A minimalist approach toward protein recognition by epitope transfer from functionally evolved beta-sheet surfaces.
A minimalist approach toward protein recognition by epitope transfer from functionally evolved beta-sheet surfaces.
复制标题
通过从功能进化的β-折叠表面转移表位来识别蛋白质的极简方法。
DOI：
10.1021/ja064885b
复制
发表时间：
2006
影响因子：
15
通讯作者：
Ghosh,Indraneel
中科院分区：
文献类型：
--
作者：
Rajagopal,Srivats;Meyer,ScottC;Goldman,Aaron;Zhou,Min;Ghosh,Indraneel
New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small β-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the β-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of β-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, β-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 Å was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the β-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 Å linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action.
