Crystal Structure of Haemophilus influenzae NadR Protein

Haemophilus influenzae NadR protein (hiNadR) has been shown to be a bifunctional enzyme possessing both NMN adenylytransferase (NMNAT; EC 2.7.7.1) and ribosylnicotinamide kinase (RNK; EC 2.7.1.22) activities. Its function is essential for the growth and survival of H. influenzae and thus may present a new highly specific anti-infectious drug target. We have solved the crystal structure ofhiNadR complexed with NAD using the selenomethionine MAD phasing method. The structure reveals the presence of two distinct domains. The N-terminal domain that hosts the NMNAT activity is closely related to archaeal NMNAT, whereas the C-terminal domain, which has been experimentally demonstrated to possess ribosylnicotinamide kinase activity, is structurally similar to yeast thymidylate kinase and several other P-loop-containing kinases. There appears to be no cross-talk between the two active sites. The bound NAD at the active site of the NMNAT domain reveals several critical interactions between NAD and the protein. There is also a second non-active-site NAD molecule associated with the C-terminal RNK domain that adopts a highly folded conformation with the nicotinamide ring stacking over the adenine base. Whereas the RNK domain of the hiNadR structure presented here is the first structural characterization of a ribosylnicotinamide kinase from any organism, the NMNAT domain ofhiNadR defines yet another member of the pyridine nucleotide adenylyltransferase family. Haemophilus influenzae NadR protein (hiNadR) has been shown to be a bifunctional enzyme possessing both NMN adenylytransferase (NMNAT; EC 2.7.7.1) and ribosylnicotinamide kinase (RNK; EC 2.7.1.22) activities. Its function is essential for the growth and survival of H. influenzae and thus may present a new highly specific anti-infectious drug target. We have solved the crystal structure ofhiNadR complexed with NAD using the selenomethionine MAD phasing method. The structure reveals the presence of two distinct domains. The N-terminal domain that hosts the NMNAT activity is closely related to archaeal NMNAT, whereas the C-terminal domain, which has been experimentally demonstrated to possess ribosylnicotinamide kinase activity, is structurally similar to yeast thymidylate kinase and several other P-loop-containing kinases. There appears to be no cross-talk between the two active sites. The bound NAD at the active site of the NMNAT domain reveals several critical interactions between NAD and the protein. There is also a second non-active-site NAD molecule associated with the C-terminal RNK domain that adopts a highly folded conformation with the nicotinamide ring stacking over the adenine base. Whereas the RNK domain of the hiNadR structure presented here is the first structural characterization of a ribosylnicotinamide kinase from any organism, the NMNAT domain ofhiNadR defines yet another member of the pyridine nucleotide adenylyltransferase family. nicotinamide mononucleotide nicotinic acid mononucleotide ribosylnicotinamide kinase, NMNAT, NMN adenylyltransferase NaMN adenylyltransferase thymidylate kinase H. influenzae NadR protein pyridine nucleotide adenylyltransferase phosphopantetheine adenylyltransferase Haemophilus influenzae, a small nonmotile Gram-negative bacterium, resides in the upper respiratory mucosa in humans and causes otitis media and respiratory tract infections, mostly in children. This organism lacks almost all of the enzymes necessary for the synthesis of NAD (1Evans N.M. Smith D.D. Wicken A.J. J. Med. Microbiol. 1974; 7: 359-365Crossref PubMed Scopus (65) Google Scholar, 2Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G.G. FitzHugh W. Fields C.A. Gocayne J.D. et al.Science. 1995; 269: 496-512Crossref PubMed Scopus (4702) Google Scholar), and it requires the presence of the so-called V-factors (NADP, NAD, NMN,1 orN-ribosylnicotinamide) in the growth medium (3Gingrich W. Schlenk F. J. Bacteriol. 