Structural analysis and insight into effector binding of the niacin-responsive repressor NiaR from Bacillus halodurans

The niacin-responsive repressor, NiaR, is transcriptional repressor of certain nicotinamide adenine dinucleotide (NAD) biosynthetic genes in response to an increase in niacin levels. NAD is a vital molecule involved in various cellular redox reactions as an electron donor or electron acceptor. The NiaR family is conserved broadly in the Bacillus/Clostridium group, as well as in the Fusobacteria and Thermotogales lineages. The NiaR structure consists of two domains: an N-terminal DNA-binding domain, and a C-terminal regulation domain containing a metal-binding site. In this paper, we report the crystal structures of apo and niacin-bound forms of NiaR from Bacillus halodurans (BhNiaR). The analysis of metal-binding and niacin-binding sites through the apo and niacin-bound structures is described. Each N- and C-terminal domain structure of BhNiaR is almost identical with NiaR from Thermotoga maritima, but the overall domain arrangement is quite different. A zinc ion is fully occupied in each subunit with well-conserved residues in the C-terminal domain. Niacin is also located at a hydrophobic pocket near the zinc ion in the C-terminal domain.

Nicotinamide adenine dinucleotide (NAD) is an essential molecule that plays an important role as both electron donor and electron acceptor in cellular metabolism [1][2][3] . The NAD molecule participates in many biochemical transformations, including several reactive centers. Nearly 88% of enzymes that rely on NAD are involved in catabolic pathways. Approximately half of these enzymes are related to the use of carbohydrates, and the remaining enzymes contributes to the oxidation of other substrates such as carboxylic acid, amino acid and fatty acid 4 . In many bacteria, NAD + is synthesized through two metabolic processes, the de novo and salvage pathways. In the de novo pathway, NAD + is synthesized from l-aspartate or l-tryptophan by the consecutive action of NadBACDE (l-aspartate oxidase, quinolinate synthetase, quinolinate phosphor-rybosyl-transferase, nicotinate mononucleotide adenylyl-transferase, and NAD synthetase) 5 . The salvage pathway is a recycling system producing NAD + from exogenous sources such as niacin (also called nicotinic acid or vitamin B 3 ), nicotinamide, nicotinamide riboside or endogenous breakdown products of NAD + by NAD + -consuming enzymes 1,6 . Despite some variations in the early steps of the two pathways, the final step of NAD + synthesis from nicotinic acid adenine dinucleotide to NAD + is highly conserved 7 .
Both de novo and salvage pathways for NAD + biosynthesis are regulated by several transcription factors, the NAD + -dependent repressor (NadR), the Nudix-related transcriptional regulators (NrtR), and the niacin-responsive repressor (NiaR). NadR in Enterobacteria acts as a repressor of certain NAD + biosynthetic genes responding to NAD + , nicotinic acid phosphoribosyltransferase (pncB) in the salvage pathway and nadBA involved in the de novo pathway 4 . In addition, NadR has enzymatic activities as nicotinamide riboside kinase and nicotinamide mononucleotide adenylyltransferase, which function in NAD + synthesis [8][9][10][11] . The NrtR acts as a regulator of NAD + biosynthesis genes and salvage genes such as, nadBACDE, nicotinamidase (pncA), pncB, and nicotinamide riboside transporter pnuC 12,13 . NiaR, previously called YrxA, was found mainly in Bacillus/Clostridium species, as well as Fusobacteria and Thermotogales 6,14 . NiaR represses transcription of certain NAD + biosynthetic genes in response to elevated niacin levels. NiaR also represses the expression of the transporter for niacin uptake (niaX, niaY, and niaP), pncA and pncB in the salvage pathway, and two operons, cysteine desulfurases (nifS) and nadBCA, sharing a promoter region, in the de novo pathway 15 . The transcriptomic analysis of Streptococcus pneumoniae NiaR showed that NiaR regulated gene expressions, including niaX, pnuC, and nadC, in response to niacin 16 . The biochemical and bioinformatic analyses provided the mechanism of NiaR and the metabolism of NAD + synthesis in Bacillus subtilis 6,17 . The NiaR regulon constitutes a transcriptional regulation system of NAD + synthesis in several groups of Gram-positive bacteria 6 .
Previous studies suggested that niacin binds to the 3-histidine domain (3H domain) of NiaR and NiaR belongs to a family of de novo NAD + synthesis pathway regulators 14 . The NiaR from Thermotoga maritima (TmNiaR) displayed metal binding and dimeric structure. The NiaR protein is composed of two domains. The N-terminal domain is a DNA-binding domain containing a helix-turn-helix motif and the C-terminal domain is a regulatory 3H domain in which three histidines are well conserved. A nickel ion is occupied in the C-terminal domain of TmNiaR and the function of this domain was assumed to involve binding to small molecules 18 .
We determined the crystal structures of BhNiaR with apo and niacin-bound forms. A zinc ion was included in both the apo and niacin-bound models. The presence of the zinc was confirmed by an inductively coupled plasma mass spectrometer (ICP-MS). The structural data of BhNiaR provides details of metal binding and ligand interaction. DNA binding affinity of BhNiaR depending on niacin or metal ions was assessed by electrophoretic mobility shift assay (EMSA).

