Extended Data Fig. 7 |. The effect of Glu407Asn on apo and pemetrexed-bound PCFT and hereditary folate malabsorption mutations in the context of the chicken PCFT structure.

a–e, Alchemical transformations show that a Glu407Asn mutation would stabilize the protein by forming hydrogen bonds with Asn166, while remaining capable of preserving the interaction with the pyrrole amine of pemetrexed. a, In the wild-type apo state Glu407 does not interact with Asn166. The Glu407Asn variant, however, can readily hydrogen-bond with Asn166 (b), resulting in the Glu407Asn variant being 1.5 kcal mol⁻¹ more stable (e) (blue). In the pemetrexed-bound state, Glu407 makes a hydrogen bond with the pyrrole amine group of pemetrexed (PMX) (c), which is also preserved in the Glu407Asn variant (d). The coordinated hydrogen-bond network among Asn166, Glu407Asn and pemetrexed further stabilizes the protein-bound state by 2 kcal mol⁻¹ compared to the apo state (e) (orange). n = 3 independent repeats with mean and s.d. plotted. f, Cartoon of chicken PCFT structure with residues involved in hereditary folate malabsorption shown as spheres. Asp164 (Asp156) and Arg384 (Arg376) are highlighted as these residues have an important role in the transport mechanism. The table shows the corresponding human residue number and the associated phenotype. PubMed identifiers for the respective studies that describe mutations associated with hereditary folate malabsorption are included.