title: Solution structures of polcalcin Phl p 7 in three ligation states: Apo-, hemi-Mg2+-bound, and fully Ca2+-bound.
abstract: Polcalcins are small EF-hand proteins believed to assist in regulating pollen-tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain-swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg(2+), or 100 μM Ca(2+). Phl p 7 is monomeric in all three ligation states. In the apo-form, both EF-hand motifs reside in the closed conformation, with roughly antiparallel N- and C-terminal helical segments. In 5.0 mM Mg(2+), the divalent ion is bound by EF-hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca(2+)-bound Phl p 7 resembles that previously reported for Bet v 4-likewise exposing apolar surface to the solvent. Occluded in the apo- and Mg(2+)-bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF-hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at -x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca(2+). When Mg(2+) resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer-sphere ligand. (15)N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA. henzlm@missouri.edu
authors.forename: Michael T
authors.initials: MT
authors.lastname: Henzl
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Sirianni
authors.forename: Wei G
authors.initials: WG
authors.lastname: Wycoff
authors.forename: Anmin
authors.initials: A
authors.lastname: Tan
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
mesh_terms: D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry* / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D020832:EF Hand Motifs; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D012996:Solutions / Q000737:chemistry
chemical_list: D052179:Antigens, Plant; D001059:Apoproteins; D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; C468525:Phl p 7 allergen; D010940:Plant Proteins; D011994:Recombinant Proteins; D012996:Solutions; D008274:Magnesium; D002118:Calcium