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Genetic, biochemical, and crystallographic characterization of Fhit-substrate complexes as the active signaling form
H C Pace1, P N Garrison, A K Robinson
1Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Abstract:
Alterations in the FHIT gene at 3p14.2 occur as early and frequent events in the development of several common human cancers. The ability of human Fhit-negative cells to form tumors in nude mice is suppressed by stable reexpression of Fhit protein. Fhit protein is a diadenosine P1,P3-triphosphate (ApppA) hydrolase whose fungal and animal homologs form a branch of the histidine triad (HIT) superfamily of nucleotide-binding proteins. Because the His-96 --> Asn substitution of Fhit, which retards ApppA hydrolase activity by seven orders of magnitude, did not block tumor-suppressor activity in vivo, we determined whether this mutation affected ApppA binding or particular steps in the ApppA catalytic cycle. Evidence is presented that His-96 --> Asn protein binds ApppA well and forms an enzyme-AMP intermediate extremely poorly, suggesting that Fhit-substrate complexes are the likely signaling form of the enzyme. The cocrystal structure of Fhit bound to Ado-p-CH2-p-ps-Ado (IB2), a nonhydrolyzable ApppA analog, was refined to 3.1 A, and the structure of His-96 --> Asn Fhit with IB2 was refined to 2.6 A, revealing that two ApppA molecules bind per Fhit dimer; identifying two additional adenosine-binding sites on the dimer surface; and illustrating that His-98 is positioned to donate a hydrogen bond to the scissile bridging oxygen of ApppA substrates. The form of Fhit bound to two ApppA substrates would present to the cell a dramatically phosphorylated surface, prominently displaying six phosphate groups and two adenosine moieties in place of a deep cavity lined with histidines, arginines, and glutamines.
Insights
The FHIT gene is crucial for suppressing tumors. Even with reduced activity, Fhit protein
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Alterations in the FHIT gene (3p14.2) are early events in human cancers.
- Fhit protein, a diadenosine P1,P3-triphosphate (ApppA) hydrolase, exhibits tumor-suppressor activity.
- Fhit belongs to the histidine triad (HIT) superfamily of nucleotide-binding proteins.
Purpose of the Study:
- Investigate the role of Fhit protein's catalytic activity in its tumor-suppressor function.
- Determine if mutations affecting ApppA hydrolase activity impact Fhit's tumor suppression in vivo.
- Elucidate the structural basis of Fhit-ApppA interactions.
Main Methods:
- Site-directed mutagenesis (His-96 --> Asn substitution) to alter Fhit activity.
- Tumorigenicity assays in nude mice with Fhit-reconstituted cells.
- Enzyme kinetics to assess ApppA hydrolase activity and intermediate formation.
- X-ray crystallography to determine the structure of Fhit-ligand complexes.
Main Results:
- The His-96 --> Asn mutation significantly reduced ApppA hydrolase activity but did not abolish tumor suppression.
- Mutant Fhit protein binds ApppA effectively but forms the enzyme-AMP intermediate poorly.
- Structural analysis revealed two ApppA molecules binding per Fhit dimer, with additional adenosine-binding sites.
- Key residues like His-98 are positioned for substrate interaction, and bound Fhit presents a phosphorylated surface.
Conclusions:
- Fhit-substrate complexes, rather than catalytic activity, likely mediate its tumor-suppressor function.
- The structural data provides insights into Fhit's mechanism of action and substrate binding.
- Understanding Fhit's interaction with ApppA may offer new therapeutic strategies for FHIT-deficient cancers.