title: Genetic, biochemical, and crystallographic characterization of Fhit-substrate complexes as the active signaling form of Fhit.
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.
authors.affiliation: Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: H C
authors.initials: HC
authors.lastname: Pace
authors.forename: P N
authors.initials: PN
authors.lastname: Garrison
authors.forename: A K
authors.initials: AK
authors.lastname: Robinson
authors.forename: L D
authors.initials: LD
authors.lastname: Barnes
authors.forename: A
authors.initials: A
authors.lastname: Draganescu
authors.forename: A
authors.initials: A
authors.lastname: Rösler
authors.forename: G M
authors.initials: GM
authors.lastname: Blackburn
authors.forename: Z
authors.initials: Z
authors.lastname: Siprashvili
authors.forename: C M
authors.initials: CM
authors.lastname: Croce
authors.forename: K
authors.initials: K
authors.lastname: Huebner
authors.forename: C
authors.initials: C
authors.lastname: Brenner
mesh_terms: D017766:Acid Anhydride Hydrolases; D000818:Animals; D018360:Crystallography, X-Ray; D019281:Dimerization; D015226:Dinucleoside Phosphates / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D000099170:Bis(5'-adenosyl)triphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D015226:Dinucleoside Phosphates; D008024:Ligands; D009363:Neoplasm Proteins; D011506:Proteins; D000099170:Bis(5'-adenosyl)triphosphatase; C011440:diadenosine triphosphate