PURIFICATION AND CRYSTALLIZATION OF COMPLEXES MODELING THE ACTIVE STATE OF THE FRAGILE HISTIDINE TRIAD PROTEIN

Fragile histidine triad protein (Fhit) is a diadenosine triphosphate ( ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors. Reintroduction of FHIT coding seque nces to cancer cell lines with FHIT deletions suppressed the ability o f these cell lines to form tumors in nude mice even when the reintrodu ced FHIT gene had been mutated to allow ApppA binding but not hydrolys is. Because this suggested that the tumor suppressor activity of FHIT protein depends on substrate-dependent signaling rather than ApppA cat abolism, we prepared two crystalline forms of Fhit protein that are ex pected to model its biologically active, substrate-bound state. Wild-t ype and the His96Asn forms of Fhit were overexpressed in Escherichia c oli, purified to homogeneity and crystallized in the presence and abse nce of ApppA and an ApppA analog. Single crystals obtained by vapor di ffusion against ammonium sulfate diffracted X-rays to beyond 2.75 Angs trom resolution. High quality native synchrotron X-ray data were colle cted for an orthorhombic and a hexagonal crystal form.