title: 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.
authors.affiliation: Unidad de Transducción de Señales, Instituto de Biomedicina de Valencia, CSIC, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Zamora-Caballero
authors.affiliation: Laboratorio de Venómica Estructural y Funcional, Instituto de Biomedicina de Valencia, CSIC, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Pérez
authors.affiliation: Laboratorio de Venómica Estructural y Funcional, Instituto de Biomedicina de Valencia, CSIC, Spain.
authors.forename: Libia
authors.initials: L
authors.lastname: Sanz
authors.affiliation: Unidad de Transducción de Señales, Instituto de Biomedicina de Valencia, CSIC, Spain. Electronic address: jbravo@ibv.csic.es.
authors.forename: Jerónimo
authors.initials: J
authors.lastname: Bravo
authors.affiliation: Laboratorio de Venómica Estructural y Funcional, Instituto de Biomedicina de Valencia, CSIC, Spain. Electronic address: jcalvete@ibv.csic.es.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Calvete
mesh_terms: D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009154:Mutation; D037121:Plant Lectins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D014461:Ultracentrifugation
chemical_list: C043014:Dioclea grandiflora lectin; D037121:Plant Lectins; D011994:Recombinant Proteins
keywords: Analytical ultracentrifugation; Dimer–tetramer equilibrium; Recombinant Diocleinae lectin; Site-directed mutagenesis; X-ray crystal structure