A New Crystal Structure of Deoxyhypusine Synthase Reveals the Configuration of the Active Enzyme and of an Enzyme·NAD·Inhibitor Ternary Complex
2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês
10.1074/jbc.m404095200
ISSN1083-351X
AutoresTimothy C. Umland, Edith C. Wolff, Myung Hee Park, David R. Davies,
Tópico(s)Porphyrin Metabolism and Disorders
ResumoDeoxyhypusine synthase catalyzes the first step in the two-step post-translational synthesis of hypusine, which is uniquely present in eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase and eIF5A are conserved throughout the eukaryotic kingdom, and both are essential for cell proliferation and survival. A previous study (Liao, D. I., Wolff, E. C., Park, M. H., and Davies, D. R. (1998) Structure 6, 23–32) of human deoxyhypusine synthase revealed four active sites of the homotetrameric enzyme located within deep tunnels. These Form I crystals were obtained under conditions of acidic pH and high ionic strength and likely contain an inactive enzyme. Each active-site entrance is blocked by a ball-and-chain motif composed of a region of extended structure capped by a two-turn α-helix. We report here at 2.2 Å a new Form II crystal of the deoxyhypusine synthase:NAD holoenzyme grown at low ionic strength and pH 8.0, near the optimal pH for enzymatic activity. The ball-and-chain motif could not be detected in the electron density, suggesting that it swings freely and thus it no longer obstructs the active-site entrance. The deoxyhypusine synthase competitive inhibitor N1-guanyl-1,7-diaminoheptane (GC7)is observed bound within the putative active site of the enzyme in the new crystal form (Form II) after exposure to the inhibitor. This first structure of a deoxyhypusine synthase·NAD·inhibitor ternary complex under physiological conditions now provides a structural context to discuss the results of previous biochemical investigations of the deoxyhypusine synthase reaction mechanism. This structure also provides a basis for the development of improved inhibitors and antiproliferative agents. Deoxyhypusine synthase catalyzes the first step in the two-step post-translational synthesis of hypusine, which is uniquely present in eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase and eIF5A are conserved throughout the eukaryotic kingdom, and both are essential for cell proliferation and survival. A previous study (Liao, D. I., Wolff, E. C., Park, M. H., and Davies, D. R. (1998) Structure 6, 23–32) of human deoxyhypusine synthase revealed four active sites of the homotetrameric enzyme located within deep tunnels. These Form I crystals were obtained under conditions of acidic pH and high ionic strength and likely contain an inactive enzyme. Each active-site entrance is blocked by a ball-and-chain motif composed of a region of extended structure capped by a two-turn α-helix. We report here at 2.2 Å a new Form II crystal of the deoxyhypusine synthase:NAD holoenzyme grown at low ionic strength and pH 8.0, near the optimal pH for enzymatic activity. The ball-and-chain motif could not be detected in the electron density, suggesting that it swings freely and thus it no longer obstructs the active-site entrance. The deoxyhypusine synthase competitive inhibitor N1-guanyl-1,7-diaminoheptane (GC7)is observed bound within the putative active site of the enzyme in the new crystal form (Form II) after exposure to the inhibitor. This first structure of a deoxyhypusine synthase·NAD·inhibitor ternary complex under physiological conditions now provides a structural context to discuss the results of previous biochemical investigations of the deoxyhypusine synthase reaction mechanism. This structure also provides a basis for the development of improved inhibitors and antiproliferative agents. Deoxyhypusine synthase (DHS) 1The abbreviations used are: DHS, deoxyhypusine synthase; eIF5A, eukaryotic initiation factor 5A; GC7, N1-guanyl-1,7-diaminoheptane; HSS, homospermidine synthase; MPD, 2-methyl-2,4-pentanediol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. catalyzes the first of two steps in the post-translational modification of a single lysine residue of the precursor eukaryotic initiation factor 5A (eIF5A(Lys)) to the unique amino acid hypusine (Nϵ-(4-aminobutyl-2-hydroxyl)lysine) (1Park M.H. Lee Y.B. Joe Y.A. Biol. Signals. 