Abstract
The reversible binding of manganese and calcium to concanavalin A determines the carbohydrate binding of the lectin by inducing large conformational changes. These changes are governed by the isomerization of a non-proline peptide bond, Ala-207-Asp-208, positioned in a beta-strand in between the calcium binding site S2 and the carbohydrate specificity-determining loop. The replacement of calcium by manganese allowed us to investigate the structures of the carbohydrate binding, locked state and the inactive, unlocked state of concanavalin A, both with and without metal ions bound. Crystals of unlocked metal-free concanavalin A convert to the locked form with the binding of two Mn(2+) ions. Removal of these ions from the crystals traps metal-free concanavalin A in its locked state, a minority species in solution. The ligation of a metal ion in S2 to unlocked concanavalin A causes bending of the beta-strand foregoing the S2 ligand residues Asp-10 and Tyr-12. This bending disrupts conventional beta-sheet hydrogen bonding and forces the Thr-11 side chain against the Ala-207-Asp-208 peptide bond. The steric strain exerted by Thr-11 is presumed to drive the trans-to-cis isomerization. Upon isomerization, Asp-208 flips into its carbohydrate binding position, and the conformation of the carbohydrate specificity determining loop changes dramatically.
Highlights
Lectins are a structurally very diverse class of proteins that bind carbohydrates with considerable specificity but moderate affinities (1)
The reversible binding of manganese and calcium to concanavalin A determines the carbohydrate binding of the lectin by inducing large conformational changes. These changes are governed by the isomerization of a non-proline peptide bond, Ala-207–Asp-208, positioned in a -strand in between the calcium binding site S2 and the carbohydrate specificity-determining loop
All Leguminosae lectins share the need for transition metal ion and calcium binding to stabilize the active conformation of these loops
Summary
Brewer et al (14) conclude that ConA occurs in essentially two conformational states, which they called the locked, containing the cis peptide, and the unlocked state, containing a usual trans peptide Both states are in equilibrium with each other, but the equilibrium constant depends on the presence or absence of metal ions. Important for our work was the observation that when Mn2ϩ is used as a replacement for Ca2ϩ, the conversion from the unlocked to the locked form is slowed down significantly, occurring on a time scale of hours or even days (at 5 °C) instead of minutes (14) This allowed us to collect x-ray data on intermediates that occur only transiently or as minority species in solution.
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