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Quaternary structure of Dioclea grandiflora lectin assessed by equilibrium sedimentation and crystallographic analysis of recombinant mutants
Author(s) -
Zamora-Caballero Sara,
Pérez Alicia,
Sanz Libia,
Bravo Jerónimo,
Calvete Juan J.
Publication year - 2015
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2015.07.020
Subject(s) - tetramer , dimer , mutant , chemistry , lectin , protein quaternary structure , crystallography , sedimentation equilibrium , stereochemistry , recombinant dna , mutation , biochemistry , enzyme , protein subunit , organic chemistry , gene
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‐ α DGL). 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‐ α DGL 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.