Coupling of prolyl peptide bond isomerization and Ca2+ binding in a C-type mannose-binding protein.

A proline residue flanked by two polar residues is a highly conserved sequence motif in the Ca2+- and carbohydrate-binding site of C-type animal lectins. Crystal structures of several C-type lectins have shown that the two flanking residues are only observed to act as Ca2+ ligands when the peptide bond 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 preceding 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 tryptophan fluorescence, and fluorescence energy transfer from tryptophan to Tb3+, reveal a slow conformational change in rat liver MBP (MBP-C) accompanying the binding of either Ca2+ or Tb3+. The Ca2+-induced increase in intrinsic tryptophan fluorescence shows biphasic kinetics: a burst phase with a rate constant greater than 1 s(-1) is followed by a slow phase with a single-exponential rate constant ranging from 0.01 to 0.05 s(-1) (36 degrees C) that depends on the concentration of Ca2+. Likewise, addition of EGTA to Ca2+-bound or Tb3+-bound MBP-C causes a decrease in intrinsic tryptophan fluorescence with biphasic kinetics consisting of a burst phase with a rate constant greater than 1 s(-1), followed by a slow phase with a single-exponential rate constant of 0.065 s(-1). In contrast, Tb3+ fluorescence produced by resonant energy transfer from MBP-C decreases in a single kinetic phase with a rate constant greater than 1 s(-1), implying that the slow change in tryptophan fluorescence monitors a conformational change that is not limited in rate by ion dissociation. The rate constants of the slow phases accompanying Ca2+ binding and release are strongly affected by temperature and are weakly accelerated by the prolyl isomerase cyclophilin. These data strongly suggest that the binding of either Ca2+ or Tb3+ to MBP-C is coupled to a conformational change that involves the cis-trans isomerization of a peptide bond. Fitting of the data to kinetic models indicates that, in the absence of Ca2+, the proline in approximately 80% of the molecules is in the trans conformation. The slow kinetics associated with cis-trans proline isomerization may be exploited by endocytic receptors to facilitate sorting of carbohydrate-bearing ligands from the receptor in the endosome.