1944; 47: 535-550Crossref PubMed Google Scholar, 4Shifrine M. Biberstein E.L. Nature. 1960; 187: 623Crossref Scopus (12) Google Scholar). Phosphorylated V-factors (NADP, NAD, and NMN) are degraded by recently identified extracellular and periplasmic hydrolases (5Kemmer G. Reilly T.J. Schmidt-Brauns J. Zlotnik G.W. Green B.A. Fiske M.J. Herbert M. Kraiss A. Schlor S. Smith A. Reidl J. J. Bacteriol. 2001; 183: 3974-3981Crossref PubMed Scopus (66) Google Scholar) toN-ribosylnicotinamide, which is likely to be the only V-factor transported across the inner membrane as an ultimate NAD precursor (6Cynamon M.H. Sorg T.B. Patapow A. J. Gen. Microbiol. 1988; 134: 2789-2799PubMed Google Scholar). Two enzymatic steps are required to convert ribosylnicotinamide to NAD in the cytoplasm: a ribosylnicotinamide kinase (RNK; EC 2.7.1.22) to catalyze the phosphorylation of nicotinamide riboside to produce NMN and a NMN adenylyltransferase (NMNAT; EC 2.7.7.1) to link NMN and the AMP moiety of ATP to generate NAD. Whereas the gene encoding RNK has not been identified in any organism until very recently, 2O. V. Kurnasov, B. M. Polanuyer, S. Ananta, R. Sloutsky, A. Tam, S. Y. Gerdes, A. L. Osterman, submitted for publication. several NMNATs and functionally related NaMNATs (EC. 2.7.7.18) have been characterized at the molecular level from many species during last few years. We will use pyridine nucleotide adenylyltransferase (PNAT) as a generic name for both NMNAT and NaMNAT as well as for enzymes with dual specificities, such as in the case of human NMN/NaMNAT. The three-dimensional structures of PNATs and their complexes with substrate/product have been solved from several sources, includingMethanococcus jannaschii (7D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar), Methanothermobacter thermautotrophicum (8Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), Escherichia coli (9Zhang H. Zhou T. Kurnasov O. Cheek S. Grishin N.V. Osterman A. Structure. 2002; 10: 69-79Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar),Bacillus subtilis (10Olland A.M. Underwood K.W. Czerwinski R.M., Lo, M.C. Aulabaugh A. Bard J. Stahl M.L. Somers W.S. Sullivan F.X. Chopra R. J. Biol. Chem. 2002; 277: 3698-3707Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and humans (11Garavaglia S. D'Angelo I. Emanuelli M. Carnevali F. Pierella F. Magni G. Rizzi M. J. Biol. Chem. 2002; 277: 8524-8530Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Werner E. Ziegler M. Lerner F. Schweiger M. Heinemann U. FEBS Lett. 2002; 516: 239-244Crossref PubMed Scopus (36) Google Scholar, 13Zhou T. Kurnasov O. Tomchick D.R. Binns D.D. Grishin N.V. Marquez V.E. Osterman A.L. Zhang H. J. Biol. Chem. 2002; 277: 13148-13154Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Since PNATs catalyze the indispensable central step in NAD biosynthesis and recycling, genes encoding these activities have been found in almost all organisms with completely sequenced genomes. In H. influenzae, the ortholog of bacterial NaMNAT (NadD gene product) is lacking, and the alternative housekeeping PNAT function is encoded in the N-terminal domain of the bifunctional NadR protein,2 which shares 54% identity to the E. coli and Salmonella typhimurium NadR proteins. The multifunctional NadR proteins contain the signature nucleotidyltransferase (H/T)IGH motif, and its NMNAT activity has been confirmed experimentally in the E. coli enzyme (14Raffaelli N. Lorenzi T. Mariani P.L. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1999; 181: 5509-5511Crossref PubMed Google Scholar) and, more recently, in H. influenzae and S. typhimurium as well.2 In addition to the NMNAT activity, NadR in S. typhimurium and E. colicontain a DNA-binding helix-turn-helix motif N-terminal to the NMNAT domain. The S. typhimurium NadR has long been demonstrated to be a NAD-dependent repressor for the transcription of genes involved in both de novo biosynthesis (nadBand nadA) and niacin salvage (pncB) (15Foster J.W. Holley-Guthrie E.A. Warren F. Mol. Gen. Genet. 