Discovery of a Nitric Oxide-Responsive Protein in Arabidopsis thaliana

Abstract

In plants, much like in animals, nitric oxide (NO) has been established as an important gaseous signaling molecule. However, contrary to animal systems, NO-sensitive or NO-responsive proteins that bind NO in the form of a sensor or participating in redox reactions have remained elusive. Here, we applied a search term constructed based on conserved and functionally annotated amino acids at the centers of Heme Nitric Oxide/Oxygen (H-NOX) domains in annotated and experimentally-tested gas-binding proteins from lower and higher eukaryotes, in order to identify candidate NO-binding proteins in Arabidopsis thaliana. The selection of candidate NO-binding proteins identified from the motif search was supported by structural modeling. This approach identified AtLRB3 (At4g01160), a member of the Light Response Bric-a-Brac/Tramtrack/Broad Complex (BTB) family, as a candidate NO-binding protein. AtLRB3 was heterologously expressed and purified, and then tested for NO-response. Spectroscopic data confirmed that AtLRB3 contains a histidine-ligated heme cofactor and importantly, the addition of NO to AtLRB3 yielded absorption characteristics reminiscent of canonical H-NOX proteins. Furthermore, substitution of the heme iron-coordinating histidine at the H-NOX center with a leucine strongly impaired the NO-response. Our finding therefore established AtLRB3 as a NO-interacting protein and future characterizations will focus on resolving the nature of this response.

Computational Identification of Functional Centers in Complex Proteins: A Step-by-Step Guide With Examples

“…exist in complex proteins with other primary domains and varied architectures (Figure 4B). Mutations to either the "H" or "Y" residues impaired heme binding and concomitantly also the ability to sense NO (Figure 4C) (Zarban et al, 2019;Wong et al, 2020). In general, candidates retrieved from the motif search that display the described structural features, will The domain organizations of ABA-interacting centers identified using a motif-based approach from Arabidopsis thaliana (At), are illustrated as 2-dimensional bars and aligned at their corresponding H-NOX centers.…”

Nitric oxide function during oxygen deprivation in physiological and stress processes

“…It therefore remains to be explained how NO positively influences the substrate degradation of the PCO branch of the PRT6 N-degron pathway. NO itself could affect the activities of enzymatic components of the pathway ( Zarban et al , 2019 ) or alter the cellular energy balance in an indirect manner ( Armstrong et al , 2019 ).…”

The plant N‐degron pathways of ubiquitin‐mediated proteolysis

“…However, in vitro assessment of the activity of PCOs (Weits et al ; White et al ; White et al ), and analysis of a reconstituted plant PCO‐regulated system for Nt‐Cys degradation in yeast (Puerta et al ) demonstrated that oxidation leading to degradation is possible in the absence of NO ( Saccharomyces cerevisiae has not been shown to produce endogenous NO under standard growth conditions). It, therefore, remains to be discovered how NO exerts a positive influence on degradation of Nt‐Cys substrates in a cellular context, that may include an effect on the activities of enzymic components of the pathway (for example it was recently shown that PRT6 contains a putative NO‐binding domain (Zarban et al )) or indirectly through alterations of cellular energy balance (Greenway and Armstrong ).…”