Background

Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs.

Methods

Various point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3.

Results

This led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (K_d = 1.5 μM vs no-binding with wild type) and LD2 peptides (K_d = 7.2 μM vs 63 μM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10⁻⁵ μm²/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10⁻³ μm²/s).

Conclusions

We observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs.

General significance

Our study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration.

中文翻译：

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LD相互作用的残基在聚焦黏附激酶的聚焦黏附靶向域上的空间排列决定了域-基序相互作用的亲和力和特异性。

背景

富含L精氨酸的A spartate基序（LD基序）是Paxillin上的分子识别基序，可识别许多粘着斑蛋白的LD-基序结合域（LDBD），以进行下游信号传导和肌动蛋白细胞骨架重塑。在这项研究中，我们确定了LDBD中影响其与Paxillin LD基序的结合亲和力的结构特征。

方法

通过将关键的赖氨酸残基移动两个和三个螺旋圈来创建粘着斑靶向激酶（FAK）的粘着斑靶向（FAT）域的各种点突变，以匹配其他两种粘着斑蛋白Vinculin在LDBD中观察到的独特构象和CCM3。

结果

这导致鉴定出FAK FAT结构域的突变体，命名为FAT（NV）（FAT结构域的Asn992被Val取代），对LD1具有显着的高亲和力（K _d  = 1.5μM，与野生型无结合）和LD2肽（K _d  = 7.2μM，而野生型为63μM）。始终如一地，表达FAK（NV）的MCF7细胞的粘着斑是高度稳定（周转率= 1.25×10 ^-5 微米² / s）的与野生型FAK转染的细胞（周转率= 1.5×10 ^-3 微米² / s）。

结论

我们观察到，LDBD表面关键LD结合氨基酸的相对位置，LD基序长亮氨酸侧链的疏水埋藏和带电表面的互补性是决定LD基序与LDBD结合亲和力的关键因素。

一般意义

我们的研究将通过调节FAK-LD基序相互作用来帮助FAK的FAT域的蛋白质工程，这对细胞黏着斑粘附和细胞迁移具有影响。