Structure of a domain-opened mutant (R121D) of the human lactoferrin N-lobe refined from a merohedrally twinned crystal form.
Structure of a domain-opened mutant (R121D) of the human lactoferrin N-lobe refined from a merohedrally twinned crystal form.
复制标题
人乳铁蛋白 N 叶的结构域开放突变体 (R121D) 的结构，由单面体孪晶形式精制而成。
DOI：
10.1107/s0907444902005127
复制
发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Baker,EdwardN
中科院分区：
文献类型：
--
作者：
Jameson,GeoffreyB;Anderson,BryanF;Breyer,WendyA;Day,CatherineL;Tweedie,JohnW;Baker,EdwardN
人乳铁蛋白是一种具有双叶结构的铁结合蛋白。每个叶包含单个 Fe3+ 离子和相关 CO_{3}^{2-} 离子的高亲和力结合位点。尽管铁结合非常紧密，但它可以在低 pH 下释放，并伴随着两个结构域分开的构象变化。为了测试Asp121侧链是否可以取代铁结合位点的CO_{3}^{2-}离子，构建了人乳铁蛋白N叶半分子的Arg121Asp（R121D）突变体。 R121D 突变蛋白以其 apo 形式结晶，因为它在结晶过程中失去了铁。该晶体也是单面孪晶，孪晶分数接近 0.5。从最初的分子替代溶液开始 [Breyer 等人。 (1999)，水晶学报。 D55, 129–138]，结构已在 3.0 Å 分辨率下精修至 R 因子 13.9%（Rfree 为 19.9%）。尽管分辨率适中，但高溶剂含量和非晶体对称性有助于获得高质量的电子密度图。 R121D 突变体的铁结合减弱可以通过 Asp 侧链封闭阴离子结合位点来解释。 apoR121D 结构中两个结构域的开放（旋转 54°）与全长乳铁蛋白中 N 叶的开放非常匹配，表明构象变化的程度取决于 N 叶固有的特性。 apoR121D 的 N 叶 C 端部分（残基 321-332）相对于闭合野生型铁结合结构的差异表明该区域在稳定开放形式中的重要性。
Human lactoferrin is an iron-binding protein with a bilobal structure. Each lobe contains a high-affinity binding site for a single Fe3+ ion and an associated CO_{3}^{2-} ion. Although iron binds very tightly, it can be released at low pH, with an accompanying conformational change in which the two domains move apart. The Arg121Asp (R121D) mutant of the N-lobe half-molecule of human lactoferrin was constructed in order to test whether the Asp121 side chain could substitute for the CO_{3}^{2-} ion at the iron-binding site. The R121D mutant protein was crystallized in its apo form as it lost iron during crystallization. The crystals were also merohedrally twinned, with a twin fraction close to 0.5. Starting from the initial molecular-replacement solution [Breyer et al. (1999), Acta Cryst. D55, 129–138], the structure has been refined at 3.0 Å resolution to an R factor of 13.9% (Rfree of 19.9%). Despite the moderate resolution, the high solvent content and non-crystallographic symmetry contributed to electron-density maps of excellent quality. Weakened iron binding by the R121D mutant is explained by occlusion of the anion-binding site by the Asp side chain. The opening of the two domains in the apoR121D structure (a rotation of 54°) closely matches that of the N-lobe in full-length lactoferrin, showing that the extent of the conformational change depends on properties inherent to the N-lobe. Differences in the C-terminal portion of the N-lobe (residues 321–332) for apoR121D relative to the closed wild-type iron-bound structure point to the importance of this region in stabilizing the open form.