Related Experiment Videos
Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein
C Brenner1, H C Pace, P N Garrison
1Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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.
Insights
The fragile histidine triad (Fhit) protein
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The fragile histidine triad (Fhit) protein is a diadenosine triphosphate (ApppA) hydrolase.
- Fhit is encoded at a human chromosome 3 fragile site frequently disrupted in tumors.
- Fhit protein's tumor suppressor activity may involve substrate-dependent signaling, not just ApppA hydrolysis.
Purpose of the Study:
- To investigate the structural basis of Fhit protein's tumor suppressor activity.
- To obtain crystalline forms of Fhit protein in its biologically active, substrate-bound state.
Main Methods:
- Overexpression and purification of wild-type and His96Asn mutant Fhit proteins in E. coli.
- Crystallization of Fhit protein in the presence and absence of ApppA and an ApppA analog.
- X-ray diffraction analysis of orthorhombic and hexagonal crystal forms to beyond 2.75 A resolution.
Main Results:
- Two crystalline forms of Fhit protein were successfully prepared.
- Crystals diffracted X-rays to high resolution, enabling structural analysis.
- Native synchrotron X-ray data were collected for both crystal forms.
Conclusions:
- The study provides structural insights into the substrate-bound state of Fhit protein.
- These findings support the hypothesis that Fhit's tumor suppressor function relies on signaling pathways.
- Further structural studies will elucidate the mechanism of Fhit's tumor suppression.