Results and discussion
Model building and quality. The apo crystal structure of BhNiaR was refined to 2.0 Å resolution with crystallographic R work and R free values of 20.18% and 22.78%, respectively. Although the refined model contained a subunit in an asymmetric unit composed of 173 residues with a zinc ion and 129 water molecules, it could be generated to a homo-dimeric structure by a symmetric subunit. The N-terminal domain is composed of residues 7-70 and C-terminal domain is composed of residues 71-179. The N-terminal region (residues 1-6), the linker www.nature.com/scientificreports/ loop region (residues 68-74) connecting the N-and C-terminal domains, and the internal region (residues 137-147) in the C-terminal domain were poorly ordered due to lack of electron-density maps. The niacin-bound BhNiaR crystals were grown by co-crystallization with 2 mM niacin. The structure of the niacin-bound form was determined at 1.8 Å resolution and refined to crystallographic R work and R free of 19.43% and 24.26%, respectively. The refined model of niacin-bound BhNiaR also contained a subunit with interpretable residues 7-179 in an asymmetric unit. The N-terminus (residues 1-6), the linker loop region (residues 68-74), and the internal region (residues 137-145) in the C-terminal domain were poorly ordered. A niacin molecule was located near a zinc ion and surrounded by a hydrophobic pocket close to the dimeric interface. All refined models for BhNiaR indicated favored or allowed regions of the Ramachandran plot. The detailed refinement statistics are summarized in Table 1.
Overall structures of the B. halodurans NiaR. Each BhNiaR structure was composed of two domains, an N-terminal DNA-binding domain (residues 1-70) and a C-terminal dimerization/substrate-binding 3H domain (residues 71-179), connected by a flexible loop (residues 66-75) (Fig. 1b). The N-terminal domain contained three α-helices and a pair of anti-parallel β-stands, which form a helix-turn-helix DNA-binding motif. The helix α2 and α3 of the helix-turn-helix motif was generally known as the site of DNA recognition and was well conserved in NiaR homologues. The C-terminal domain contained three α-helices and four anti-parallel β-stands, forming layered α-helices and β-stands of the 3H domain with an order of secondary structural elements in βαββαβα, which is a feature of histidine-containing phosphor-carrier (HPr)-like proteins. Three highly conserved histidines (His83, His150, and His152) and a glutamate (Glu91) form a binding site for the zinc ion and niacin. A dimeric BhNiaR structure was generated by a crystallographic twofold symmetry ( Fig. 1c and Fig. S1). The buried surface area of the dimer is about 1990 Å 2 , approximately 19% of the monomer surface area (PDBePISA protein-protein interaction server: http://www.ebi.ac.uk/msd-srv/prot_int/). The dimeric structure is stabilized by the hydrogen bonds and hydrophobic interactions centered around a faced β5 strand forming an anti-parallel A dimer is also consistent with results from size-exclusion chromatography with multi-angle light scattering (SEC-MALS) (Fig. S2). The niacin-bound structure of BhNiaR is almost identical to the apo structure with a root-mean-square deviation (r.m.s.d.) value of 0.2 Å. The most noticeable structural difference was the loop region between the N-terminal and C-terminal domains (Fig. S3).
Metal-binding site. Previous work suggested Ni 2+ , Cu 2+ , or Zn 2+ as the most probable metals in the TmNiaR structure based on coordination geometry, anomalous difference Fourier peaks, refinement results, and similar ligating residues in the Metalloprotein Database 14,19 . A zinc ion fully occupied the metal-binding site in the apo and niacin-bound forms of BhNiaR, which was confirmed by ICP-MS and also was verified by a peak on an omit map at the counter levels even at 9σ (Fig. S4). In addition, a nickel ion was partially occupied with BhNiaR (~ 5%). The zinc ion is located between layered α-helices and a β-sheet of the C-terminal domain. The metal-binding site appeared to be fully occupied, with the temperature factors for a zinc ion being 43.95 Å 2 and 22.81 Å 2 in the apo and niacin-bound forms, respectively.
In the apo structure of TmNiaR, it has been reported that four conserved residues (His79, Glu87, His146, and His148) and a water molecule coordinated with a nickel ion in octahedral geometry 14 . However, in the apo structure of BhNiaR, three residues (Glu91, His150, and His152), corresponding to TmNiaR residues (Glu87, His146, and His148), and a water molecule coordinated with a zinc ion in tetrahedral geometry: Glu91 Oε1, His150 Nε1, His152 Nε2, and Wat108 O. The His83 residue was not involved directly in metal coordination, with a distance of 3.8 Å. In addition, in the niacin-bound structure, the coordinated water molecule was replaced by a carboxyl oxygen (O2) of the niacin molecule and the His83 residue formed a hydrogen bond with a second carboxyl oxygen (O1) of the niacin molecule (Fig. 2).

