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

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.