Zhao D, Gilfoyle D J, Smith A T, Loew G H
Molecular Research Institute, Palo Alto, California 94304, USA.
Proteins. 1996 Oct;26(2):204-16. doi: 10.1002/(SICI)1097-0134(199610)26:2<204::AID-PROT10>3.0.CO;2-T.
In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)-differing mainly in the structure of a long untemplated insertion-were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca2+ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures.
在本研究中，对辣根过氧化物酶（HRP-C）的两种替代性三维（3D）模型进行了优化、系统评估，并用于进行预测，这些预测既能指导未来的实验研究，又能接受其检验。这两种模型主要在一个长的无模板插入结构上有所不同。模型构建过程中的关键第一步是基于结构保守区域和关键保守残基（包括为两个Ca2+结合位点提供配体的侧链）进行多序列比对的程序。此处报道的模型优化包括：（1）侧链构象的优化；（3）使用与模板无关的程序添加结构水；（2）利用能量标准和核磁共振数据对位于F螺旋和G螺旋之间的34个氨基酸的无模板插入进行结构优化；（4）对优化后的模型进行无约束能量优化。通过这些程序，获得了HRP-C的两种优化结构，主要区别在于这个长插入结构的构象。该插入结构中存在可能与结合底物相互作用的残基，这表明其可能具有与III类过氧化物酶与多种底物形成稳定1:1复合物的一般能力相关的功能作用。通过多种标准对模型的结构有效性进行了系统评估。最值得注意的是，两个HRP-C模型的ProsaII z评分和Profiles 3D评分表明，它们明显优于以任何已知结构为模板通过简单氨基酸替换获得的模型。然后，利用这两个3D HRP-C模型预测二维核磁共振研究中先前记录的NOESY交叉峰归属的候选残基。具体而言，这些残基包括异亮氨酸X、苯丙氨酸A、苯丙氨酸B、脂肪族残基Q和异亮氨酸T。还确定了候选底物结合位点，并与基于实验的预测进行了比较。这项工作很及时，因为预计新的X射线结构将有助于验证这些程序。
