MUTATION OF ARGININE-121 IN LACTOFERRIN DESTABILIZES IRON-BINDING BY DISRUPTION OF ANION-BINDING - CRYSTAL-STRUCTURES OF R121S AND R121E MUTANTS

A conserved arginine residue helps to form the synergistic anion bindi ng site in transferrins. To probe the importance of this residue for a nion binding and iron binding, Arg 121 has been mutated to Ser and Glu in the N-terminal half-molecule of human lactoferrin. The two mutants , R121S and R121E, have been expressed, purified, and crystallized. Th eir three-dimensional structures have been determined by X-ray diffrac tion at 2.3 and 2.5 Angstrom resolution, respectively. The structures were determined by molecular replacement and were refined by restraine d least squares methods to final R values of 0.185 and 0.204. Both mut ants still bind iron but with decreased stability. The crystal structu res show that destabilization of iron binding probably results from di sruption of the anion binding site; mutation of Arg 121 removes one wa ll of the anion binding pocket and causes the synergistic carbonate io n to be displaced 0.5 Angstrom from its position in the wild-type prot ein. In the process it becomes partially detached from the helix N-ter minus that forms the rest of the anion binding site.