1987; 208: 279-287Crossref PubMed Scopus (13) Google Scholar, 16Penfound T. Foster J.W. J. Bacteriol. 1999; 181: 648-655Crossref PubMed Google Scholar). It was also suggested that NadR may interact with an integral membrane transporter PnuC protein and directly participate in the uptake of exogenous NAD precursors (17Zhu N. Olivera B.M. Roth J.R. J. Bacteriol. 1991; 173: 1311-1320Crossref PubMed Google Scholar). H. influenzae NadR lacks the helix-turn-helix DNA binding domain, which correlates with the absence of any genes of de novo NAD biosynthesis or niacin salvage in this organism. Recently, we have predicted and experimentally verified that previously uncharacterized RNK activity resides within the C-terminal domain of NadR. The essentiality of NadR for the growth and survival of H. influenzae has also been established.2 Here we report the crystal structure of H. influenzae NadR protein (hiNadR) complexed with NAD. This structure reveals that whereas the NMNAT domain is mostly similar to the archaeal NMNAT, the C-terminal RNK domain is structurally similar to the yeast thymidylate kinase and several other P-loop-containing nucleotide and nucleoside kinases. The cloning, expression, and purification of hiNadR protein is described elsewhere.2 Briefly, the gene encoding residues 52–421 of sequence NADR_HAEIN (gi‖1171638) was PCR-amplified from H. influenzae genomic DNA and was cloned into a pET-derived vector containing a T7 promoter, His6 tag, and TEV protease cleavage site (gift from Dr. Meg Philips, UT Southwestern Medical Center). The resulting plasmid was transformed into the E. coli strain BL21(DE3) (Invitrogen) for expression. The overexpressed hiNadR protein was first purified with a Ni2+-nitrilotriacetic acid-agarose (Qiagen) column, followed by a Superdex 200 gel filtration column (AmershamBiosciences). The selenomethionine hiNadR was expressed in the met− auxotrophic strain B834(DE3) (Novagen), grown in minimal medium supplemented with selenomethionine and other nutrients (18Doublie S. Methods Enzymol. 1997; 276: 523-530Crossref PubMed Scopus (798) Google Scholar), and purified using the same procedure as the native protein. The hiNadR crystals were grown at 20 °C using the hanging drop vapor diffusion method. 10 mg/ml hiNadR in 100 mm Hepes, pH 7.2, 0.3 m NaCl, and 1 mm dithiothreitol was first incubated with 3 mm of NAD and 3 mm of ATP and then mixed with an equal volume of the reservoir solution (0.1m MES, pH 6.0, 1.0 m ammonia sulfate) and equilibrated against the reservoir. Large diamond-shaped crystals appeared after 2–7 days. These hiNadR co-crystals belong to a hexagonal crystal system with cell dimensions a =b = 106.9 Å, c = 174.9 Å.α = β = 90°, andγ = 120°. The exact space group of these crystals was later determined to be P6422. There is onehiNadR molecule in the asymmetric unit. These crystals diffract to 3.0-Å resolution on a rotating anode x-ray generator and about 2.7 Å at synchrotron. The selenomethionine hiNadR crystals were grown at similar conditions and are of same quality as the native crystals. For data collection at 100 K, crystals were transferred stepwise to a cryoprotection solution containing all components of the reservoir and additional 30% ethylene glycol. A 3.3-Å resolution multiwavelength anomalous diffraction data set and a 2.9-Å resolution native data set were collected at beamline X12B (National Synchrotron Light Source, Brookhaven National Laboratory). All diffraction data were processed and scaled with the HKL2000 package (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The statistics for all data sets are listed in Table I.Table ICrystal data and refinement statistics of hiNadRSeMet hiNadRNAD complexPeakInflectionRemoteWavelength (A)0.97850.97930.96110.9840Resolution (A)30–3.330–3.330–3.330–2.9Total observations372,894373,956373,285183,179Unique reflections94779446947113,327Completeness (outer shell)100% (100%)100% (99.7%)100% (100%)98.0% (97.3%)Rsym (outer shell)a0.104 (0.587)0.086 (0.504)0.098 (0.569)0.041 (0.409)I/ς9.010.09.017.6Figure of merit0.64Refinement Rwork1-bRwork = Σhkl‖Fo −Fc‖/Σhkl‖Fo‖, where Fo and Fc are the observed and calculated structure factors, respectively.23.6% Rfree1-cRfree is the R factor calculated for a randomly selected 10% of the reflections that were omitted from the refinement.29.8% Protein atoms2842 Hetero groups2NAD, 5SO4 r.m.s.deviation bond length0.011A r.m.s.deviation bond angle1.56° Average B factors (A) Protein atoms78.5 LigandsNAD81.4SO4120.9 Ramachandran Plot Most favored region (%)79.7 Additionally allowed region (%)14.8 Generously allowed region (%)5.2 Disallowed region (%)0.31-aRsym = Σhkl[(Σj‖Ij − ‖)/Σj‖Ij‖].1-b Rwork = Σhkl‖Fo −Fc‖/Σhkl‖Fo‖, where Fo and Fc are the observed and calculated structure factors, respectively.1-c Rfree is the R factor calculated for a randomly selected 10% of the reflections that were omitted from the refinement. Open table in a new tab 1-aRsym = Σhkl[(Σj‖Ij − ‖)/Σj‖Ij‖]. The initial phases of thehiNadR crystal structure were solved by the multiwavelength anomalous diffraction phasing method using SOLVE (20Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. Cryst. Chem. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Six selenium sites out of a total of eight were located, and the resulting phases have a figure of merit of 0.64. Density modification was performed with RESOLVE (21Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. Cryst. Chem. 2000; 56: 965-972Crossref PubMed Scopus (1636) Google Scholar), which resulted in a clearly interpretable electron density map. The hiNadR polypeptide chain was manually built into this map using O (22Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The refinement of hiNadR structure was carried out using CNS (23Bru¨nger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. Cryst. Chem. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) with simulated annealing protocol. The first round of refinement resulted in an Rwork of 32.5% andRfree of 38.2%. Several rounds of manual rebuilding and refinements improved the R factors to a value of 27.3% for the Rwork and 33.9% for theRfree. At this point, densities for two NAD molecules became evident: one at the active site of the NMNAT domain and the other associated with the C-terminal RNK domain but not in its active site. The starting coordinates for the NAD at the active site of the NMNAT domain were derived from the coordinates of NAD in theM. thermoautotrophicum NMNAT·NAD complex structure (8Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The second NAD molecule adopts a highly compact conformation with the nicotinamide ring stacking over the adenine base. The model for this NAD molecule was built manually to fit the density, and its conformation was optimized with CNS (23Bru¨nger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. Cryst. Chem. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Several putative sulfate molecules were also included in the subsequent rounds of refinement. Further rounds of refinement with these ligands included in the model resulted in a decrease in Rwork to 23.6% andRfree to 29.8%. The current refinement statistics are listed in Table I. Sedimentation equilibrium experiments were performed with a Beckman XL-I analytical ultracentrifuge and using both scanning absorption and Rayleigh interference optics. Wavelengths used were 295 nm in the absorbance mode and 675 nm for Rayleigh interference. We used an An-60Ti rotor with double sector cells of 1.2-cm path length at 4 °C for all experiments. The sedimentation equilibrium was carried out at 9,000 r.p.m. and 13,000 r.p.m. Two different concentrations of the protein (1.2 and 0.4 mg/ml) dissolved in 100 mm Hepes buffer, pH 7.2, containing 0.3 m NaCl, 2 mmdithiothreitol, 1 mm EDTA were used in the study. All centrifugation data were analyzed with the OptimaTM data analysis software. The overexpressed recombinant hiNadR protein contains residues 52–421 of the NADR_HAEIN sequence (total of 370 amino