Low resolution structure of the human α4 protein (IgBP1) and studies on the stability of α4 and of its yeast ortholog Tap42

The yeast Tap42 and mammalian alpha 4 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 a4 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 a4 C-terminal deletion mutants at amino acids 222 (alpha 4 Delta 222), 236 (alpha 4 Delta 236) and 254 (alpha 4 Delta 254) were expressed in E. coli. alpha 4 Delta 254 undergoes proteolysis, producing a fragment similar to the one generated by full-length a4, whereas alpha 4 Delta 222 and alpha 4 Delta 236 are highly stable proteins. alpha 4 and Tap42 show alpha-helical circular dichroism spectra, as do their respective N-terminal proteolysis resistant products. The cloned truncated proteins alpha 4 Delta 222 and alpha 4 Delta 236, however, possess a higher content of a-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, alpha 4 Delta 222 and alpha 4 Delta 236 showed thermal unfolding kinetics similar to the full-length alpha 4. Based on small angle X-ray scattering (SAXS), the calculated radius of gyration for alpha 4 and Tap42 were 41.2 +/- 0.8 angstrom and 42.8 +/- 0.7 angstrom and their maximum dimension similar to 142 angstrom and similar to 147 angstrom, respectively. The radii of gyration for alpha 4 Delta 222 and alpha 4 Delta 236 were 21.6 +/- 0.3 angstrom and 25.7 +/- 0.2 angstrom, respectively. Kratky plots show that all studied proteins show variable degree of compactness. Calculation of model structures based on SAXS data showed that alpha 4 Delta 222 and alpha 4 Delta 236 proteins have globular conformation, whereas alpha 4 and Tap42 exhibit elongated shapes. (c) 2006 Elsevier B.V. All rights reserved.