Metal ion binding and conformational transitions in concanavalin a: A structure-function study

Abstract

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn2+ and Ca2+, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: A “locked” form and an “unlocked” form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn2+ and Ca2+. They also demonstrated that Mn2+ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25°C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin. Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (~ 22 kcal M-1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied. We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn2+ and saccharide, and have determined both metal ion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them. The main finding is that saccharide binds very weakly (Kd~2 M) to unlocked apo-Con A and very tightly to the locked ternary Mn2+’Con A complex (Kd~ 10-4 M). Saccharide binding increases along the various pathways connecting these two species in a nonadditive fashion. Thus, both conformation and metal ion binding determine the saccharide affinity of each complex, although the specificity of saccharide binding of the various species is maintained throughout. © Taylor & Francis Group, LLC.