2 ' Halo-ATP and -GTP analogues: Rational phasing tools for protein crystallography

The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2'-halogenated nucleotides have been reported as potential universal phasing tools for nuc leotide binding proteins. However, only limited data are available dealing with the effect of 2'-substitution on recognition by the protein. We have d etermined equilibrium dissociation constants of 2'-halogenated ATP analogue s for the ATP binding proteins UMP/CMP kinase and the molecular chaperone D naK. Whereas the affinities to UMP/CMP kinase are of the same order of magn itude as for unsubstituted ATP, the affinities to DnaK are drastically decr eased to undetectable levels. For 2'-halogenated GTP analogues, the kinetic s of. interaction were determined for the small CTPases p2(ras)(Y32W) (fluo rescent mutant) and Rab5. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, w hereas the rates of dissociation were accelerated by factors of similar to 100 (p21(ras)) or similar to 10(5) (Rab5), and the resulting equilibrium di ssociation constants are in the nm or mu M range, respectively. The data de monstrate that 2'halo-ATP and -GTP are substrates or Ligands for all protei ns tested except the chaperone DnaK;. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 10(5)-f old decrease in affinity as observed for Rab5 places the equilibrium dissoc iation constant in the mu M range, so that they are still well suited for c rystallization of the G-protein:nucleotide complex.