Figure 3. Structural and functional insights into the binding of MsNrtR to its cognate DNA target.
(A) Structural analysis of the predicted DNA-binding motif through structural modeling of M. smegmatis NrtR (http://swissmodel.espasy.org/). The image shows the superposition of M. smegmatis NrtR with the Shewanella oneidensis NrtR-DNA complex (PDB: 3GZ6). MsNrtR is highlighted in cyan and soNrtR is indicated in gray. Double-stranded DNA is denoted by two orange lines. (B) Structural prediction of the critical DNA-binding residues of the M. smegmatis NrtR. The six residues (D167, T169, N170, R173, K179, and R196) that are implicated in direct or indirect contact with cognate DNA are labeled in red. (C) Electrophoretic mobility shift assay (EMSA)-based visualization of the interaction of MsNrtR with the nrtR probe. The amount of NrtR protein incubated with the DNA probe is in each lane is (left to right): 0, 0.5, 1, 2, 5, 10, 20, and 40 pmol. (D) Surface plasmon resonance (SPR) measurements of M. smegmatis NrtR binding to the nrtR promoter. NrtR protein at various concentrations (typically 15.625–500 nM) were injected over the immobilized DNA probe comprising of the NrtR palindrome of nrtR gene. KD, kd/ka, ka, association constant; kd, dissociation constant; RU, response units.
Figure 3—figure supplement 1. Characterization of the M. smegmatis NrtR.
(A) Fast protein liquid chromatography (FPLC) profile for the M. smegmatis NrtR. The protein sample was analyzed in a Superdex 75 column run by AKTA Purifier. The inset shows the SDS-PAGE gel of the recombinant 6 × His NrtR. The apparent mass of the recombinant NrtR of M. smegmatis is ~30 kDa. Note that M. smegmatis NrtR can easily form dimers in solution. OD280, optical density at 280 nm; AU, absorbance units. (B) Chemical cross-linking analyses of the purified NrtR protein. The samples were separated by 12% SDS-PAGE following the chemical cross-linking assays. The ethylene glycol bissuccinimidylsuccinate (EGS) cross-linking reagent was added at the concentrations shown at the top. M, protein standard marker. FadR is a known dimeric protein that was used as a positive control. (C) Mass spectrometry identification of the recombinant M. smegmatis NrtR. The peptide fragments that were matched are shown in bold and underlined (87.33%).
Figure 3—figure supplement 2. The M. smegmatis NrtR cannot bind to an unrelated DNA, the vprA probe.
The vprA probe (56 bp), an unrelated DNA (Supplementary file 2), acts here as a negative control. Above the gel image, the minus ‘–' sign indicates no addition of NrtR protein, and the triangle on right refers to the addition of increasingly levels of NrtR protein (0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 pmols). Although it binds the nrtR probe (57 bp, Supplementary file 2), the NrtR of M. smegmatis can’t interact with the Vibrio cholerae vprA probe, an unrelated DNA. This verifies that the physical interplay between NrtR and the nadADC operon depends on specific binding to the nrtR probe located in the intergenic region (Figures 1A and 3C).
Figure 3—figure supplement 3. Mapping of NrtR-DNA interactions.
(A) 12% SDS-PAGE profile of the purified NrtR and its mutants. (B) Enlarged view of the predicted DNA-binding residues of M. smegmatis NrtR. (C) Binding ability of wild-type NrtR to the nrtR probe evaluated by EMSA. (D) Binding ability of the D167A point mutant of NrtR to the nrtR probe evaluated by EMSA. (E) Loss of binding of the T169A point mutant of NrtR to the nrtR probe evaluated by EMSA. (F) Loss of binding of the N170A point mutant of NrtR to the nrtR probe evaluated by EMSA. (G) Loss of binding of the R173A point mutant of NrtR to the nrtR probe evaluated by EMSA. (H) Loss of binding of the R196A point mutant of NrtR to the nrtR probe evaluated by EMSA. (I) Impairment of binding of the K179A point mutant of NrtR to the nrtR probe evaluated by EMSA. Minus ‘--' signs denote no addition of NrtR protein, and the triangles to the right of these signs refer to the addition of increasing levels of NrtR protein (2, 5, and 10 pmols).
Figure 3—figure supplement 4. Identification of the ADPR pyrophosphatase activity of the M. smegmatis NrtR and its mutant (Q54E, K58E and D60G).
(A) Multiple sequence alignments of the Nudix domain of M. smegmatis NrtR and its homologous proteins. The multiple alignment was conducted using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the final output was expressed through processing by the ESPript 2.2 program (Robert and Gouet, 2014). Identical residues are in white letters with red background, similar residues are in red letters with white background, varied residues are in black letters, and dots represent gaps. GenBank accession numbers and organisms are as follows: VCA0097, Vibrio cholerae; Tlet0901, Thermotoga lettinagae; ACIAD0962, Acinetobacter sp.; MSMEG_3198, M. smegmatis; MCON0143, Methanosatea concilli; Mthe1467, Methanosatea themophila; DR0192, Deinococcus radiodurans; SCO1767, Streptomyces coelicotor; and SCO5817, Streptomyces coelicotor. The Nudix motif signature is shown below the sequences as GX5EX7REUXEEXGU (U: Ile, Leu, or Val). (B) The mutations (Q54E, K58E and D60G) present in M. smegmatis nrtR revealed by direct DNA sequencing. The mutation of Q54E and K58E and D60G at DNA level denotes the genotype of the mutant protein. (C) The SDS-PAGE gel of the purified NrtR and its mutant (Q54E and K58E and D60G). (D) Schematic diagram of the hydrolyzation of ADP-ribose by ADP-ribose pyrophosphatase which belongs to the Nudix hydrolase superfamily. (E) Identification of the reaction product upon hydrolysis of ADP-ribose by NrtR. Null, before the addition of the recombinant NrtR or its mutant; MsNrtR, after incubation with M. smegmatis NrtR for 30 min; MsNrtR Mutant, after incubation with NrtRQ54E&K58E&D60G.
Figure 3—figure supplement 5. The NAD metabolite ADP-ribose interferes with the binding of MsNrtR to cognate DNA. The image shows the electrophoretic mobility of the nrtR (0.2 μM) probe incubated alone (lane 1) or with purified MsNrtR (5 μM) in the absence (lane 2) and in the presence of 50 (lane 3) and 75 (lane 4) mM of ADP-ribose. The volume of the EMSA reaction system is 20 μl.
