The C-Domain of the NAC Transcription Factor ANAC019 Is Necessary for pH-Tuned DNA Binding through a Histidine Switch in the N-Domain

Abstract

The affinity of transcription factors (TFs) for their target DNA is a critical determinant of gene expression. Whether the DNA-binding domain (DBD) of TFs alone can regulate binding affinity to DNA is an important question for identifying the design principle of TFs. We studied ANAC019, a member of the NAC TF family of proteins in Arabidopsis, and found a well-conserved histidine switch located in its DBD, which regulates both homodimerization and transcriptional control of the TF through H135 protonation. We found that the removal of a C-terminal intrinsically disordered region (IDR) in the TF abolished the pH-dependent binding of the N-terminal DBD to DNA. We propose a mechanism in which long-range electrostatic interactions between DNA and the negatively charged C-terminal IDR turns on the pH dependency of the DNA-binding affinity of the N-terminal DBD.

Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence

“…In this study we find that mutations in these three residues of NAM-A1 lead to complete loss of protein interaction ability and delayed peduncle senescence. These results are consistent with the predicted role of these residues in stabilising the formation of the NAC dimers found in Kang et al and provides strong evidence for their biological relevance in planta (25). Our ability to both recapitulate and support these findings in wheat also highlights the fact that the functional domains, and essential residues, of NAC transcription factors are likely to be highly conserved across species.…”

“…Further investigation identified a pair of residues essential for protein dimerization in ANAC019 ( Figure 1A, subdomain i) (24). More recently, various residues within the ANAC019 NAC domain have been predicted to have a role in pH-dependent stabilisation of the NAC domain (subdomain i, highlighted in green) (25). However, to our knowledge no other residues of the NAC domain have been shown experimentally to be required for protein dimerization, nor have the above residues been shown to have a biologically significant role in planta.…”

“…Three of the mutations we studied here are located within the dimerization domain highlighted in Kang et al (Figure 1A; green highlight in subdomain i) (25). Two residues, P38S (K3661) and R41Q (K2615), correspond directly to residues predicted to form stabilising bonds in the "perfect" dimer state (P16 and R19 in ANAC019, respectively).…”

“…During the course of this study, Kang et al investigated the role of a specific residue in stabilising protein structure at varying pH levels in the Arabidopsis protein ANAC019 (25). These authors report that the protonation status of the H135 histidine residue of ANAC019 ( Figure 1A; highlighted green in subdomain iv) defines the dimer form of the protein and its ability to form a complex with DNA.…”

“…Previous work has tested the role of specific residues in protein binding (subdomain i) and DNA binding (subdomains iii to v); residues which were required for the corresponding process are underlined in red, those that were not required are underlined in blue (24). Kang et al showed that a histidine residue in subdomain iv plays a role in protein dimer stabilisation through interactions with part of the protein dimerization domain (subdomain i; both highlighted in green) (25). The consensus NAC domain sequence obtained from this alignment is shown below the conservation scores (first line).…”