TPR-Mediated Self-Association of Plant SGT1

Abstract

The tetratricopeptide repeat (TPR) domain mediates inter-protein associations in a number of systems. The domain is also thought to mediate oligomerization of some proteins, but this has remained controversial, with conflicting data appearing in the literature. By way of investigating such TPR-mediated self-associations we used a variety of biophysical techniques to characterize purified recombinant Sgt1, a TPR-containing protein found in all eukaryotes that is involved in a broad range of biological processes, including kinetochore assembly in humans and yeast and disease resistance in plants. We show that recombinant Sgt1 from Arabidopsis, barley, and yeast self-associates in vitro while recombinant human Sgt1 does not. Further experiments on barley Sgt1 demonstrate unambiguously a TPR-mediated dimerization, which is concentration- and ionic-strength-dependent and results in a global increase in helical structure and stability of the protein. Dimerization is also redox sensitive, being completely abolished by the formation of an intramolecular disulfide bond where the contributing cysteines are conserved in plant Sgt1s. The dimer interface was mapped through cross-linking and mass spectrometry to the C-terminal region of the TPR domain. Our study, which provides the first biophysical characterization of plant Sgt1, highlights how TPR domains can mediate self-association in solution and that sequence variation in the regions involved in oligomerization affects the propensity of TPR-containing proteins to dimerize.

Structural and Functional Analysis of SGT1 Reveals That Its Interaction with HSP90 Is Required for the Accumulation of Rx, an R Protein Involved in Plant Immunity

“…These data support the idea that SGT1 may form higher order oligomers. Consistent with such a model, we recently described how plant SGT1 forms a TPR-mediated dimer that increases the stability of the protein in vitro (Nyarko et al, 2007).…”

AIP and its interacting partners

“…The self-association of AIP is very likely mediated by the TPR domain, as demonstrated for other TPR-containing proteins (Das et al 1998, Taylor et al 2001, Nyarko et al 2007). This suggests that AIP can homodimerise without the association of other auxiliary proteins or at least others than AhR or hsp90 and also that more than one molecule of AIP could be present in the AhR complex.…”

“…If these ratios are correct, the authors suggested that the multimeric complexes of AIP act as a reservoir able to regulate the amount of available monomeric AIP that can be included into the AhR complex or maybe in the other complexes with which AIP has been shown to interact. Furthermore, the TPR-mediated self-association of AIP might be a mechanism to specifically regulate its biological functions, as reported for the TPR-containing proteins PP5 and Sgt1 (Yang et al 2005, Nyarko et al 2007. For example, the phosphatase activity of PP5 is suppressed by an autoinhibited conformation maintained by the TPR domaincatalytic domain interaction (Yang et al 2005).…”

Crystal Structure of the N-terminal Domain of Anaphase-promoting Complex Subunit 7

“…Moreover, nApc7 was found to form a dimer in solution. This homodimerization via its N-terminal dimerization motif is the first example of homodimerization that is mediated by the N terminus of TPR protein, although the TPR motif is known to mediate self-association for many TPRcontaining proteins, such as OGT (40), Tom70p (41), and SGT (42).…”