1997; 6: 115-123Crossref PubMed Scopus (193) Google Scholar). DHS converts lysine to deoxyhypusine (Nϵ-(4-aminobutyl)lysine) by a NAD-dependent transfer of the butylamine moiety of spermidine. In the second step of the modification, deoxyhypusine hydroxylase catalyzes the hydroxylation of deoxyhypusine, yielding hypusine. A highly interesting aspect of this post-translational modification is the extreme specificity of it. The only known endogenous occurrence of hypusine is a single modified lysine in the mature form of eIF5A. The importance of this post-translational modification is underscored by the conservation of DHS and eIF5A in all eukaryotes and archaea (2Gordon E.D. Mora R. Meredith S.C. Lee C. Lindquist S.L. J. Biol. Chem. 1987; 262: 16585-16589Abstract Full Text PDF PubMed Google Scholar, 3Bartig D. Lemkemeier K. Frank J. Lottspeich F. Klink F. Eur. J. Biochem. 1992; 204: 751-758Crossref PubMed Scopus (59) Google Scholar, 4Chen K.Y. Liu A.Y. Biol. Signals. 1997; 6: 105-109Crossref PubMed Scopus (102) Google Scholar) and the requirement of active DHS and eIF5A for cell proliferation. eIF5A(Lys) is likely modified directly following translation, as there is little or no accumulation of eIF5A(Lys) in cultured cells (1Park M.H. Lee Y.B. Joe Y.A. Biol. Signals. 1997; 6: 115-123Crossref PubMed Scopus (193) Google Scholar). Specific inhibitors of DHS or deoxyhypusine hydroxylase result in cellular growth arrest and alter tumor cell proliferation and differentiation (5Hanauske-Abel H.M. Park M.H. Hanauske A.R. Popowicz A.M. Lalande M. Folk J.E. Biochim. Biophys. Acta. 1994; 1221: 115-124Crossref PubMed Scopus (132) Google Scholar, 6Park M.H. Wolff E.C. Lee Y.B. Folk J.E. J. Biol. Chem. 1994; 269: 27827-27832Abstract Full Text PDF PubMed Google Scholar, 7Chen Z.P. Yan Y.P. Ding Q.J. Knapp S. Potenza J.A. Schugar H.J. Chen K.Y. Cancer Lett. 1996; 105: 233-239Crossref PubMed Scopus (58) Google Scholar, 8Jansson B.P. Malandrin L. Johansson H.E. J. Bacteriol. 2000; 182: 1158-1161Crossref PubMed Scopus (56) Google Scholar, 9Nishimura K. Ohki Y. Fukuchi-Shimogori T. Sakata K. Saiga K. Beppu T. Shirahata A. Kashiwagi K. Igarashi K. Biochem. J. 2002; 363: 761-768Crossref PubMed Google Scholar). Furthermore, the hypusine residue of eIF5A has been reported to be critical for in vitro RNA binding (10Xu A. Chen K.Y. J. Biol. Chem. 2001; 276: 2555-2561Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and the interaction with exportin 4 (11Lipowsky G. Bischoff F.R. Schwarzmaier P. Kraft R. Kostka S. Hartmann E. Kutay U. Gorlich D. EMBO J. 2000; 19: 4362-4371Crossref PubMed Scopus (161) Google Scholar). The multistep reaction catalyzed by DHS requires two substrates, spermidine and eIF5A(Lys), plus the cofactor NAD. It is proposed to proceed in four steps, employing two imine intermediates and a transient hydride (12Wolff E.C. Park M.H. Folk J.E. J. Biol. Chem. 1990; 265: 4793-4799Abstract Full Text PDF PubMed Google Scholar, 13Wolff E.C. Folk J.E. Park M.H. J. Biol. Chem. 1997; 272: 15865-15871Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Wolff E.C. Wolff J. Park M.H. J. Biol. Chem. 2000; 275: 9170-9177Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar): 1) spermidine + NAD → dehydrospermidine + NADH, 2) dehydrospermidine + enzyme → enzyme(Lys)-imine intermediate + diaminopropane, 3) enzyme(Lys)-imine intermediate + eIF5A(Lys) → eIF5A(Lys)-imine intermediate + enzyme, and 4) eIF5A-imine intermediate + NADH → eIF5A-deoxyhypusine intermediate + NAD. Evidence for each of these steps has been reported based on biochemical and fluorescence studies (1Park M.H. Lee Y.B. Joe Y.A. Biol. Signals. 1997; 6: 115-123Crossref PubMed Scopus (193) Google Scholar, 12Wolff E.C. Park M.H. Folk J.E. J. Biol. Chem. 1990; 265: 4793-4799Abstract Full Text PDF PubMed Google Scholar, 13Wolff E.C. Folk J.E. Park M.H. J. Biol. Chem. 1997; 272: 15865-15871Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Wolff E.C. Wolff J. Park M.H. J. Biol. Chem. 2000; 275: 9170-9177Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The first two steps can proceed in the absence of eIF5A(Lys) yielding diaminopropane and Δ1-pyrroline. The structure of human DHS complexed with NAD was reported previously by this laboratory (15Liao D.I. Wolff E.C. Park M.H. Davies D.R. Structure. 