Niacin-binding site. A niacin molecule is bound to the C-terminal domain of
BhNiaR near the metalbinding site. The pyridine moiety of niacin is buried in a hydrophobic pocket surrounded by highly conserved residues (Met88, Leu92, Val105, Leu119, Phe130, Met134, Leu141, Leu142, and Tyr112′) (Fig. 2d,e). The carboxyl group of niacin is located at the position of the water molecule near the zinc ion in apo form. Therefore, the O1 and O2 atoms of the niacin carboxyl group coordinated with His83 Nε2 and the zinc ion, respectively. Coordination of the carboxyl group with the zinc ion and the hydrogen bond with the His83 residue contributed to the stability of niacin binding, as did van der Waals attractions involving nonpolar residues (Met88, Leu92, Phe130, Met134, Leu141, Leu142, and Tyr112′).

DNA binding affinity.
NiaR binds to the promoter region containing the consensus sequence in the presence of niacin 6 . The DNA motif of NiaR proteins is classified into two distinct types. Type I contains the consensus sequence, TGT-N 4 -ACA, characterized in the Fusobacteria lineage and the Bacillus/Clostridium groups. Type II contains consensus sequence, ACA-N 5 -TGT, found in the Thermotogales lineage. DNA-binding affinities of type I and II DNA motifs have been investigated by EMSA using NiaR proteins from B. subtilis and T. maritima 6 .
To confirm the DNA-binding affinity of BhNiaR with its predicted cognate DNA segments, we performed EMSA using the type I DNA, 34-bp oligonucleotide (5′-CTC ATT TACAT ATGT CTTG ACA TCT ATA TTT ACA -3′) containing the nifS-nadB promoter region. The DNA was mixed with various amount of BhNiaR either in the absence or presence of niacin. BhNiaR bound to its cognate DNA with a dissociation constant (Kd) of ~ 0.5 µM in the presence of niacin, whereas the DNA-binding ability was decreased in the absence of niacin (Fig. 3a). BhNiaR moderately reduced DNA-binding to non-cognate sequences either with or without niacin (Fig. 3b). These results are consistent with homologous NiaR proteins from B. subtilis and T. maritima 6 .
To further assess whether the metal ion affects the DNA-binding affinity of BhNiaR, a BhNiaR H152A mutant was generated by site-directed mutagenesis resulting in metal-free BhNiaR, which was confirmed by an ICP-MS. The BhNiaR H152A mutant showed a severe defect in DNA-binding affinity regardless of the presence or absence of niacin (Fig. 3c). These results indicate that the metal ion, as well as niacin, plays an important role in DNA binding. TmNiaR formed a dimer through the C-terminal domains alone. The apo and niacin-bound forms of BhNiaR are almost identical. These structural arrangements made it difficult to bind DNA because the N-terminal DNAbinding domains were located at both sides of the C-terminal domains and the helix-turn-helix motifs were not exposed for binding to DNA. The distance between the recognition helices α3 (at residue Arg38) in the TmNiaR dimer was 40 Å, whereas in BhNiaR (at residue Arg40) the distance was 57 Å (Fig. S6). It seems that both the apo and niacin-bound BhNiaR structures display non-DNA bound conformations because of the crystal packing and interactions between the N-and C-terminal domains of each subunit in the dimer. In order to obtain the biologically relevant structure for BhNiaR, the DNA-bound form is required.

