INSIGHTS INTO TYROSINE PHOSPHORYLATION CONTROL OF PROTEIN-PROTEIN ASSOCIATION FROM THE NMR STRUCTURE OF A BAND-3 PEPTIDE INHIBITOR BOUND TOGLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

A protein-protein association regulated by phosphorylation of tyrosine is examined by NMR structural studies and biochemical studies, Bindin g of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and aldolase to the N-terminus of human erythrocyte anion transporter, band 3, inhibit s enzyme activity. This inhibition is reversed upon phosphorylation of band 3 Y8, as shown by kinetic studies on purified components, as wel l as in vivo studies. Thus, tyrosine phosphorylation mediates against the intermolecular protein-protein association, in contrast to the pos itive control involving SH2 and PTB domains where phosphorylation is r equired for binding. To elucidate the basis of recognition and negativ e control by tyrosine phosphorylation, the structure of a synthetic pe ptide, B3P, corresponding to the first 15 residues of band 3 (MEELQDDY EDMMEEN-NH2), bound to G3PDH has been determined using the exchange-tr ansferred nuclear Overhauser effect. The G3PDH-bound B3P structure was found to be very similar to the structure recognized by aldolase. A h ydrophobic triad forms from side chains within a loop structure of res idues 4 through 9 in both bound species. Another structural feature st abilizing the loop, in the case of the B3P-G3PDH complex, is a hydroge n bond between the side chains of Y8 and D10 associated with a beta-tu rn of residues 8-11. Based on the structure of this phosphorylation se nsitive interaction (PSI) loop, it is suggested that tyrosine phosphor ylation disrupts protein-protein association, in part, by intramolecul ar electrostatic destabilization. The inhibition by B3P is competitive with respect to the coenzyme NAD(+) and noncompetitive with the subst rate analog arsenate, Specific binding of B3P to G3PDH is demonstrated by reversion of the NMR spectral properties of bound B3P to those of the free peptide upon addition of coenzyme and substrate analog. The s toichiometry of binding for the B3P-G3PDH complex was determined from Sephadex G-50 displacement experiments to be 4:1, Collectively, these results are consistent with B3P binding the active site of G3PDH.