1998; 6: 23-32Abstract Full Text Full Text PDF PubMed Google Scholar) and will be referred to as the Form I crystal. These DHS crystals were grown in the presence of high ionic strength (1.7 m sodium/potassium phosphate) and at an acidic pH (∼4.5) at which DHS is inactive in vitro. This structure revealed that the entrance to each of the four tunnel-like active sites of the DHS homotetramer was sealed from the external milieu by a ball-and-chain motif composed of a two-turn α-helix (residues Ala11–Val17) attached to an extended loop. This compact helix obstructs the active-site entrance with the helical axis approximately perpendicular to the pathway down the tunnel. Furthermore, in the previous study the competitive DHS inhibitor 1,7-diaminoheptane was included in the crystallization mother liquor, but it was not observed in the refined crystal structure. The failure to observe bound 1,7-diaminoheptane was attributed to the obstructed active-site entryway, the acidic pH, and high ionic strength. The inaccessibility of the DHS active site suggests that the Form I structure is unlikely to be the fully active form of the enzyme. This observation prompted continued screening for alternate crystallization conditions. We report here a new crystal form (Form II) of the human DHS:NAD holoenzyme obtained under significantly different crystallization conditions, i.e. low ionic strength and pH 8.0. Importantly, in vitro assays have demonstrated that DHS is enzymatically active at this pH (16Murphey R.J. Gerner E.W. J. Biol. Chem. 1987; 262: 15033-15036Abstract Full Text PDF PubMed Google Scholar, 17Lee Y.B. Joe Y.A. Wolff E.C. Dimitriadis E.K. Park M.H. Biochem. J. 1999; 340: 273-281Crossref PubMed Scopus (33) Google Scholar, 18Lee C.H. Park M.H. Biochem. J. 2000; 352: 851-857Crossref PubMed Google Scholar). In this new Form II crystal, the ball-and-chain motif has undergone a dramatic structural change allowing accessibility to the active sites. This structure is likely to be more representative of the active form of DHS. Furthermore, DHS Form II crystals soaked in the potent DHS competitive inhibitor, N1-guanyl-1,7-diaminoheptane (GC7) (19Jakus J. Wolff E.C. Park M.H. Folk J.E. J. Biol. Chem. 1993; 268: 13151-13159Abstract Full Text PDF PubMed Google Scholar), yielded the crystal structure of the first DHS·NAD·inhibitor ternary complex. The observation of the spermidine analog GC7 bound in the DHS active site provides a new structural perspective to discuss mutational data, the proposed mode of spermidine binding, and the reaction mechanism. Protein Purification—Recombinant human DHS was overexpressed in Escherichia coli and purified as described previously (13Wolff E.C. Folk J.E. Park M.H. J. Biol. Chem. 1997; 272: 15865-15871Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Liao D.I. Wolff E.C. Park M.H. Davies D.R. Structure. 1998; 6: 23-32Abstract Full Text Full Text PDF PubMed Google Scholar, 20Joe Y.A. Wolff E.C. Park M.H. J. Biol. Chem. 1995; 270: 22386-22392Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Crystallization—The new crystal form (Form II) of DHS was initially observed while using the Hampton Research 2-methyl-2,4-pentanediol (MPD) grid screen. Optimized crystallization conditions were 4.5–7.0 mg/ml DHS in 3 mm NAD, 50 mm Tris·HCl, pH 7.8, and 100 mm KCl combined in a 1:1 ratio with the reservoir solution (64–70% (v/v) MPD and 0.1 m Tris·HCl, pH 8.0). A typical initial droplet size was 3.5 μl. The crystallization droplets were equilibrated via vapor diffusion against 1 ml of the reservoir solution at room temperature. Well formed but small crystals (∼0.1-mm maximum each dimension) grew within several days. Selected crystals were soaked in a stabilizing solution containing 64% (v/v) MPD, 2 mm GC7, 0.11 m Tris·HCl, pH 8.0, for several days to form the inhibitor-enzyme complex. GC7 was synthesized as described previously (19Jakus J. Wolff E.C. Park M.H. Folk J.E. J. Biol. Chem. 1993; 268: 13151-13159Abstract Full Text PDF PubMed Google Scholar). Form II crystals were washed well with a stabilizing solution devoid of DHS, dissolved in water, and subjected to MALDI-TOF and SDS-PAGE analysis (data not shown). This analysis confirmed the presence of only full-length DHS in the Form II crystals. Fresh Form I crystals were grown following the published protocol (15Liao D.I. Wolff E.C. Park M.H. Davies D.R. Structure. 