The 7-fold mutation Cys201Met/Cys332Tyr/Lys344Asp/Tyr345Ile/Val351Ala/Tyr391Lys/Arg395Ser converts the cofactor specificity of Escherichia coli isocitrate dehydrogenase from a 7000-fold preference for NADP+ to a 200-fold preference for NAD+, with overall activity comparable to that of wild-type NAD+- dependent isocitrate dehydrogenases. The structure of the NAD+-dependent mutant has been determined and refined to a working R-factor of 0.186 at 1.9 Å resolution. The structure shows that NADP+ affinity is destroyed by removing favorable interactions between the 2'-phosphate and Tyr345, Tyr391, and Arg395 and by adding a repulsive interaction with Asp344. NAD+ affinity is enhanced by adding hydrogen bonds between Asp344 and the free 2'-hydroxyl. The favorable Asp344-2'-OH interaction requires a change in the pucker of the ribose to C2' endo and a shift in the adenine ring. The ring shift is made possible by a series of changes in steric packing interactions. The linchpin for repacking in the adenosine binding site is residue 351. The side chain of this 'second layer' residue dictates packing of the surrounding 'first layer' residues which interact with the 2' moiety and, in turn, directly determine specificity.