Quaternary structure of Dioclea grandiflora lectin assessed by equilibrium sedimentation and crystallographic analysis of recombinant mutants

Abstract

The structural basis of the pH dependency of the dimer–tetramer transition exhibited by Brinda’s type II Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of recombinant wild-type and site-directed single and double mutants of the pH-stable tetrameric Dioclea grandiflora lectin (r-αDGL). Releasing the peripheral site interdimeric contact between R60 and D78 rendered a mutant displaying dimer–tetramer equilibrium in the pH range equivalent to pKa±1 of the γ-COOH. Mutation of both histidines 51 and 131, but not the mutation of each His separately, abolished the formation of the Diocleinae canonical tetramer in the pH range 2.5–8.5. The X-ray structure of the double mutant r-αDGL H51G/H131N suggests that H131 plays a crucial role in networking loop 114–125 residues from all four subunits at the central cavity of the tetrameric lectin, and that H51 maintains the central cavity loops in a proper spatial orientation to make H131-mediated interdimer contacts.