1998; 6: 23-32Abstract Full Text Full Text PDF PubMed Google Scholar) excluding 1,7-diaminoheptane. Crystals of both types were grown using DHS from the same sample aliquot. Form II crystals contain two DHS monomers/asymmetric unit, as opposed to the single monomer/asymmetric unit observed within the Form I crystal. X-ray Diffraction Data Collection—Crystals were flash-cooled using liquid propane. No additional cryoprotectant was required because of the high concentration of MPD. Data collection was conducted at 95 K. Initial diffraction data sets were obtained using CuKα radiation produced from a Rigaku RU-200 rotating anode and a R-Axis IIC detector. Final data sets were collected at Beamline X-9B at the National Synchrotron Light Source using monochromatic x-rays with a wavelength of 0.98 Å and an ADSC Quantum 4 CCD detector. The data were processed with the HKL package (21Otwinowski Z. Minor W. Carter Jr., C.W. Sweet R.M. Methods in Enzymology. 276. Academic Press, Orlando1997: 307-326Google Scholar). Please see Table I for data collection and refinement statistics.Table IData collection and refinement statisticsDHS·NAD (Form I - this study)DHS·NAD (Form II)DHS·NAD·GC7 (Form II)Space groupP4212P3221P3221Cell dimensions (Å)a = b108.6104.8104.8c69.8159.7159.3Resolution (Å)2.12.23.0No. of observations10430919623968751No. of unique reflections223845096320171Completeness (%)aNumbers in parenthesis refer to statistics for highest resolution shell89.5 (67.4)99.9 (99.3)96.3 (92.5)Rsym (%)aNumbers in parenthesis refer to statistics for highest resolution shell5.8 (17.4)8.0 (30.3)9.9 (25.4)Refinement statisticsNo. of reflections (I/σ(I) ≥ 0.0)220194878719545Working207244649218603Test12952295942R (Rfree)0.199 (0.245)0.177 (0.199)0.196 (0.232)No. of protein atoms267851445154No. of NAD atoms448888No. of GC7 atoms24No. of waters138423〈B-factor〉 (Å2)Monomer A16.929.646.1Monomer B27.542.3NAD34.526.340.2GC719.0Waters24.838.7Root mean square deviation from idealityBond lengths (Å)0.00900.00950.0104Bond angles (degrees)1.81.41.4a Numbers in parenthesis refer to statistics for highest resolution shell Open table in a new tab Structure Determination and Refinement—The structure of the new Form II crystal of the DHS·NAD binary complex was determined using the molecular replacement program AMORE (22Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar). The search model was the DHS·NAD complex from the Form I crystal (PDB code 1DHS) (15Liao D.I. Wolff E.C. Park M.H. Davies D.R. Structure. 1998; 6: 23-32Abstract Full Text Full Text PDF PubMed Google Scholar). The unambiguous results from the rotation and translation functions were used to position two copies of the search model within the Form II asymmetric unit. CNS (23Brunger 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. 1998; 54: 905-921Crossref PubMed Scopus (16991) Google Scholar) and O (24Jones T.A. Cowan S. Zou J.-Y. Kjeldgaard M. Acta Cryst. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13016) Google Scholar) programs were used for refinement and model building, respectively. A test set of reflections (2295, 4.4% of total) was randomly selected prior to refinement to calculate Rfree, to monitor the refinement progress. Bulk solvent correction and an overall anisotropic temperature factor correction were performed. Non-crystallographic symmetry 2-fold restraints were employed, with the weighting scheme guided by Rfree. Initially the noncrystallographic symmetry restraints were strong, and as the model improved, the restraints were weakened to allow the model to reflect structural discrepancies observed in the electron density. Waters were included in the model at later stages of refinement, with positions located based on peaks ≥3σ in Fo - Fc difference maps and possessing reasonable hydrogen bonding geometry. The final model included individual isotropic temperature factors. Crystals of the DHS·NAD·GC7 ternary complex were isomorphous to the Form II