SOLUTION STRUCTURE OF A BAND-3 PEPTIDE INHIBITOR BOUND TO ALDOLASE - A PROPOSED MECHANISM FOR REGULATING BINDING BY TYROSINE PHOSPHORYLATION

Human erythrocyte band 3 inhibits glycolytic enzymes, including aldola se, by binding these cytoplasmic enzymes at its N-terminus. Phosphoryl ation of Y8 disrupts inhibition, and there is evidence that in vivo gl ycolysis levels in erythrocytes are regulated in part by a phosphoryla tion/dephosphorylation signaling pathway. The structural basis for con trol by phosphorylation has been investigated by NMR studies on a comp lex between aldolase and a synthetic peptide corresponding to the firs t 15 residues of band 3 (MEELQDDYEDMMEEN-NH2). The structure of this b and 3 peptide (B3P) when it is bound to rabbit muscle aldolase was det ermined using the exchange-transferred nuclear Overhauser effect (ET-N OE). Two hundred NMR structures for B3P were generated by simulated an nealing molecular dynamics with NMR-derived distance restraints and ex cluding electrostatic terms, Twenty structures were further refined ag ainst a force field including full partial charges, The important conf ormational feature of B3P in the bound state is a folded loop structur e involving residues 4-9 and M12 that surrounds Y8 and is stabilized b y a hydrophobic cluster with the ring of Y8 sandwiched between the met hyl groups of L4 and M12, Differential line broadening indicates that this loop structure binds aldolase in a relatively specific manner, wh ile terminal regions are structurally heterogeneous. To better underst and B3P inhibition of aldolase and the mechanism of phosphorylation co ntrol, a complex was modeled by docking B3P into the active site of al dolase and optimizing the fit using restrained molecular dynamics and energy minimization. The B3P loop is complementary in conformation to the beta-barrel central core containing the aldolase active site resid ues. Binding is electrostatic in nature with numerous ionic and hydrog en-bonding interactions involving several conserved lysine and arginin e residues of aldolase, How phosphorylation of band 3 could disrupt in hibition was considered by modeling a phosphoryl moiety onto Y8 of B3P . An energetic analysis with respect to rigid phosphate rotation sugge sts that aldolase inhibition is reversed primarily because of electros tatic repulsion between B3P residues that destabilizes the B3P loop fo rmed in the complex. This proposed intramolecular mechanism for blocki ng protein-protein association by electrostatic repulsion with the pho sphoryl group may be applicable to other protein-protein signaling com plexes.