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

MARÍA E. PRIMO; MAURICIO P. SICA; VALERIA A. RISSO; MARTÍN E. NOGUERA; MARIO R. ERMÁCORA; EDGARDO POSKUS

Rosario, Santa Fe, Argentina

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

Sociedad Argentina de Biofísica (SABiofísica)

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.