Fig 1.
Stability of NadR is increased by small molecule ligands.
(A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). All DSC profiles are representative of triplicate experiments.
Table 1.
Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments.
Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments.
Table 2.
Data collection and refinement statistics for NadR structures.
Fig 2.
The crystal structure of NadR in complex with 4-HPA.
(A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). For simplicity, secondary structure elements are labelled for chain B only. Red dashes show hypothetical positions of chain B residues 88–90 that were not modeled due to lack of electron density. (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix [38]. The map is contoured at 1σ and the figure was prepared with a density mesh carve factor of 1.7, using Pymol (www.pymol.org).
Fig 3.
Analysis of the NadR dimer interface.
(A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. The WT and most of the mutants show a single elution peak with an absorbance maximum at 17.5 min. Only the mutation L130K has a noteworthy effect on the oligomeric state, inducing a second peak with a longer retention time and a second peak maximum at 18.6 min. To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. The curves plotted correspond to Absorbance Units (mAU) at 280nm wavelength (green), light scattering (red), and refractive index (blue).
Fig 4.
Atomic details of NadR/HPA interactions.
A) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA. Green and blue ribbons depict NadR chains A and B, respectively. 4-HPA is shown in yellow sticks, with oxygen atoms in red. A water molecule is shown by the red sphere. H-bonds up to 3.6Å are shown as dashed lines. The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum [44] and verified manually). Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36. Bond distances for interacting polar atoms are provided in Table 3. Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36.
Table 3.
List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds.
Fig 5.
Structural differences of NadR in ligand-bound or free forms.
(A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two ‘mirrored’ arrows. The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation.
Fig 6.
NMR spectra of NadR in the presence and absence of 4-HPA.
(A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). The spectra acquired at 10°C are excluded from panel A for simplicity.
Fig 7.
Overall apo- and holo-NadR structures are similar.
(A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. Here, larger differences are observed in the α6 helices (top).
Fig 8.
Structural comparisons of NadR and modelling of interactions with DNA.
(A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). The α4 helices aligned closely, Cα rmsd 0.2Å for 14 residues. (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. Dashes indicate the Ala77 Cα atoms, in the most highly shifted region of the ‘non-fixed’ α4 helix. (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. The proteins share ~30% amino acid sequence identity. For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove.
Fig 9.
Structure-based point mutations shed light on ligand-induced regulation of NadR.
Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control).
Fig 10.
NadR shows a ligand binding site distinct from other MarR homologues.
(A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation.