Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein |
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Authors: | Brenner C; Pace HC; Garrison PN; Robinson AK; Rosler A; Liu XH; Blackburn GM; Croce CM; Huebner K; Barnes LD |
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Affiliation: | Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA. |
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Abstract: | 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.
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