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 specific 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 regulator associated with focal-adhesion kinase (GRAF). It shares approximately 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 been expressed, purified and crystallized. Trigonal crystals, of space group P3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 A were grown from solutions of PEG 6000. Data to 2.15 A were collected from a flash-frozen sample on an R-AXIS IV imaging-plate detector mounted on a rotating anode X-ray generator.
Sheffield
P J
PJ
Department of Physiology, University of Virginia, Charlottesville 22908, USA.
Derewenda
U
U
Taylor
J
J
Parsons
T J
TJ
Derewenda
Z S
ZS
D000595:Amino Acid Sequence; D000818:Animals; D015815:Cell Adhesion Molecules / Q000737:chemistry; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D015815:Cell Adhesion Molecules; D020690:GTPase-Activating Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020558:GTP Phosphohydrolases