pH modulates the binding of early growth response protein 1 transcription factor to DNA

Abstract

EGR1 transcription factor orchestrates a plethora of signaling cascades involved in cellular homeostasis and its down-regulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with increasing pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as H382 by virtue of the fact that its substitution to non-ionizable residues abolishes pH-dependence of the binding of EGR1 to DNA. Notably, H382 inserts into the major groove of DNA and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, H382 is predominantly conserved across other members of EGR1 family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating protein-DNA interactions central to this family of transcription factors. Collectively, our findings uncover an unexpected but a key step in the molecular recognition of EGR1 family of transcription factors and suggest that they may act as sensors of pH within the intracellular environment.

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions

“…Even though the extra RRM3 ␣-helix (␣ 1 ) fails in binding directly to RNA oligonucleotides, its relative orientation regarding ␣ 3 and ␤ 4 could facilitate stable RNA binding (30). A similar effect has been recently reported for the transcription factor EGR1 (early growth response protein 1) in its binding to DNA (31). His-382 is well conserved and modulates protein/ DNA binding, acting as a pH-dependent molecular switch.…”

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

“…A 2 J(NÀH) coupling-based NMR approachi sp roposed to unveil, at the molecular level, the contribution of the imidazole groups of the histidines from D/RBPs (in general), and His96 from TIA-1 RRM2 (in particular) on the modulation of binding to nucleic acids by pH. The NMR experiment goes beyondt he conventional 1 NH SQC spectra at different pH values (Figure 2). The changes in the resonances were specific of certain residues surrounding His96, the only histidine residue of the RRM2 domain that showedt he highest CSP.N otably,r esidues near to His96 (Val97, Asp123,P he143, and Asn145;F igure 2C)w ere affected, but to al esser extent.…”

“…However,i ti sw idely accepted that well-conserved residues can modulate different protein functions like protein/RNA-DNA interactions, acting as pH-dependentm olecular switches. [1][2][3][4] An extensively studied molecular switch is the hemagglutinin fusion peptideo ft he influenza virus, which is responsible for viral penetration into host cells by membrane fusion. Changes in the protonation state of its acidic residues from pH 7t o4g enerate different conformations that control the fusion process.…”

“…The affinity by DNA increases at basic pH values and the His382 (conserved across the EGR family) stabilizes the interaction. [1] Such protonation/deprotonation eventsh ave recently been monitored on the histidine residues of human T-cell intracellular antigen-1 (TIA-1), aD /RBP that controls relevant transcriptional and post-transcriptional events between the nucleus and the cytoplasm of the cell. [5,6] TIA-1 regulates pre-mRNA alternative splicing in the nucleus [7,8] and modulates the translation of several mRNAs in the cytoplasm under cellular stress conditions.…”

“…The changes in the resonances were specific of certain residues surrounding His96, the only histidine residue of the RRM2 domain that showedt he highest CSP.N otably,r esidues near to His96 (Val97, Asp123,P he143, and Asn145;F igure 2C)w ere affected, but to al esser extent. The analysis of CSPs as afunction of pH values gave rise to sigmoidal curves for perturbed residues.M ost of them could be fitted to single protonation events with pK a values associated ranging from 4.99 to 5.23 for both 1 Ha nd 15 Nd imensions (Figure 2D and Table S1 in the Supporting Information).…”