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.