Genetic, biochemical, and crystallographic characterization of Fhit-substrate complexes as the active signaling form of Fhit

Proc Natl Acad Sci U S A. 1998 May 12;95(10):5484-9. doi: 10.1073/pnas.95.10.5484.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acid Anhydride Hydrolases*
  • Animals
  • Crystallography, X-Ray
  • Dimerization
  • Dinucleoside Phosphates / metabolism
  • Humans
  • Ligands
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Neoplasm Proteins*
  • Protein Conformation
  • Proteins / chemistry*
  • Proteins / genetics
  • Proteins / metabolism
  • Static Electricity


  • Dinucleoside Phosphates
  • Ligands
  • Neoplasm Proteins
  • Proteins
  • fragile histidine triad protein
  • diadenosine triphosphate
  • Acid Anhydride Hydrolases

Associated data

  • PDB/1FHI
  • PDB/2FHI