Complete mapping of divergent amino acids responsible for differential ligand binding of folate receptors alpha and beta

The folate receptor (FR) type a may be distinguished from FR-b 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-b (Shen, F., Zheng, X., Wang, H., and Ratnam, M. (1997) Biochemistry 36, 6157–6163); however, the results also indicated that the minimum requirement for conversion of FR-b to the functional equivalent of FR-a should include amino acid substitution(s) downstream of residue 92 in addition to mutation of L49A. To pinpoint those residues, chimeric FR-bL49A/FR-a constructs including progressively shorter segments of FR-adownstream of position 92 as well as selected point mutants were studied. Simultaneous substitution of Leu-49, Phe-104, and Gly-166 in FR-b with the corresponding FR-a residues Ala, Val, and Glu, respectively, reconstituted the ligand binding characteristics of FR-a. The results also exclude a role for other residues in FR-ain determining its functional divergence. A homology model of FR-abased 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.