 and
Hsp90, which feature a highly conserved EEVD motif at their C-termini. This interaction
requires the Sgt2TPR domain, and likely modulates Hsp70 and Hsp90 function in protein
folding by affecting the ATPase activity and substrate interaction. Sgt2 was also shown to
bind to Hsp104 (3), a ring-forming ATP-dependent molecular chaperone, which has the
remarkable ability to rescue stress-damaged proteins from a previously aggregated state.
Interestingly, yeast Hsp104 features a unique C-terminal domain (CTD), which is not found
in the bacterial homolog ClpB, and is reminiscent but distinct from the EEVD motif present in
Hsp70 and Hsp90 chaperones. The structure of the Hsp104CTD remains elusive.
To provide a molecular understanding of Sgt2’s role in Hsp70 and Hsp104 chaperone
function, we wished to determine the high-resolution crystal structures of Sgt2TPR bound to
Hsp104CTD and Hsp70CTD, respectively. Bacterial expression constructs were generated by
PCR cloning from the full-length gene and featured an N-terminal Tobacco Etch Virus (TEV)
protease cleavable His6-tag. Proteins were overexpressed in E. coli and purified to near
homogeneity using immobilized-metal affinity chromatography. Using a pull-down assay, we
confirmed the interaction of yeast Sgt2TPR with human Hsp70CTD and yeast Hsp104CTD,
respectively. Sgt2TPR formed a 1:1 complex with either Hsp70CTD or Hsp104CTD as
determined by analytical size-exclusion chromatography.
Crystals of Sgt2TPR in the presence of Hsp104CTD were obtained by the hanging drop vapor
diffusion technique. Crystals diffracted to 2.6-Å resolution on a home X-ray source, and
belonged to the orthorhombic space group, C2221 with unit cell dimensions a= 72.378. Å, b=
81.413 Å, c= 109.349 Å, and α=β=γ=90°. The crystal structure was determined by molecular
replacement and contained two molecules of Sgt2 in the crystallographic asymmetric unit.
However, Hsp104CTD was not present. Further crystallization experiments with both
Hsp70CTD and Hsp104CTD are on going. Several new conditions were found recently, which
yielded crystals but which are still too small for structural analysis. We anticipate that the
outcome of this research will provide new mechanistic insight into the structure-function
relationship of Sgt2.
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