Conclusion
The crystal structures of the niacin-responsive repressor, BhNiaR, which negatively regulates the expression of genes involved in the NAD + synthesis pathway, were determined to be the apo and niacin-bound forms. Our results show the coordination of a zinc ion and the binding of the effector molecule, niacin, in the NiaR structure.
In the apo form, the zinc ion is coordinated by three residues (Glu91, His150, and His152) conserved in HPr-like proteins with a water molecule in tetrahedral geometry. Another conserved residue, His83, is not involved in zinc ion coordination but is involved in niacin binding. The niacin bound to the C-terminal domain of BhNiaR is located near a zinc ion, His83, and a well-conserved hydrophobic pocket. Both the niacin and the metal ion cooperatively affect DNA-binding affinity of BhNiaR, and the EMSA results show that the absence of a metal ion causes a significant defect in the DNA-binding affinity of BhNiaR. The frozen pellets were placed in a lysis solution containing buffer A (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10% (v/v) glycerol) and 1 mM phenylmethylsulfonyl fluoride, homogenized by an ultrasonic processor (Sonics Vibra Cell, VCX750) and separated from the insoluble fraction by centrifugation at 31,000g for 60 min at 277 K. The recombinant BhNiaR protein in soluble fraction was loaded on a nickel-charged His-trap immobilized metal affinity chromatography (IMAC) column (GE Healthcare, UK) and eluted with buffer B (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10% (v/v) glycerol, and 300 mM imidazole). Enzymatic removal of the His-tag from the BhNiaR proteins was achieved by overnight incubation with PreScission protease. The uncleaved His-tagged proteins were removed from the native proteins by applying to a nickel-charged His-trap IMAC. The next purification step was size-exclusion chromatography on a Superdex-75 column (GE healthcare) with elution buffer (20 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 5% (v/v) glycerol, 1 mM dithiothreitol and 1 mM MgCl 2 ). The purified BhNiaR protein was concentrated to 15 mg ml −1 using Centricon YM-10 (Millipore).
Site-directed mutagenesis. Site-directed mutagenesis of BhNiaR was performed by PCR using a recombinant plasmid containing the wild-type niaR gene as a template. Two complementary primers were designed such that the encoded His152 (CAC) was replaced by GCA-encoded alanine (Forward primer: 5′-GTG TTC ATATG GCA ACG TTA GAAGC-3′, and Reverse primer: 5′-GCT TCT AACGT TGC CAT ATG AACAC-3′). PCR products were treated with DpnI for digestion of preexisting recombinant plasmids containing the wild-type niaR gene. PCR products were introduced into E. coli DH5α, and then the mutant plasmids were isolated and Structure determination and refinement. The crystal structures of both forms of BhNiaR were solved by molecular replacement using the program PHASER MR from the CCP4 program suite 23 using the TmNiaR structure (PDB code 1J5Y) as a search model 14 . The structures of apo and niacin-bound forms built by COOT 24 and were refined by PHENIX. All refined structures of BhNiaR were evaluated by MolProbity 25 and have been deposited in the Protein Data Bank. The figure was generated using the computer program PyMol (Version 2.3.2, Schrödinger, LLC, https ://www.pymol .org) 26 . The refinement statistics of BhNiaR are shown in Table 1.
Electrophoretic mobility shift assay. To confirm the DNA-binding affinity of BhNiaR, 34-bp doublestranded DNA (Forward : 5′-CTC ATT TAC ATA TGT CTT GAC ATC TATA TTT ACA -3′ and Reverse : 5′-TGT AAA TA TAG ATG TCA AGA CAT ATG TAAAT GAG -3′) containing own promoter region (nifS-nadB) was prepared by Oligo Synthesis (Macrogen, Republic of Korea). The reaction mixture was made by mixing the native BhNiaR (0-3 µM) and the reaction buffer (20 mM Tris, pH 8.0, 100 mM KCl, 1 mM MgCl 2 , 1 mM dithiothreitol, and 5% (v/v) glycerol with or without 1 mM niacin) prior to mixing the double-stranded DNA (80 nM). All reaction mixtures were incubated for 30 min at room temperature, after which each mixture was loaded on a pre-chilled 6% native polyacrylamide gel in 0.5 X Tris-Borate, pH 8.3. Electrophoresis was performed at 277 K and the gel was visualized using SYBR Green (ThermoFisher Scientific).

Size-exclusion chromatography with multi-angle light scattering (SEC-MALS). SEC-MALS
experiments were performed using a fast protein liquid chromatography system (GE Healthcare) connected to a Wyatt DAWN Heleos II instrument and a Wyatt Optilab T-Rex differential refractometer. A Superdex-200 10/300 GL (GE Healthcare) gel filtration column pre-equilibrated with 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1 mM DTT was normalized using ovalbumin (40 kDA) as the protein standard. The BhNiaR protein was injected (10-12 mg ml −1 , 0.1 ml) at a flow rate of 0.5 ml min− 1 . The data were evaluated using the Zimm model for static light-scattering data fitting and represented using an EASI graph with a UV peak in the ASTRA V software (Wyatt).