Three-Dimensional Structure of Full-Length NtrX, an Unusual Member of the NtrC Family of Response Regulators

Abstract

Bacteria sense and adapt to environmental changes using two-component systems. These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator (RR), which finally modulates the transcription of target genes. The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response. NtrX is a modular protein with REC, AAA+, and DNA-binding domains, an architecture that classifies it among the NtrC subfamily of RRs. However, it lacks the signature GAFTGA motif that is essential for activating transcription by the mechanism proposed for canonical members of this subfamily. In this article, we present the first crystal structure of full-length NtrX, which is also the first structure of a full-length NtrC-like RR with all the domains solved, showing that the protein is structurally similar to other members of the subfamily. We also report that NtrX binds nucleotides and the structures of the protein bound to ATP and ADP. Despite binding ATP, NtrX does not have ATPase activity and does not form oligomers in response to phosphorylation or nucleotide binding. We also identify a nucleotide sequence recognized by NtrX that allows it to bind to a promoter region that regulates its own transcription and to establish a negative feedback mechanism to modulate its expression. Overall, this article provides a detailed description of the NtrX RR and supports that it functions by a mechanism different to classical NtrC-like RRs.