acids), which includes the NMNAT and RNK domains. We chose to carry out crystallographic study of this segment, since the multiple alignment of all NadR homologs has shown that the reliable sequence similarity between hiNadR and other NadR homologs starts only from the NMNAT domain. Overexpression of the extended version of hiNadR containing an N-terminal His6 tag fused at position Met15results in two protein products, suggesting an alternative translation initiation at Met52.2 This possibility is further strengthened by the fact that it is more consistent with the predicted starts of NadR homologs in several other species, such asMycobacterium tuberculosis (gi‖92220269), Nostoc punctiforme (gi‖91112614), and Lactococcus lactis(gi‖15673969). The N-terminally truncated version ofhiNadR (residues 52–421) possesses full NMNAT and RNK activities and is functionally indistinguishable from the full-length protein.2 The current crystal structure model of hiNadR contains residues 57–345 and 357–411 (among the expressed segment 52–421). The first 5 residues along with the N-terminal His6 tag region, a loop containing residues 346–356, and the last 10 residues at the C terminus are disordered in the crystal structure. Additionally, there are two NAD and five sulfate molecules that are included in the model. Although 3 mm ATP was present in the crystallization solution, no ATP molecule could be located in the density map; therefore, the current structure must be discussed as ahiNadR·NAD complex. The structure of the hiNadR monomer revealed the presence of two distinct domains connected by a loop (Fig.1). The N-terminal NMNAT domain (residues 52–224) adopts a Rossmann-like three-layered α/β/α fold with the central five-stranded parallel β-sheet having the strand order 32145. The structure of this domain closely resembles archaeal NMNATs (7D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar, 8Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) with r.m.s. deviation of 2.6 Å for 147 C-α atoms when superimposed with the M. janaschii NMNAT (Protein Data Bank code 1f9a). The C-terminal RNK domain (residues 225–421) also adopts a three-layered α/β/α fold with a five-stranded central parallel β-sheet of strand order 23145. Note that the strand order in the central β-sheet of the C-terminal RNK domain differs from that in the NMNAT domain. Search of the protein structure data base (PDB) using DALI (24Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1291) Google Scholar) revealed that the RNK domain is structurally similar to several typical P-loop kinases, such as yeast thymidylate kinase (Protein Data Bank code 3tmk, Z score of 14.8, r.m.s. deviation of 3.1 Å over 164 C-α atoms), Bacillus stearothermophilus adenylate kinase (Protein Data Bank code 1zin,Z score of 10.3, r.m.s. deviation of 3.2 Å over 136 C-α atoms), and herpes simplex virus type 1 thymidine kinase (Protein Data Bank code 1qhi, Z score of 8.3, r.m.s. deviation of 3.3 Å over 146 C-α atoms). Superpositions of hiNadR with archaeal NMNAT and yeast thymidylate kinase are shown in Fig.2.Figure 2Comparison of hiNadR with archaeal NMNAT and TMK. a, superposition of the C-α trace of hiNadR NMNAT domain (dark blue) with mthNMNAT (1EJ2) (dark red). b, superposition of the C-α trace ofhiNadR RNK domain (blue) with yeast TMK (3TMK) (red). c, structure-based alignment ofhiNadR with mthNMNAT and yeast TMK. The color and letter coding of secondary structural elements in the sequence is the same as in Fig. 1. The (H/T)IGH motif of NMNAT, the P-loop (Walker-A motif), Walker-B motif, and the "LID" of the RNK domain areboxed. The sequences in regions that are not superimposed are in lowercase type, and the disordered segments are initalic lowercase type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although there is only onehiNadR molecule in the asymmetric unit, inspection of crystal packing indicated that four symmetry-related hiNadR monomers are tightly associated, and the protein is likely to exist as