Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein.
Fragile histidine triad protein (Fhit) is a diadenosine triphosphate (ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors. Reintroduction of FHIT coding sequences to cancer cell lines with FHIT deletions suppressed the ability of these cell lines to form tumors in nude mice even when the reintroduced FHIT gene had been mutated to allow ApppA binding but not hydrolysis. Because this suggested that the tumor suppressor activity of Fhit protein depends on substrate-dependent signaling rather than ApppA catabolism, we prepared two crystalline forms of Fhit protein that are expected to model its biologically active, substrate-bound state. Wild-type and the His96Asn forms of Fhit were overexpressed in Escherichia coli, purified to homogeneity and crystallized in the presence and absence of ApppA and an ApppA analog. Single crystals obtained by vapor diffusion against ammonium sulfate diffracted X-rays to beyond 2.75 A resolution. High quality native synchrotron X-ray data were collected for an orthorhombic and a hexagonal crystal form.
Brenner
C
C
Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Pace
H C
HC
Garrison
P N
PN
Robinson
A K
AK
Rosler
A
A
Liu
X H
XH
Blackburn
G M
GM
Croce
C M
CM
Huebner
K
K
Barnes
L D
LD
D017766:Acid Anhydride Hydrolases; D000645:Ammonium Sulfate; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D009363:Neoplasm Proteins / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D000645:Ammonium Sulfate; D005982:Glutathione Transferase; D009363:Neoplasm Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000099170:Bis(5'-adenosyl)triphosphatase