The interaction of folylpolyglutamates with deoxyhemoglobin. Identification of the binding site.
Previous studies have shown that pteroylheptaglutamate (PteGlu7) can form a 1:1 complex with deoxyhemoglobin. The solution and crystallographic studies reported in this paper delineate the nature of the PteGlu7 binding site. We find that the three structural elements of PteGlu7 (the pteridine moiety, the p-aminobenzoyl portion, and the glutamate groups) each contribute to the binding energy by interacting with residues in the central cavity between the beta subunits and with residues at the alpha 1 beta 1 interface. Identification of the 2,3-diphosphoglycerate (DPG) binding site as part of the PteGlu7 binding site was accomplished in two ways; first by the demonstration of reduced PteGlu7 binding to hemoglobin selectively modified by pyridoxylation at this site, and second by the finding that DPG and PteGlu7 bind to deoxyhemoglobin in a competitive manner. In addition, since analogs of PteGlu7 in which the pteridine moiety is modified display reduced binding, it can be concluded that the pteridine group also contributes significantly to the binding energy. The crystallographic studies are completely consistent with the results determined in solution. A difference electron density image at 4.3 A resolution shows that the pteridine and p-aminobenzoyl groups are nestled against an interior edge of the alpha 1 beta 1 interface with the pteridine ring interacting with Phe 36 alpha 1 and the p-aminobenzoyl group positioned against a portion of the H helix between residues Lys 132 beta 1 and Ala 135 beta 1. The difference density for the glutamate residues is less well resolved (for reasons described in the text), but it is clear that some of the carboxylate side chains must interact with residues at the DPG binding site.
A
A
Arnone
P H
PH
Rogers
R E
RE
Benesch
R
R
Benesch
S
S
Kwong
D001665:Binding Sites; D005492:Folic Acid / Q000031:analogs & derivatives*; D006454:Hemoglobins / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism*; D013329:Structure-Activity Relationship
D006454:Hemoglobins; D011624:Pteroylpolyglutamic Acids; D005492:Folic Acid