crystals that did not undergo the inhibitor soak, and so additional phasing information was not required to calculate an initial electron density map. However, the soaked crystals exhibited weaker diffraction intensities. GC7 molecules were built into the strong (>3σ) Fo - Fc difference density present in each active site of the DHS tetramer. The refinement scheme was similar to that described above. The test data set used to calculate Rfree was composed of reflections having the same indices as those in the test set used in the Form II DHS·NAD binary complex refinement, minimizing the bias in Rfree. No waters were included in the model of the inhibited holoenzyme because of the modest resolution of the data. The Form I DHS crystal structure was refined using newly collected data from crystals grown in parallel with the Form II crystals. This experiment was done to ensure that structural differences between the DHS Form I and the Form II crystal structures were truly because of the dissimilar crystallization conditions (e.g. pH and ionic strength) and not because of the use of different DHS sample preparations. The original Form I structure was the starting model, and refinement commenced using simulated annealing protocols in CNS. DHS in the Form II crystals exhibited several regions of structural disorder as judged by the absence of observed electron density following refinement. These regions include the N terminus (Met1-Glu27) and the C terminus (His364-Asp369) for both monomers within the asymmetric unit, and Ser78-Arg92 within monomer B only. The loop containing Ser78-Arg92 was also disordered in the Form I structure. The geometry of all final models was analyzed with PROCHECK (25Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar). Figures were created using RIBBONS (26Carson M. J. Appl. Cryst. 1991; 24: 958-961Crossref Scopus (784) Google Scholar) and GRASP (27Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5318) Google Scholar). Homospermidine Synthase (HSS) Homology Model—A model of the three-dimensional structure of HSS monomer from Senecio vulgaris was constructed by the Swiss-Model Server (28Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Crossref PubMed Scopus (901) Google Scholar) using DHS as the homology template (58.1% identical, 73.8% similar). Overall Structure—DHS is present as a homotetramer in the new Form II crystal (Fig. 1A) as it was in the Form I crystal (Fig. 1B) (15Liao D.I. Wolff E.C. Park M.H. Davies D.R. Structure. 1998; 6: 23-32Abstract Full Text Full Text PDF PubMed Google Scholar). The only species observed corresponded to the expected molecular mass of a DHS tetramer (164 kDa) in size exclusion chromatography and dynamic light scattering solution studies (1Park M.H. Lee Y.B. Joe Y.A. Biol. Signals. 1997; 6: 115-123Crossref PubMed Scopus (193) Google Scholar, 12Wolff E.C. Park M.H. Folk J.E. J. Biol. Chem. 1990; 265: 4793-4799Abstract Full Text PDF PubMed Google Scholar, 17Lee Y.B. Joe Y.A. Wolff E.C. Dimitriadis E.K. Park M.H. Biochem. J. 1999; 340: 273-281Crossref PubMed Scopus (33) Google Scholar). 2T. C. Umland and D. R. Davies, unpublished data. Thus the tetramer is highly likely to be biologically relevant and not a crystal-packing artifact. The tetramer in the Form II crystal exhibits pseudo-222 symmetry, governed by a single crystallographic 2-fold and two non-crystallographic 2-fold rotational symmetry elements; whereas, the Form I tetramer exhibits strict 222 symmetry. The two unique monomers within the Form II crystallographic asymmetric unit are denoted A and B, respectively. The tetramer may be considered as a dimer of dimers. This distinction is based upon the observation that each catalytic site is comprised of residues from two monomers. Each fundamental dimeric subunit of two closely associated monomers (Fig. 1A, A1 with B1 and A2 with B2) form two complete but antiparallel active sites at their interface, with 2830 Å2/monomer buried in the Form II crystal. These two crystallographically identical dimers form a looser association, creating interfaces between monomers A1 and B2, and between monomers B1 and A2. This second interface buries 2470 Å2/monomer in the Form II crystal. No additional active sites are created by