Complete mapping of divergent amino acids responsible for differential ligand binding of folate receptors α and β

Abstract

The folate receptor (FR) type α may be distinguished from FR-β by its higher affinity for the circulating folate coenzyme, (6S)-5- methyltetrahydrofolate (5-CH3H4folate), and its opposite stereospecificity for reduced folate coenzymes. Previous studies showed that a single leucine to alanine substitution at position 49 of the mature protein sequence is responsible for the functional divergence of FR-β (Shen, F., Zheng, X., Wang, H., and Ratham, M. (1997) Biochemistry 36, 6157-6163); however, the results also indicated that the minimum requirement for conversion of FR-β to the functional equivalent of FR-α should include amino acid substitution(s) downstream of residue 92 in addition to mutation of L49A. To pinpoint those residues, chimeric FR-β(L49A)/FR-α constructs including progressively shorter segments of FR-α downstream of position 92 as well as selected point mutants were studied. Simultaneous substitution of Leu-49, Phe-104, and Gly-166 in FR-β with the corresponding FR-α residues Ala, Val, and Glu, respectively, reconstituted the ligand binding characteristics of FR-α. The results also exclude a role for other residues in FR-α in determining its functional divergence. A homology model of FR-α based on the three-dimensional structure of the chicken riboflavin-binding protein is used to show the position of residues 49, 104, and 166 in relation to the hydrophobic cleft corresponding to the riboflavin-binding pocket.