Origins of specificity in the binding of small molecules to dihydrofolate reductase

Dihydrofolate reductase is the target for the therapeutically important 'anti-folate' drugs such as methotrexate and trimethoprim. Methotrexate is a powerful inhibitor of the enzyme, binding up to 10,000 times more tightly than the structurally similar substrate, folate. Two contributions to this striking difference in affinity have been identified: the two ligands bind in different charge states, and there are conformational differences between the two complexes. The origins of the tight binding of methotrexate have been explored further by studying the binding of 2,4-diaminopyrimidine and p-aminobenzoyl-L-glutamate, which may be considered as 'fragments' of methotrexate. These two compounds bind simultaneously but also cooperatively, the binding of one 'fragment' leading to a 50-fold increase in the affinity for the other. Studies of structural analogues of these fragments show that the specificity as well as the strength of binding can be altered by the presence of the other 'fragment'; both positive and negative cooperativity are observed. The relation of these observations to methotrexate binding, and the notion of intramolecular cooperativity in ligand binding are discussed.