Expression, purification and structural characterization of the type 1-specific ATP binding site of IP3 receptor (IP3R1-ATPA).

The inositol 1,4,5-triphosphate receptor (IP 3 R), an IP 3 -gated Ca 2+ release channel on the endoplasmic reticulum (ER) membrane, plays a critical role in maintaining cytosolic Ca 2+ homeostasis in cells. Particularly, ATP increases IP 3 R activity by binding to ATPA, a putative glycine-rich Walker A-type motif (GXGXXG) specific to type 1 IP 3 R (IP 3 R1). Here, we established an efficient process to produce the ATPA containing domain of IP 3 R1 (IP 3 R1-ATPA) using a chaperone co-expression system in Escherichia coli . The recombinant protein was well expressed as a soluble form and showed a high thermostability. Circular dichroism results indicated a mainly α-helical conformation of the purified protein. Additionally, model structures of IP 3 R1-ATPA were calculated and validated using different modeling algorithms. The structural models of IP 3 R1-ATPA not only supported the observed high thermostability, but also suggested a potential ATP binding site. [ABSTRACT FROM AUTHOR]