Influence of a C-terminal His-tag on the stability and crystallization behavior of IA-2ec449-576

PRIMO MARÍA EVANGELINA; SICA MAURICIO PABLO; RISSO VALERIA A.; NOGUERA MARTÍN EZEQUIEL; ERMÁCORA MARIO ROBERTO; POSKUS EDGARDO

Rosario

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

The 106 kDa insulinoma?associated protein (IA?2), a member of the receptor protein tyrosine phosphatase superfamily, is located in the membrane of secretory granules (SGs) of neural, pituitary and pancreatic islet cells [1,2]. IA 2 post translational processing mainly consists in a proteolytic cleavage at position 448 449, yielding a mature extracellular domain (IA 2ec449 576), attached to the intracellular domain (IA-2ic) through a single transmembrane region (residues 576 600). Additionally, in beta cells, SGs exocytosis promotes the Ca2+ dependent cleavage of the intracellular domain by m-calpain, releasing a fragment that translocates to the nucleus and promotes the expression of insulin [3,4]. From this evidence, it has been speculated that, upon exposure on the cell surface, binding of as yet unknown ligands to IA 2ec449 576 might further regulate the intracellular cleavage of IA 2ic. The fact that IA-2 3D structure is unknown and the lack of sequence similarity between IA 2ec449 576 and proteins preclude further elaboration on its biological function. Recently, in order to study its structural properties, our laboratory has expressed a C terminal His tag variant of IA 2ec449 576 H6. Our results demonstrated that IA 2ec449 576 is a very stable autonomous folding domain in physiological conditions [5], which has encouraged the attempts to elucidate its 3D structure by X ray crystallography. In the present work, we have studied the influence of the C terminal His tag in the stability and crystallization rate of IA 2ec449 576 H6. Far UV and near UV CD spectra at room temperature indicated that the His tag does not affect the secondary and tertiary structure content of the domain at pH 4.6 or 7.0. To further characterize its thermodynamic stability at these pH conditions, both variants were unfolded by linear increasing of temperature from 4 to 90 °C and the CD signal at 220 nm was monitored. Interestingly, results show that the His tag destabilizes the domain by six and four degrees at pH 4.7 and pH 7.0, respectively. These results are in good agreement with our observations that, in two different conditions, IA 2ec449 576 H6 yielded crystals tenfold more slowly with respect to the wild type domain. In conclusion, we have demonstrated that His tag, although does not present any effect on the overall structure of the domain, has a negative impact in the IA 2ec449 576 thermodynamic stability, which is directly related to its crystal forming propensity. [1] Solimena M, Dirkx R, Hermel JM, Pleasic-Williams S, Shapiro JA, Caron L and Rabin DU, Embo J, 1996, 15, 2102-14. [2] Dirkx, R., Jr., Hermel J.M., Rabin D.U. and Solimena M., Adv Pharmacol, 1998, 42, 243-6. [3] Trajkovski M, Mziaut M, Altkruger A, Ouwendijk J, Knoch KP, Muller S and Solimena M, J Cell Biol, 2004,167, 1063-74. [4] Mziaut H, Trajkovski M, Kersting S, Ehninger A, Altkruger A, and Solimena M, Nat Cell Biol., 2006, 8, 435-45. [5] Primo, M.E., Sica M.P., Risso V.A., Poskus E. and Ermacora M.R., Biochim Biophys Acta., 2006, 1764, 174-81.