ジャーナル: Proteins / 年: 2013 タイトル: Solution structures of polcalcin Phl p 7 in three ligation states: Apo-, hemi-Mg2+-bound, and fully Ca2+-bound. 著者: Michael T Henzl / Arthur G Sirianni / Wei G Wycoff / Anmin Tan / John J Tanner / 要旨: 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 ...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.
内容: 3 mM [U-98% 13C; U-98% 15N] Phl p 7, 150 mM sodium chloride, 10 mM MES, 10 % [U-99% 2H] D2O, 0.1 % sodium azide, 0.1 mM calcium chloride, 90% H2O/10% D2O 溶媒系: 90% H2O/10% D2O
試料
濃度 (mg/ml)
構成要素
Isotopic labeling
Solution-ID
3mM
Phl p 7-1
[U-98% 13C; U-98% 15N]
1
150mM
sodium chloride-2
1
10mM
MES-3
1
10 %
D2O-4
[U-99% 2H]
1
0.1 %
sodium azide-5
1
0.1mM
calcium chloride-6
1
試料状態
イオン強度: 160 / pH: 6.0 / 圧: ambient / 温度: 293 K
-
NMR測定
NMRスペクトロメーター
タイプ
製造業者
モデル
磁場強度 (MHz)
Spectrometer-ID
Varian INOVA
Varian
INOVA
600
1
Varian INOVA
Varian
INOVA
600
2
Varian INOVA
Varian
INOVA
600
3
-
解析
NMR software
名称
開発者
分類
VNMR
Varian
collection
NMRPipe
Delaglio, Grzesiek, Vuister, Zhu, PfeiferandBax
解析
Sparky
Goddard
chemicalshiftassignment
Sparky
Goddard
データ解析
Sparky
Goddard
peakpicking
TALOS
Cornilescu, DelaglioandBax
データ解析
CYANA
Guntert, MumenthalerandWuthrich
構造決定
CYANA
Guntert, MumenthalerandWuthrich
精密化
精密化
手法: torsion angle dynamics / ソフトェア番号: 1
NMR constraints
NOE constraints total: 2176 / NOE intraresidue total count: 489 / NOE long range total count: 470 / NOE medium range total count: 623 / NOE sequential total count: 594
代表構造
選択基準: closest to the average
NMRアンサンブル
コンフォーマー選択の基準: target function / 計算したコンフォーマーの数: 100 / 登録したコンフォーマーの数: 20
NMR ensemble rms
Distance rms dev: 0.046 Å / Distance rms dev error: 0.002 Å