Expression, purification and crystallization of a BH domain from the GTPase regulatory protein associated with focal adhesion kinase

Signaling by small GTPases is down-regulated by GTPase activating proteins (GAPs) which enhance the rate of GTP hydrolysis. The activity of GAPs speci fic for Rho GTPases resides in the BH domain, many homologues of which are found in any mammalian genome. One of them was identified in the GTPase reg ulator associated with focal-adhesion kinase (GRAF). It shares approximatel y 20% sequence identity with p50RhoGAP. This GAP activates RhoA and Cdc42Hs , but not Rac. In order to dissect the molecular basis of this specificity, a 231-residue-long fragment corresponding to the BH domain of GRAF has bee n expressed, purified and crystallized. Trigonal crystals, of space group P 3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 Angstr om were grown from solutions of PEG 6000. Data to 2.15 Angstrom were collec ted from a flash-frozen sample on an R-AXIS IV imaging-plate detector mount ed on a rotating-anode X-ray generator.