TPR-Mediated self-association of plant SGT1

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 Sgtl, 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 Sgtl from Arabidopsis, barley, and yeast self-associates in vitro while recombinant human Sgtl does not. Further experiments on barley Sgtl 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 Sgtls. 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 Sgtl, 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.