Coupling of prolyl peptide bond isomerization and Ca2+ binding in a C-typemannose-binding protein

A proline residue flanked by two polar residues is a highly conserved seque nce motif in the Ca2+- and carbohydrate-binding site of C-type animal lecti ns, Crystal structures of several C-type lectins have shown that the two fl anking residues are only observed to act as Ca2+ ligands when the peptide b ond preceding the proline residue is in the cis conformation. In contrast, structures of the apo- and one-ion forms of mannose-binding proteins (MBPs) reveal that, when the Ca2+-binding site is empty, the peptide bond precedi ng the proline can adopt either the cis or trans conformation, and distinct structures in adjacent regions are associated with the two proline isomers . In this work, measurements of Ca2+-induced changes in intrinsic tryptopha n fluorescence, and fluorescence energy transfer from tryptophan to Tb3+, r eveal a slow conformational change in rat liver MBP (MBP-C) accompanying th e binding of either Ca2+ or Tb3+. The Ca2+-induced increase in intrinsic tr yptophan fluorescence shows biphasic kinetics: a burst phase with a rate co nstant greater than 1 s(-1) is followed by a slow phase with a single-expon ential rate constant ranging from 0.01 to 0.05 s(-1) (36 degrees C) that de pends on the concentration of Ca2+. Likewise, addition of EGTA to Ca2+-boun d or Tb3+-bound MBP-C causes a decrease in intrinsic tryptophan fluorescenc e with biphasic kinetics consisting of a burst phase with a rate constant g reater than 1 s(-1), followed by a slow phase with a single-exponential rat e constant of 0.065 s(-1). In contrast, Tb3+ fluorescence produced by reson ant energy transfer from MBP-C decreases in a single kinetic phase with a r ate constant greater than 1 s(-1), implying that the slow change in tryptop han fluorescence monitors a conformational change that is not limited in ra te by ion dissociation. The rate constants of the slow phases accompanying Ca2+ binding and release are strongly affected by temperature and are weakl y accelerated by the prolyl isomerase cyclophilin. These data strongly sugg est that the binding of either Ca2+ or Tb3+ to MBP-C is coupled to a confor mational change that involves the cia-trans isomerization of a peptide bond . Fitting of the data to kinetic models indicates that, in the absence of C a2+, the proline in approximately 80% of the molecules is in the trans conf ormation. The slow kinetics associated with cis-trans proline isomerization may be exploited by endocytic receptors to facilitate sorting of carbohydr ate-bearing ligands from the receptor in the endosome.