Deciphering the structural and functional impact of missense mutations in Egr1-DNA interacting interface: an integrative computational approach

Early growth response-1 (Egr1) is a zinc-finger transcription factor that plays a critical role in controlling cell growth, proliferation, differentiation, angiogenesis, and apoptosis. Egr1 is induced by many growth factors, cytokines, and stress signals and is also known to be involved in several pathological conditions like cancer, neurological and ocular disorders. The DNA binding domain of Egr1 is a highly conserved Cys2His2 (C2H2) zinc finger (ZNF) domain which specifically binds to GC-rich consensus sequence GcG (G/T) GGGCG and activates transcription. As the C2H2 domain specifically recognizes its DNA target, the mutations spanning this region shall perturb DNA recognition and may hinder transcription of target genes. Therefore, in this study, the missense mutations occurring specifically at the DNA binding domain (DBD) of Egr1 were probed by computational approaches involving <i>in silico</i> screening of pathogenic and functional mutants coupled with extensive molecular dynamics simulations, to determine the mutants that affect its structural stability and interactions with DNA. From the pathogenicity analysis of 38 missense mutations spanning Egr1-DBD, 17 were predicted as pathogenic, and 7 amongst these were found to have functional impact on Egr1. On combined analysis of molecular dynamics simulation, Residue interaction analysis and Egr1<i>-</i>DNA interaction analysis results, the mutants R371C and R375C showed least impact, whilst, H382R tend to increase the structural stability, whereas R360H, H390R, E393V, and H414Y conferred greater impact by altering the structural stability and DNA interactions. Hence, this study exposes the prospects of considering these 4 deleterious mutations for clinical significance, but needs further experimental validation.Highlights<i>Egr1’s</i> DNA binding domain is a highly conserved Cys2His2 (C2H2) zinc finger domain that specifically recognizes its DNA target.Mutations spanning in the DNA binding domain shall perturb DNA recognition and may hinder transcription.Among the missense mutations, mutants R360H, H390R, E393V, and H414Y were inferred to have a greater impact on <i>Egr1</i> by altering the structural stability and DNA interactions. <i>Egr1’s</i> DNA binding domain is a highly conserved Cys2His2 (C2H2) zinc finger domain that specifically recognizes its DNA target. Mutations spanning in the DNA binding domain shall perturb DNA recognition and may hinder transcription. Among the missense mutations, mutants R360H, H390R, E393V, and H414Y were inferred to have a greater impact on <i>Egr1</i> by altering the structural stability and DNA interactions.

早期生长应答因子1（Early growth response-1，Egr1）是一种锌指转录因子（zinc-finger transcription factor），在调控细胞生长、增殖、分化、血管生成及细胞凋亡过程中发挥关键作用。Egr1可被多种生长因子、细胞因子及应激信号诱导表达，且已知其参与癌症、神经系统疾病与眼部疾病等多种病理过程。Egr1的DNA结合域（DNA binding domain, DBD）为高度保守的Cys2His2（C2H2）型锌指（zinc finger, ZNF）结构域，可特异性结合富含GC的共有序列GcG(G/T)GGGCG并激活转录。由于C2H2结构域可特异性识别其DNA靶序列，该区域发生的突变会干扰DNA识别过程，进而阻碍靶基因的转录。因此，本研究针对Egr1 DNA结合域（DBD）上发生的错义突变，采用包含致病性与功能性突变体in silico筛选、结合大规模分子动力学模拟的计算方法进行探究，以鉴定影响其结构稳定性及与DNA相互作用的突变体。通过对覆盖Egr1-DBD的38个错义突变进行致病性分析，预测其中17个突变为致病性突变，且其中7个被发现对Egr1具有功能性影响。综合分子动力学模拟、残基相互作用分析及Egr1-DNA相互作用分析的结果可知，突变体R371C与R375C的影响最小；而H382R可提升结构稳定性，R360H、H390R、E393V及H414Y则通过改变结构稳定性与DNA相互作用，产生更为显著的影响。因此，本研究提示可将这4个有害突变纳入临床意义相关研究的考量范围，但仍需进一步的实验验证。研究亮点：Egr1的DNA结合域为高度保守的Cys2His2（C2H2）型锌指结构域，可特异性识别其DNA靶序列。DNA结合域内发生的突变会干扰DNA识别过程，进而阻碍靶基因的转录。在错义突变中，R360H、H390R、E393V及H414Y这几个突变体可通过改变结构稳定性与DNA相互作用，对Egr1产生更为显著的影响。Egr1的DNA结合域为高度保守的Cys2His2（C2H2）型锌指结构域，可特异性识别其DNA靶序列。DNA结合域内发生的突变会干扰DNA识别过程，进而阻碍靶基因的转录。在错义突变中，R360H、H390R、E393V及H414Y这几个突变体可通过改变结构稳定性与DNA相互作用，对Egr1产生更为显著的影响。