Genetic, biochemical, and crystallographic characterization of Fhit–substrate complexes as the active signaling form of Fhit |
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| Authors: | Helen C. Pace, Preston N. Garrison, Angela K. Robinson, Larry D. Barnes, Alexandra Draganescu, Angelika R sler, G. Michael Blackburn, Zurab Siprashvili, Carlo M. Croce, Kay Huebner, Charles Brenner |
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| Affiliation: | Helen C. Pace, Preston N. Garrison, Angela K. Robinson, Larry D. Barnes, Alexandra Draganescu, Angelika Rösler, G. Michael Blackburn, Zurab Siprashvili, Carlo M. Croce, Kay Huebner, and Charles Brenner |
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| 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 Å, and the structure of His-96 → Asn Fhit with IB2 was refined to 2.6 Å, 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. |
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