Structural Analysis of Redox-sensing Transcriptional Repressor Rex from Thermotoga maritima

Abstract

The cellular redox state of organisms continues to fluctuate during the metabolism. All organisms have various sensors that help detect and adapt to changes in the redox state. Nicotinamide adenine dinucleotides (NAD+/NADH), which are involved in various cellular metabolic activities as cofactors, have been revealed as the key molecules sensing the intra-cellular redox state. The Rex family members are well conserved transcriptional repressors that regulate the expression of respiratory genes by sensing the redox state according to the intra-cellular NAD+/NADH balance. Herein, we reported crystal structures of apo and NAD+- and NADH-bound forms of Rex from Thermotoga maritima to analyse the structural basis of transcriptional regulation depending on either NAD+ or NADH binding. The different orientation of the reduced nicotinamide group to helix α9 caused the rearrangement of N-terminal DNA binding domain, thus resulting in closed form of Rex to dissociate from cognate DNA. The structural data of Rex from T. maritima also support the previous redox-sensing mechanism models of Rex homologues.

Hydrogenases and H2 metabolism in sulfate-reducing bacteria of the Desulfovibrio genus

“…Rex orthologues were identified in proteobacteria in the delta subdivision, the desulfovibrionales species (Ravcheev et al, 2012). In D. fructosovorans, a rex gene (locus-tag DesfrDRAFT_2623) encodes a 24.8 kDa protein that shows 47 % identity with the Rex repressor from T. maritima whose crystallographic structure has been recently solved (Park, Jang, Joo, & Lee, 2018). In T. saccharolyticum, a Rex putative binding site in genes coding for the Ech and Hfs hydrogenases was identified suggesting that Rex may play an important role in T. saccharolyticum H 2 metabolism (T. Zheng, Lanahan, Lynd, & Olson, 2018).…”

How Bacterial Redox Sensors Transmit Redox Signals via Structural Changes

“…Various structural studies of Rex in complex with NADH, NAD + , its cognate DNA or in the apo state revealed how Rex translates the NADH:NAD+ ratio into a structural conformational change to control the binding affinity between the protein and DNA [ 95 , 96 , 97 , 98 , 99 ] ( Table 1 ). The structural organization and overall structural characteristics of Rex in various organisms are very similar to each other.…”

“…These extensive hydrophobic interactions stabilize the homodimeric structure of Rex. In the structures of Rex in complex with NAD+ or NADH, the NAD+/NADH-binding pocket can be divided into two parts: (i) a P-loop containing a hydrophobic pocket where the ADP moiety binds, and (ii) a pocket near the dimeric interface formed by the N-terminus of the domain-swapped α-helix and the α5/β4 loop of the opposing subunit, where nicotinamide and the N-ribose moiety bind [ 95 , 96 , 97 , 98 ]. In both NAD+- and NADH-bound structures, the ADP moiety is fixed stably in the pocket and remains essentially the same, whereas the nicotinamide and N-ribose moieties show different binding patterns ( Figure 8 ).…”

“…In both NAD+- and NADH-bound structures, the ADP moiety is fixed stably in the pocket and remains essentially the same, whereas the nicotinamide and N-ribose moieties show different binding patterns ( Figure 8 ). In the structure of the NAD+–Rex complex, the carboxamide nitrogen atom and N-ribose group for hydrogen bonds with the highly conserved Ala and Tyr residues of the other subunit, respectively (Ala 94 in T. aquaticus Rex and Ala96 in T. maritima Rex; Tyr98 in T. aquaticus Rex and Tyr100 in T. maritima Rex) [ 95 , 98 ]. In comparison, the reduced nicotinamide of NADH is flipped when the carboxamide group forms hydrogen bonds with the main chain atoms of the N-terminal residues in the domain-swapped α-helix (Phe189 in T. aquaticus Ile190, 192 in T. maritima Rex) [ 96 , 98 ].…”

“…In the structure of the NAD+–Rex complex, the carboxamide nitrogen atom and N-ribose group for hydrogen bonds with the highly conserved Ala and Tyr residues of the other subunit, respectively (Ala 94 in T. aquaticus Rex and Ala96 in T. maritima Rex; Tyr98 in T. aquaticus Rex and Tyr100 in T. maritima Rex) [ 95 , 98 ]. In comparison, the reduced nicotinamide of NADH is flipped when the carboxamide group forms hydrogen bonds with the main chain atoms of the N-terminal residues in the domain-swapped α-helix (Phe189 in T. aquaticus Ile190, 192 in T. maritima Rex) [ 96 , 98 ]. As the domain-swapped helix interacts with the N-terminal DNA-binding domain of the other subunit, the structural rearrangement in the NAD+/NADH-binding pocket affects the DNA binding domain, ultimately altering the conformation regulating the DNA-binding properties ( Figure 8 ).…”