Acharya K R, Stuart D I, Walker N P, Lewis M, Phillips D C
Laboratory of Molecular Biophysics, University of Oxford, U.K.
J Mol Biol. 1989 Jul 5;208(1):99-127. doi: 10.1016/0022-2836(89)90091-0.
The solution of the structure of alpha-lactalbumin from baboon milk (Papio cynocephalus) at 4.5 A resolution using the isomorphous replacement method has been reported previously. Initial refinement on the basis of these low-resolution studies was not successful because of the poor isomorphism of the best heavy-atom derivative. Because of the striking similarity between the structure of lysozyme and alpha-lactalbumin, a more cautious molecular replacement approach was tried to refine the model. Using hen egg-white lysozyme as the starting model, preliminary refinement was performed using heavily constrained least-squares minimization in reciprocal space. The model was further refined using stereochemical restraints at 1.7 A resolution to a conventional crystallographic residual of 0.22 for 1141 protein atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A, and for angle distances it is 0.027 A. The refinement was carried out using the human alpha-lactalbumin sequence and "omit maps" calculated during the course of refinement indicated eight possible sequence changes in the baboon alpha-lactalbumin X-ray sequence. During the refinement, a tightly bound calcium ion and 150 water molecules, of which four are internal, have been located. Some of the water molecules were modelled for disordered side-chains. The co-ordination around the calcium is a slightly distorted pentagonal bipyramid. The Ca-O distances vary from 2.2 A to 2.6 A, representing a tight calcium-binding loop in the structure. The calcium-binding fold only superficially resembles the "EF-hand" and presumably has no evolutionary relationship with other EF-hand structures. The overall structure of alpha-lactalbumin is very similar to that of lysozyme. All large deviations occur in the loops where all sequence deletions and insertions are found. The C terminus appears to be rather flexible in alpha-lactalbumin compared to lysozyme. The experimental evidence supports the earlier predictions for the alpha-lactalbumin structure that were based upon the assumption that alpha-lactalbumin and lysozyme have similar three-dimensional structures, with minimal deletions and insertions. A detailed comparison of the two structures shows striking features as well as throwing some light on the evolution of these two proteins from a common precursor.
此前已有报道使用同晶置换法在4.5埃分辨率下解析狒狒奶中α-乳白蛋白（Papio cynocephalus）的结构。基于这些低分辨率研究的初步精修并不成功，原因是最佳重原子衍生物的同晶性较差。由于溶菌酶和α-乳白蛋白的结构极为相似，因此尝试采用更为谨慎的分子置换方法来精修模型。以鸡蛋白溶菌酶作为起始模型，在倒易空间中使用高度受限的最小二乘最小化进行初步精修。该模型在1.7埃分辨率下使用立体化学约束进一步精修，对于1141个蛋白质原子，常规晶体学残余为0.22。在最终模型中，键长与理想值的均方根偏差为0.015埃，键角偏差为0.027埃。精修使用的是人类α-乳白蛋白序列，精修过程中计算的“省略图”表明狒狒α-乳白蛋白X射线序列中有八个可能的序列变化。在精修过程中，定位到了一个紧密结合的钙离子和150个水分子，其中四个是内部水分子。一些水分子是为无序侧链建模的。钙周围的配位是一个略有扭曲的五角双锥。Ca-O距离在2.2埃到2.6埃之间变化，代表结构中一个紧密的钙结合环。钙结合折叠仅表面上类似于“EF手”，推测与其他EF手结构没有进化关系。α-乳白蛋白的整体结构与溶菌酶非常相似。所有大的偏差都出现在发现所有序列缺失和插入的环中。与溶菌酶相比，α-乳白蛋白的C末端似乎相当灵活。实验证据支持了早期基于α-乳白蛋白和溶菌酶具有相似三维结构且缺失和插入最少这一假设对α-乳白蛋白结构的预测。对这两种结构的详细比较显示出显著特征，也为这两种蛋白质从共同前体的进化提供了一些线索。