tetramer (Fig. 3). To investigate the oligomeric status of the protein in solution, we performed analytical ultracentrifugation studies at two different hiNadR concentrations (1.2 mg/ml and 0.4 mg/ml) and two different speeds (9,000 and 13,000 r.p.m.). The apparent molecular masses obtained are 170 kDa (1.2 mg/ml) and 150 kDa (0.4 mg/ml), which are somewhat lower than the calculated molecular mass of hiNadR tetramer (180 kDa), indicating the presence of lower molecular weight species. The data collected at the two different protein concentrations and two different speeds allow us to fit either a dimer/tetramer or a monomer/tetramer model for hiNadR, both of which yielded dissociation constants in the low micromolar range (KD of 1–10 μm for the dimer-tetramer model, data not shown). This indicates that tetramer is the major oligomerization species of hiNadR in solution at higher than micromolar concentrations. The hiNadR tetramer in crystal displays a 222 symmetry. Two distinct types of interfaces between individual monomers are observed in the tetramer. Monomers A and B (or C and D) form a "handshake-like" association that buries 1384 Å2 of surface area per monomer at the interface. The C-terminal RNK domain from one monomer sits snugly in a hinge grove between the two domains of the second monomer, making extensive contacts with both domains of the second monomer (Fig. 3a). The other type of dimer interface is between monomers A and D (or B and C) and is less extensive, burying a surface area of only 894 Å2 per monomer. The resulting tetramer has the appearance of a closed barrel with the hinge between the two domains of each monomer located at the middle of the barrel. This creates a cavity at the center of the barrel with a diameter of ∼18 Å. A narrow channel extends from this central cavity and runs through the whole length of the tetramer, exiting between the less densely packed dimer (Fig. 3b). The electrostatic potential mapped on the surface of the tetramer revealed a strikingly positively charged cluster in the middle and along the central channel of the barrel (Fig. 3c). Amino acids Lys215, Lys219, Arg222, His296, His298, and Lys299 from each of the four monomers contribute to this positively charged patch, which is located at the interface between monomers in the tetramer. It was thus not surprising that several sulfate molecules were found in this region. The active sites of both NMNAT and RNK domains are located on the outside of the barrel, facing away from the central cavity (Fig.3). Four non-active-site-bound NAD molecules associated with the C-terminal RNK domains of the four hiNadR monomers were found aggregated inside the central cavity of the tetramer (Fig. 3). Each of these NAD molecules adopts a highly folded conformation with the nicotinamide ring stacked over the adenine base in a nearly parallel fashion (Fig.4a). The distance between the adenine C-6 and nicotinamide C-2 atoms, which has been used in the literature as a general measure of compactness of the bound NAD(P) (25Bell C.E. Yeates T.O. Eisenberg D. Protein Sci. 1997; 6: 2084-2096Crossref PubMed Scopus (64) Google Scholar), is 4.2 Å for this NAD molecule. Whereas most of the enzyme-bound NAD molecules adopt extended conformation such as the one bound in the active site of NMNAT domain of hiNadR, there is only one other example of enzyme-bound NAD that adopts a similarly highly folded conformation. In the crystal structure of flavin reductase P complexed with its inhibitor NAD, the NAD molecule adopts a compact conformation with a distance between adenine C-6 and nicotinamide C-2 atoms of 3.9 Å (26Tanner J.J., Tu, S.C. Barbour L.J. Barnes C.L. Krause K.L. Protein Sci. 1999; 8: 1725-1732Crossref PubMed Scopus (40) Google Scholar). However, the conformation of the flavin reductase P-bound NAD differs drastically from the second NAD in the hiNadR·NAD complex structure (Fig. 4b), reflecting the remarkable flexibility of NAD molecule. Notably, nuclear magnetic resonance studies and molecular dynamics simulation have indicated that, in contrast to the protein-bound forms, NAD in aqueous and some organic solutions adopts compact folded conformations in which the distance between the nicotinamide and adenine rings is 4–5 Å (27Sarma R.H. Ross V. Kaplan N.O. Biochemistry. 