the dimer-dimer interaction. An essential question regarding DHS is why is it present as a tetramer when the fundamental catalytic unit of the enzyme appears to be a dimer? Data indicate the possible presence of negative cooperativity upon the successive binding of ligands, and the DHS tetramer appears to bind only a single eIF5A(Lys) monomer (14Wolff E.C. Wolff J. Park M.H. J. Biol. Chem. 2000; 275: 9170-9177Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 17Lee Y.B. Joe Y.A. Wolff E.C. Dimitriadis E.K. Park M.H. Biochem. J. 1999; 340: 273-281Crossref PubMed Scopus (33) Google Scholar). Fluorescence studies monitoring the conversion between NAD and NADH demonstrated that a certain population of the enzyme-bound NADH may not be readily available to reduce the eIF5A-imine intermediate in the final step of the proposed mechanism (14Wolff E.C. Wolff J. Park M.H. J. Biol. Chem. 2000; 275: 9170-9177Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). These data have led to speculation that the DHS tetramer may undergo an asymmetric conformational change upon binding, either singularly or in combination, the cofactor and one or both of the substrates. The relaxed symmetry requirements for the DHS tetramer in the new Form II crystal coupled with the ability of DHS to bind GC7 within these crystals allowed for further analysis of the interplay between monomers within the tetramer. Intimate contacts formed between each monomer with each of the remaining three partners within the DHS tetramer provide additional evidence that the tetramer is required for either a regulatory mechanism or enhanced stability. For example, consider monomer A1. It interacts with monomers B1 and B2, forming the two types of dimer interfaces discussed above. More interestingly, monomer B2 contributes an N-terminal ball-and-chain motif (Fig. 1) that is capable of blocking one of the active sites formed at the A1-B1 dimer interface, as observed in the Form I crystal (Fig. 1B). Monomer A1 lacks extensive contacts with the fourth monomer, monomer A2, but the limited interactions are distinctive. First, monomer A2 contributes a ball-and-chain motif capable of blocking the entrance to the second active site formed at the A1-B1 interface. Secondly, Phe54 of monomer A2 makes extensive contacts to a loop in monomer A1 (Asp313 and Gly314) and a loop in monomer B1 (Asn106), both of which interact extensively with the NAD adenine moiety. Phe54 is present in a hairpin turn connecting two helices, possessing the unusual strained main chain geometry of ϕ ∼ 42° and Ψ ∼ -135° in both crystal forms, and is well defined by electron density. These interactions allow for a potential transmission path for a regulatory conformation change. The root mean squared deviation is 0.17 Å between the Cα atoms of the two independent monomers in the Form II crystal of the DHS:NAD holoenzyme, superimposable by a rotation of 180.0° coupled with a translation of 0.16 Å. Each NAD binding site is occupied to a comparable degree based upon a visual inspection of the electron density and the refined temperature factors. Similarly, the root mean squared deviation between the Cα atoms of the two independent monomers in the Form II crystal of the DHS·NAD·GC7 ternary complex is also 0.17 Å, and each of the NAD and the GC7 binding sites is equally occupied. Furthermore, the Form II crystals exhibited only two limited regions of the main chain possessing significant conformational differences between the two independent monomers. First, there is a lack of electron density to define the position of residues His364-Asp369, and presumably they are highly mobile. Met363 is the last residue of the chain defined by electron density, assuming different conformations in the independent monomers. Second, the loop composed of residues Ser78–Arg92 is observed within monomer A but not within monomer B of the Form II crystals. It is also unobserved within the Form I crystal structure. No function has been identified with either one of these two regions, but both human and Saccharomyces cerevisiae DHS require the presence of the five C-terminal residues for full activity (20Joe Y.A. Wolff E.C. Park M.H. J. Biol. Chem. 1995; 270: 22386-22392Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29Kang