Low resolution structure of the human alpha4 protein (IgBP1) and studies on the stability of alpha4 and of its yeast ortholog Tap42

The yeast Tap42 and mammalian alpha4 proteins belong to a highly conserved family of regulators of the type 2A phosphatases, which participate in the rapamycin-sensitive signaling pathway, connecting nutrient availability to cell growth. The mechanism of regulation involves binding of Tap42 to Sit4 and PPH21/22 in yeast and binding of alpha4 to the catalytic subunits of type 2A-related phosphatases PP2A, PP4 and PP6 in mammals. Both recombinant proteins undergo partial proteolysis, generating stable N-terminal fragments. The full-length proteins and alpha4 C-terminal deletion mutants at amino acids 222 (alpha4Delta222), 236 (alpha4Delta236) and 254 (alpha4Delta254) were expressed in E. coli. alpha4Delta254 undergoes proteolysis, producing a fragment similar to the one generated by full-length alpha4, whereas alpha4Delta222 and alpha4Delta236 are highly stable proteins. alpha4 and Tap42 show alpha-helical circular dichroism spectra, as do their respective N-terminal proteolysis resistant products. The cloned truncated proteins alpha4Delta222 and alpha4Delta236, however, possess a higher content of alpha-helix, indicating that the C-terminal region is less structured, which is consistent with its higher sensitivity to proteolysis. In spite of their higher secondary structure content, alpha4Delta222 and alpha4Delta236 showed thermal unfolding kinetics similar to the full-length alpha4. Based on small angle X-ray scattering (SAXS), the calculated radius of gyration for alpha4 and Tap42 were 41.2 +/- 0.8 A and 42.8 +/- 0.7 A and their maximum dimension approximately 142 A and approximately 147 A, respectively. The radii of gyration for alpha4Delta222 and alpha4Delta236 were 21.6 +/- 0.3 A and 25.7 +/- 0.2 A, respectively. Kratky plots show that all studied proteins show variable degree of compactness. Calculation of model structures based on SAXS data showed that alpha4Delta222 and alpha4Delta236 proteins have globular conformation, whereas alpha4 and Tap42 exhibit elongated shapes.