Structure of the L-leucine-binding protein refined at 2.4 A resolution and comparison with the Leu/Ile/Val-binding protein structure.
Structure of the L-leucine-binding protein refined at 2.4 A resolution and comparison with the Leu/Ile/Val-binding protein structure.
复制标题
以 2.4 A 分辨率精修的 L-亮氨酸结合蛋白结构以及与 Leu/Ile/Val 结合蛋白结构的比较。
DOI：
10.1016/0022-2836(89)90532-9
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发表时间：
1989
影响因子：
5.6
通讯作者：
Quiocho,FA
中科院分区：
文献类型：
--
作者：
Sack,JS;Trakhanov,SD;Tsigannik,IH;Quiocho,FA
以Leu/Ile/Val结合蛋白（344个残基）的精细结构为模型结构，用分子置换法测定了大肠杆菌主动转运组分亮氨酸结合蛋白（36，900 M和346个残基）的三维X射线结构。这两种氨基酸结合蛋白具有80%的序列同一性，尽管两者在相同的空间群中结晶，但它们具有非常不同的晶胞尺寸。旋转函数产生一个显著的峰值，随后导致单个自洽平移函数解。该模型首先通过约束最小二乘法进行改进，其中分子的两个结构域中的每一个分别处理，以允许两个结构域的相对取向的任何微小变化。然后修改该模型以反映从Leu/Ile/Val结合蛋白序列到I-亮氨酸结合蛋白序列的氨基酸侧链的72个变化和两个插入。最后的结构精修，使用约束最小二乘法，导致一个R-因子为0·20的13,797反射到2.4毫米的分辨率。该模型由2600个蛋白质原子和91个溶剂分子组成，与Leu/Ile/Val结合蛋白的结构非常相似，均为无配体构象，两个结构域之间的裂缝敞开，易于接近。结构的叠加产生317个等效残基的α-碳原子的均方根差为0.68 π。亮氨酸结合蛋白结构的五个区域与Leu/Ile/Val结合蛋白结构相差超过1.6 μ m，远离配体结合位点的主要部分，其位于双叶蛋白的一个结构域中。在结构之间，形成底物结合位点的主要部分的氨基酸侧链存在三个差异。这些取代本身不能清楚地解释支链脂肪族氨基酸特异性的差异。
The three-dimensional X-ray structure of the leucine-binding protein (36,900Mrand 346 residues), an active transport component ofEscherichia coli, has been determined by the method of molecular replacement, using the refined structure of the Leu/Ile/Val-binding protein (344 residues) as the model structure. The two amino acid-binding proteins have 80% sequence identity and, although both crystallize in the same space group, they have very different unit cell dimensions. The rotation function yielded one significant peak, which subsequently led to a single self-consistent translation function solution. The model was first refined by the constrained least-squares method, with each of the two domains of the molecule treated separately to allow for any small change in the relative orientation of the two domains. The model was then modified in order to reflect the 72 changes in amino acid side-chains and two insertions in going from the Leu/Ile/Val-binding protein sequence to that of thel-leucine-binding protein. Final structure refinement, using the restrained least-squares technique, resulted in anR-factor of 0·20 for 13,797 reflections to a resolution of 2.4 Å. The model is comprised of 2600 protein atoms and 91 solvent molecules.Thel-leucine-binding protein structure is, as expected, very similar to the Leu/Ile/Val-binding protein structure; both are in the unliganded conformation with the cleft between the two domains wide open and easily accessible. The superimposing of the structures yields a root-mean-square difference of 0.68 Å in the α-carbon atoms of the 317 equivalent residues. The five regions of the leucine-binding protein structure that differ by more than 1.6 Å from the Leu/Ile/Val-binding protein structure are far from the major portion of the ligand-binding site, which is located in one domain of the bilobate protein. Between the structures, there are three differences in the amino acid side-chains that form the major portion of the substrate-binding sites. These substitutions, by themselves, fail to clearly explain the differences in the specificities for branched aliphatic amino acids.