Pex5p binding affinities for canonical and noncanonical PTS1 peptides
Pex5p binding affinities for canonical and noncanonical PTS1 peptides
复制标题
DOI：
10.1002/prot.20112
复制
发表时间：
2004-06-01
影响因子：
2.9
通讯作者：
Berg, JM
中科院分区：
文献类型：
--
作者：
Maynard, EL;Gatto, GJ;Berg, JM
大多数靶向过氧化物酶体腔的蛋白质含有由过氧化物酶Pex 5 p结合的C末端过氧化物酶体靶向信号-1（PTS 1）。PTS 1通常被认为是一个C-末端三肽，它附着于共有序列（S/A/C）（K/R/H）（L/M）。先前，我们研究了YQX（-3）X（-2）X（-1）形式的肽与人Pex 5 p的肽结合结构域（称为Pex 5 p-C）的结合亲和力。发现YQSKL的最佳亲和力，其以200 +/- 40 nM的亲和力结合。为了扩展这项工作，我们研究了含有酰基辅酶A氧化酶（RHYLK-PLQSKL）的最后9个残基的肽的性质，并发现它以1.4 +/- 0.4 nM的解离常数与Pex 5 p-C结合，比YQSKL紧密180倍。进一步的分析表明，增强的亲和力主要是由于（-5）位亮氨酸的存在。此外，发现对应于荧光素酶C末端的肽（YKGGKSKL）与Pex 5 p-C的结合比YQSKL紧密约20倍。这种效应的大部分是由于在位置（4）具有赖氨酸。过氧化氢酶含有一个非典型的PTS 1（-AREKANL）。发现YQANL的亲和力为3600 +/-400 nH。这种相对较弱的结合与先前通过将-ANL融合到其C-末端（-GGA-ANL）来将氯霉素乙酰转移酶引导到过氧化物酶体的不成功尝试一致。肽YKANL、YEKANL、YREKANL和YAREKANL都以比YQANL更高的亲和力结合Pex 5 p-C，但亲和力仍然低于在其他情况下对应于功能靶向信号的肽。由于过氧化氢酶和Pex 5 p都是四聚体（与我们研究中使用的单体Pex 5 p-C和肽相反），因此应考虑Pex 5 p和其他寡聚蛋白质之间结合亲和力的多齿效应。我们的研究提供了直接的热力学数据，揭示肽与Pex 5 p-C结合受到（4）位的赖氨酸和（-5）位的亮氨酸的青睐。我们的结果表明，在（4）和/或（-5）位置具有优化残基的肽或蛋白质可以以比YQSKL的亲和力大至少两个数量级的亲和力结合Pex 5 p，并且这种稳定性可以补偿否则弱结合的PTSI。(C)2004 Wiley-Liss，Inc.
The majority of proteins targeted to the peroxisomal lumen contain a C-terminal peroxisomal targeting signal-1 (PTS1) that is bound by the peroxin Pex5p. The PTS1 is generally regarded as a C-terminal tripeptide that adheres to the consensus (S/A/C)(K/R/H)(L/M). Previously, we studied the binding affinity of peptides of the form YQX(-3)X(-2)X(-1) to the peptide-binding domain of human Pex5p (referred to as Pex5p-C). Optimal affinity was found for YQSKL, which bound with an affinity of 200 +/- 40 nM. To extend this work, we investigated the properties of a peptide containing the last 9 residues of acyl-CoA oxidase (RHYLK-PLQSKL) and discovered that it binds to Pex5p-C with a dissociation constant of 1.4 +/- 0.4 nM, 180 times tighter than YQSKL. Further analysis revealed that the enhanced affinity is primarily due to the presence of leucine in the (-5) position. In addition, a peptide corresponding to the luciferase C-terminus (YKGGKSKL) was found to bind Pex5p-C about 20 times tighter than YQSKL. The majority of this effect results from having lysine in position (4). Catalase contains a noncanonical PTS1 (-AREKANL). The affinity of YQANL was found to be 3600 +/- 400 nH. This relatively weak binding is consistent with previous unsuccessful attempts to direct chloramphenicol acetyltransferase to the peroxisome by fusing -ANL to its C-terminus (-GGA-ANL). The peptides YKANL, YEKANL, YREKANL, and YAREKANL all bound Pex5p-C with higher affinities than did YQANL, but the affinities are still lower than peptides that correspond to functional targeting signals in other contexts. Because both catalase and Pex5p are tetramers (as opposed to the monomeric Pex5p-C and the peptides used in our studies), multidentate effects on binding affinity between Pex5p and other oligomeric proteins should be considered. Our study provides direct thermodynamic data revealing that peptide binding to Pex5p-C binding is favored by lysine in the (4) position and leucine in the (-5) position. Our results suggest that peptides or proteins with optimized residues in the (4) and/or (-5) positions can bind to Pex5p with affinities that are at least two orders of magnitude greater than that of YQSKL, and that this stabilization can compensates for otherwise weakly binding PTSIs. (C) 2004 Wiley-Liss, Inc.