1968; 7: 3052-3062Crossref PubMed Scopus (69) Google Scholar, 28Smith P.E. Tanner J.J. J. Am. Chem. Soc. 1999; 121: 8637-8644Crossref Scopus (27) Google Scholar). There are several specific interactions between the non-active-site NAD molecule andhiNadR tetramer. The adenine and nicotinamide rings are sandwiched between the side chains of Tyr292 and Trp256 (Fig. 4a). The pyrophosphate moiety of the NAD points outward and is in contact with the Lys126side chain of an adjacent monomer in the tetramer. Additionally, the oxygen of the nicotinamide carboxyamide group is within the hydrogen bonding range of the main chain amide of Trp256, and the N-6 of the adenine appears to be in contact with main chain carbonyl of Tyr289. Although we cannot rule out the possibility that binding of the non-active-site NAD molecule is a crystallization artifact, the specific interactions between this NAD molecule andhiNadR tetramer indicate that it may have biological implications. In E. coli and S. typhimurium, the NadR protein represses three genes involved in NAD biosynthesis in a NAD-dependent manner (15Foster J.W. Holley-Guthrie E.A. Warren F. Mol. Gen. Genet. 1987; 208: 279-287Crossref PubMed Scopus (13) Google Scholar, 16Penfound T. Foster J.W. J. Bacteriol. 1999; 181: 648-655Crossref PubMed Google Scholar). It is possible that the NAD molecule as an effector may bind to a site different from the active site of NMNAT domain. Binding of the NAD effector at this regulatory site presumably induces a conformational change and leads to the binding of NadR to operator DNA. It is thus tempting to speculate that the second nonactive NAD binding site in hiNadR may represent the NAD effector binding site in E. coli andS. typhimurium NadR. Although hiNadR do not possess repressor function, the sequence identities between the NMNAT and RNK domains of hiNadR and that of E. coliNadR and S. typhimurium NadR are high (∼54%). Therefore,hiNadR must have retained many structural and functional features of E. coli NadR and S. typhimurium NadR. More biochemical and structural studies are needed to investigate this hypothesis. The NAD molecule that was found in the active site of the N-terminal NMNAT domain of hiNadR adopts an extended conformation similar to that bound to the mthNMNAT (8Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) (Fig.5). The adenine nucleotide binding site includes the (H/T)IGH motif, the preceding loop between the (H/T)IGH motif, and the end of the first β-stand, which is also highly conserved, the N terminus of α-helix E that contains the ISSTXXR motif, and a loop connecting β-strande and helix E that interacts with the adenine base. A sulfate molecule found in this active site is superimposable with that in the mthNMNAT·NAD complex (8Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and appears to occupy the site that would normally be occupied by the β and γ phosphates of ATP, as was shown in the structure of the M. janaschii NMNAT·ATP complex (7D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar). Similar to the archaeal NMNATs, in hiNadR the loop connecting strand c to helix C (residues 138–149) plays a central role in the binding of the nicotinamide portion of the substrate. We therefore term this loop the "nicotinamide recognition loop." The exocyclic carboxyamide group of the nicotinamide interacts with several main chain groups from this nicotinamide recognition loop (Fig. 5); the carboxyl group lies in close proximity to the main chain amide of Trp149, whereas the amide group is close to the main chain carbonyls of Pro143 and Ser144. Trp152, which is aligned with Trp84 in MthNMNAT, appears to play the same role in forming a stacking interaction with