Structural Basis of Redox-Sensing Transcriptional Repressor Rex with Cofactor NAD+ and Operator DNA

Kang Hwa Jeong,Jae Young Lee,Hyun Jin Lee,Young Woo Park

doi:10.3390/ijms23031578

Abstract

The transcriptional repressor Rex plays important roles in regulating the expression of respiratory genes by sensing the reduction–oxidation (redox) state according to the intracellular NAD+/NADH balance. Previously, we reported on crystal structures of apo, NAD+-bound, and NADH-bound forms of Rex from Thermotoga maritima to analyze the structural basis of transcriptional regulation depending on either NAD+ or NADH binding. In this study, the crystal structure of Rex in ternary complex with NAD+ and operator DNA revealed that the N-terminal domain of Rex, including the helix-turn-helix motif, forms extensive contacts with DNA in addition to DNA sequence specificity. Structural comparison of the Rex in apo, NAD+-bound, NADH-bound, and ternary complex forms provides a comprehensive picture of transcriptional regulation in the Rex. These data demonstrate that the conformational change in Rex when binding with the reduced NADH or oxidized NAD+ determines operator DNA binding. The movement of the N-terminal domains toward the operator DNA was blocked upon binding of NADH ligand molecules. The structural results provide insights into the molecular mechanism of Rex binding with operator DNA and cofactor NAD+/NADH, which is conserved among Rex family repressors. Structural analysis of Rex from T. maritima also supports the previous hypothesis about the NAD+/NADH-specific transcriptional regulation mechanism of Rex homologues.

Highlights

Bacteria monitor and respond to environmental oxygen levels and intracellular reduction–oxidation states by controlling the transcription of genes involved in respiratory pathways [1]
Rex monitors the redox state according to the intracellular Nicotinamide adenine dinucleotide (NAD)+ /NADH ratio and controls the Rex regulon involved in various pathways, including cellular respiration, fermentation, oxidative stress response, and biofilm formation [2,5,8–10]
The relation between the metabolic pathway under Rex control and pathogenesis has been suggested in some bacteria, such as Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Streptococcus suis serotype 2 (SS2) [11–14]

Summary

Introduction

Bacteria monitor and respond to environmental oxygen levels and intracellular reduction–. Oxidation (redox) states by controlling the transcription of genes involved in respiratory pathways [1]. Intracellular redox reactions are involved in many biological processes, such as cellular respiration, enzymatic reactions, and energy metabolism [2–4]. NAD+ /NADH ratio can be a critical indicator of the intracellular redox state corresponding to environmental oxygen levels. Rex monitors the redox state according to the intracellular NAD+ /NADH ratio and controls the Rex regulon involved in various pathways, including cellular respiration, fermentation, oxidative stress response, and biofilm formation [2,5,8–10]. The Rex regulon in Thermotoga maritima has been identified in 12 operons that include 40 genes involved in central carbohydrate metabolism and hydrogen production [15]. Rex DNA operator sites in T. maritima have been experimentally demonstrated by in vitro binding assays, including whether

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