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

Abstract

A useful (2) J(N-H) coupling-based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA-binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T-cell intracellular antigen-1 (TIA-1) protein. The pKa values of the His96 ionizable groups were substantially higher in the complexes with short U-rich RNA and T-rich DNA oligonucleotides than those of the isolated TIA-1 RRM2. Herein, the methodology applied to determine changes in pKa of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA-binding proteins that shuttle among different cellular compartments.

TIA-1 RRM23 binding and recognition of target oligonucleotides

“…Hydrogen bond interactions were observed that help to explain the preferential binding of U-rich or T-rich oligonucleotides. In particular, an interaction between His96 and the third thymine base provided structural verification of a previous observation that the pKa of this histidine sidechain is impacted by RNA binding (28,29). We further probed this using NMR and observed increased signal intensities of the sidechain N-H signals upon DNA binding, again consistent with the formation of an oligonucleotide interaction with His96.…”

“…This interaction likely explains the previously reported rise in p K a value of His96 that occurs upon oligonucleotide binding by TIA-1 RRM2 (28). The direct hydrogen bonding of His96 Nδ 1 would be expected to increase the nucleophilicity of the Nε 2 atom of the imidazole and thus raise the p K a .…”

“…In order to further investigate this interaction we performed 2D 1 H- 15 N HSQC experiments that could provide a direct readout of the protonation state of the TIA-1 RRM2 His96 sidechain and backbone nitrogens before and after forming a complex with a 5΄-TTTTT-3΄ oligonucleotide. At pH 5 (which is well below the His96 p K a value of 5.3 for apo-TIA-1 RRM2 and His96 p K a of 5.6 for TIA-1 RRM2 bound to DNA; (28,29) the 1 H- 15 N HSQC spectra show a 2-fold increase in intensity for the Nε 2 –Hε 2 cross-peaks when the complex with DNA is formed (integrated value of 1.075 for free peaks versus 2.167 for DNA-bound signals in arbitrary units) (Figure 7). This is consistent with its increased nucleophilicity.…”

“…15 N labeled TIA-1 RRM2 S (residues 95–184) was prepared for NMR studies as previously described (28). 15 N labeled RRM2 S was expressed in E. coli BL21(DE3) cells as a His-tagged protein and was purified by nickel affinity chromatography (Ni Sepharose 6 Fast Flow, GE Healthcare) according to earlier protocols (26,29).…”

A putative RNA binding protein from Plasmodium vivax apicoplast

“…Eukaryotic RRMs are often found as multiple copies within a protein, and together with other protein domains, they confer different affinity and specificity for the RNA sequences . RRMs are also found in prokaryotes, where they tend to occur as single domains in small proteins, typically around 100 amino acids in length (Pfam ID PF0076 and InterPro ID IPR000504 ).…”