SEQUENTIAL STRUCTURAL-CHANGES UPON ZINC AND CALCIUM-BINDING TO METAL-FREE CONCANAVALIN-A

The lectin concanavalin A (ConA) sequentially binds a transition metal ion in the metal-binding site S1 and a calcium ion in the metal-bindi ng site S2 to form its saccharide-binding site. Metal-free ConA crysta ls soaked with either Zn2+ (apoZn-ConA) or Co2+ (apoCo-ConA) display p artial binding of these ions in the proto-transition metal-binding sit e, but no further conformational changes are observed, These structure s can represent the very first step in going from metal-free ConA towa rd the holoprotein. In the co-crystals of metal-free ConA with Zn2+ (Z n-ConA), the zinc ion can fully occupy the S1 site, The positions of t he carboxylate ligands Asp(10) and Asp(19) that bridge the S1 and S2 s ites are affected, The ligation to Zn2+ orients Asp(10) optimally for calcium ligation and stabilizes Asp(19) by a hydrogen bond to one of i ts water ligands, The neutralizing and stabilizing effect of the bindi ng of Zn2+ in S1 is necessary to allow for subsequent Ca2+ binding in the S2 site. However, the S2 site of monometallized ConA is still disr upted, The co crystals of met al-free ConA with both Zn2+ and Ca2+ con tain the active holoprotein (ConA ZnCa). Ca2+ has induced large confor mational changes to stabilize its hepta-coordination in the S2 site, w hich comprise the trans to cts isomerization of the Ala(207)-Asp(208) peptide bond accompanied by the formation of the saccharide-binding si te, The Zn2+ Ligation in ConA ZnCa is similar to Mn2+, Cd2+, Co2+, or Ni2+ ligation in the S1 site, in disagreement with earlier extended x- ray absorption fine structure results that suggested a lower coordinat ion number for Zn2+.