K.R. Chung S.I. Exp. Mol. Med. 1999; 31: 210-216Crossref PubMed Scopus (10) Google Scholar). It should be emphasized that the weights of the non-crystallographic symmetry restraints employed during refinement were carefully selected so as not to artificially force the monomers to be similar, and 2Fo - Fc omit maps calculated following a short simulated annealing protocol were used to reduce model bias. Thus, there is no structural indication from the Form II crystals that NAD alone or the combination of NAD and GC7, when present in excess stimulates negative cooperativity within DHS because of an asymmetric conformational change lowering the affinity for the successive binding of ligands. However, any potential conformational changes to the DHS tetramer resulting from the binding of eIF5A(Lys) or from less than equivalent concentrations of NAD or GC7 remains to be addressed. NAD Binding Site—The NAD cofactor binds to a Rossmann dinucleotide-binding motif present within the core of the DHS monomer (Fig. 1C). A subdomain is inserted into this motif following strand β2, partially forming the active site. The DHS polypeptide chain is extended on both the N- and C-terminal sides of the dinucleotide-binding motif. The NAD cofactor is bound in the canonical manner to the Rossmann fold of monomer A but is also subjected to an interaction with monomer B involving a loop (Thr308–Lys329) following strand β5. This loop also contributes crucial residues to the catalytic active site. The NAD buried surface area due to interactions with monomer A and B are ∼476 and ∼294 Å2, respectively. The majority of the NAD-binding interactions are similar to those observed in the Form I crystal. However, in the Form I crystal Oϵ-2 of Glu137 interacts with the amide nitrogen of the nicotinamide ring. In the Form II holoenzyme crystal, this residue assumes a different rotamer in each of the two independent monomers. In monomer A this side chain points away from the nicotinamide ring, hydrogen bonding to Oζ of Tyr176 and several waters, whereas Glu137 of monomer B resembles that seen in the Form I crystal. In the Form II holoenzyme·inhibitor complex, Glu137 of monomer A interacts weakly with both the nicotinamide ring and the hydroxyl group of Tyr176, and again this glutamate residue in monomer B resembles that observed in the Form I crystal. Only the nicotinamide ring of NAD has any appreciable solvent accessibility (∼30 Å2) in the absence of a bound substrate or inhibitor and is completely solvent-inaccessible in the presence of GC7. Active Site—The observation of an enzyme-imine intermediate (Enz–Lys–N=CH(CH2)3NH2) identified Lys329 of human DHS and Lys350 of yeast DHS as the residue to which the butylamine moiety of spermidine is transferred in the second reaction step (13Wolff E.C. Folk J.E. Park M.H. J. Biol. Chem. 1997; 272: 15865-15871Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 30Wolff E.C. Park M.H. Yeast. 1999; 15: 43-50Crossref PubMed Scopus (12) Google Scholar). The Form I crystal structure (15Liao D.I. Wolff E.C. Park M.H. Davies D.R. Structure. 1998; 6: 23-32Abstract Full Text Full Text PDF PubMed Google Scholar) suggested further residues as having a role in the active site based on their proximity to Lys329 (14Wolff E.C. Wolff J. Park M.H. J. Biol. Chem. 2000; 275: 9170-9177Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 30Wolff E.C. Park M.H. Yeast. 1999; 15: 43-50Crossref PubMed Scopus (12) Google Scholar, 31Lee C.H. Um P.Y. Park M.H. Biochem. J. 2001; 355: 841-849Crossref PubMed Scopus (25) Google Scholar, 32Joe Y.A. Wolff E.C. Lee Y.B. Park M.H. J. Biol. Chem. 1997; 272: 32679-32685Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The active site is contained within a deep narrow tunnel present at a dimer interface. The specificity is conferred by the large number of charged residues lining the tunnel walls and entrance coupled with the inaccessibility (see Figs. 3 and 4A). The tunnel is ∼17 Å deep, as measured between the main chain carboxyl of Gly314(A) to Oδ-2 of Asp243(B). The entrance to the active-site tunnel is a broad funnel that is ∼7 Å deep, resulting in the bottom of the tunnel being
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