Ultsch M H, Somers W, Kossiakoff A A, de Vos A M
Department of Protein Engineering Genetech, Inc, South San Francisco, CA 94080.
J Mol Biol. 1994 Feb 11;236(1):286-99. doi: 10.1006/jmbi.1994.1135.
A variant of human growth hormone (hGH), in which 15 mutations were introduced with phage display mutagenesis to improve receptor binding affinity by 400-fold, yielded two related crystal forms diffracting to high resolution. The structure of this variant was determined in both crystal forms, one at 2.0 A resolution and one at 2.4 A resolution, using molecular replacement with wild-type hGH taken from the receptor complex structure as a search model. Crystallographic refinement of the 2 A structure gave an R-value R-value of 18.5% for data in the resolution range 8 to 2 A. The final model consists of residues 1 to 128 and 155 to 191, with three side-chains modeled in alternative conformations, together with 77 water molecules. Comparison of the structure with wild-type hGH shows that most of the secondary structural elements are unchanged. The exception is the first turn of the third helix in the four-helix bundle core, which is unraveled in the present variant. Analysis of the two related packing environments suggests that this change is caused by crystal packing forces. A large change in the orientation of a short segment of helix found in the connection between the first two core helices is interpreted as evidence for rigid-body variability of this helical segment. Analysis of the mutations in light of the structure of the wild-type hGH/receptor complex shows that six of the mutations are buried in the hormone, whereas the remaining nine involve residues that interact with the receptor in the complex.
一种人生长激素（hGH）变体，通过噬菌体展示诱变引入了15个突变，使受体结合亲和力提高了400倍，产生了两种衍射至高分辨率的相关晶体形式。使用来自受体复合物结构的野生型hGH作为搜索模型，通过分子置换确定了该变体在两种晶体形式下的结构，一种分辨率为2.0 Å，另一种分辨率为2.4 Å。对2 Å结构进行晶体学精修，对于8至2 Å分辨率范围内的数据，R值为18.5%。最终模型由1至128位和155至191位残基组成，三个侧链以替代构象建模，还有77个水分子。将该结构与野生型hGH进行比较表明，大多数二级结构元件未发生变化。例外的是四螺旋束核心中第三个螺旋的第一圈，在当前变体中它解开了。对两种相关堆积环境的分析表明，这种变化是由晶体堆积力引起的。在前两个核心螺旋之间的连接处发现的一小段螺旋的方向发生了很大变化，这被解释为该螺旋段刚体变异性的证据。根据野生型hGH/受体复合物的结构对突变进行分析表明，其中六个突变埋藏在激素中，而其余九个涉及复合物中与受体相互作用的残基。
