Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, N-21, W-11, Sapporo 001-0021, Japan.
J Biomol NMR. 2012 May;53(1):53-63. doi: 10.1007/s10858-012-9623-8. Epub 2012 Apr 10.
Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein-protein and protein-ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.
假接触位移 (PCSs) 是由固定在蛋白质框架中的顺磁镧系离子诱导产生的，可提供远程距离和角度信息，对于蛋白质-蛋白质和蛋白质-配体复合物的结构确定非常有价值。我们一直在开发一种通过肽键和二硫键将镧系元素结合肽标签（以下简称 LBT）固定在两个点上的方法，以锚定到目标蛋白上。然而，磁导率张量具有对称性，这可能导致仅基于 PCSs 的结构计算中出现多个简并解。在这里，我们展示了一种通过改变 LBT 和目标蛋白之间的间隔长度来解决这种简并性的简便方法。我们将这种方法应用于 FKBP12-雷帕霉素复合物和 mTOR FRB 结构域之间基于 PCS 的刚体对接，并证明使用两种不同间隔长度的两点锚定 LBT 获得的 PCS 约束可以解决简并性。本策略将显著提高两点锚定 LBT 用于蛋白质复合物结构确定的有用性。
