J. Am. Chem. SOC.1981, 103,907-914
907
Crystal and Molecular Structure of the Lithium Salt of Nicotinamide Adenine Dinucleotide Dihydrate (NAD+, DPN’, Cozymase, Codehydrase I) B. S. Reddy, W. Saenger,* K. Miihlegger, and G . Weimann Contribution from the Abteilung Chemie, Max- Planck- Institut fur Experimentelle Medizin, 0 - 3 4 Gottingen, Federal Republic of Germany. Received May 14, 1980 Abstract: The lithium salt of the coenzyme nicotinamide adenine dinucleotide (NAD’) crystallizes from slightly acidic, 40% aqueous methanol as a dihydrate in the orthorhombic space group P212121with a = 10.073 A, b = 15.839 A, c = 17.821 A, and Z = 4. Owing to the small crystal size only 1365 X-ray data up to 1.09-A resolution could be measured by means of
a four-circle diffractometer. The structure was solved by direct methods and Fourier analyses and refined by full-matrix least squares to R = 0.10. Both nucleotides occur in preferred conformations,with anti orientation of the nucleo bases, sugar puckering C(2’)-endo for andenosine and C(3’)-endo for nicotinamide riboside, and (+)-gauche conformation for both C(3’)-C(4’)C(5’)-O(5’) bonds. Torsion angles about C(5’)-0(5’) bonds are trans and angles about 0-P bonds in nicotinamide ribotide (+)-gauche but eclipsed (-125’ and 133’) in the AMP moiety. The latter conformation appears to be due to Li’ coordination which links N(7) of adenine with nicotinamide phosphate and also the pyrophosphate free oxygens to form a tetrahedral coordination scheme. The molecular structure of NAD’ shows an extended form with nicotinamide and adenine nearly perpendicular to each other at 12-A separation. A similar extended conformation of NAD’ had been observed earlier in dehydrogenase-NAD’ complexes and had been proposed for NAD’ on the basii of several nucleoside diphosphatecrystal structures. This conformation is similar to the one proposed from spectroscopic data for NAD’ in aqueous solution at low pH or under addition of alcohol but differs from the “folded” NAD’ conformation observed in aqueous solution around pH 7. This “folded” form requires intramolecular, antiparallel stacking between adenine and nicotinamide, an interaction which is found between symmetry-related NAD’ molecules in this crystal structure.
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Introduction In biological redox processes associated with dehydrogenases as catalysts, hydride is transferred from one substrate to another. This transfer is mediated by coenzymes of which nicotinamide adenine dinucleotide (NAD’, Figure l), formerly called DPN’, coenzyme I, codehydrase I, or cozymase, plays a dominant role. Isolated and characterized in the 1930s by von Euler, Warburg, Christian, and Theorell’ NAD+ has since then intensively been studied in order to clarify its properties when free in aqueous solution and bound to the enzyme active site. Aqueous solution experiments include spectroscopic investigations such as UV absorption: fluorescence,*5 circular dichroism,6 and proton, 13Cand 31PNMR on NAD’ and on its deri~atives.’~The results suggest that NAD+ can occur in three different conformations which are in equilibrium. In two of these conformations, existing to about 40% under physiological condition^,'^ NAD’ is “folded” with the adenine and nicotinamide residues stacked on top of each other to form left- or right-handed helical structures while in the “extended” form, the two heterocycles are separated by 10-12 A.8 (1) P. D. Boyer, Ed., “The Enzymes, XI, Oxidation-Reduction”,Academic Press, New York, 1975. (2) J. M. Siegel, G.A. Montgomery, and R. M. Boch, Arch. Biochem., Biophys., 82, 288-299 (1959). (3) H. Sund in “Biological Oxidations”, T. P. Singer, Ed., Interscience, New York, 1968, pp 603-639. (4) G. Weber, Nature (London), 180, 1409-1410 (1957). (5) S. F. Velick, J . Biol. Chem., 233, 1455-1467 (1958). (6) D. W. Miles and D. W. Urry, J . Biol. Chem., 243,4181-4188 (1968). (7) 0. Jardetzky and N. G. Wade Jardetzky, J. Biol. Chem., 241, 85-91 (1966). (8) R. H. Sarma and R. J. Mynott In “Proceedings of the Jerusalem Symposium, V”, E. D. Bergmann and B. Pullman, Eds., Academic Press, New York, 1973, pp 591-626. (9) N. J. Oppenheimer, L. J. Arnold, and N. 0. Kaplan, Proc. Natl. Acad. Sci. U.S.A.,68, 3200-3205 (1971). (10) J. Jacobus, Biochemistry, 10, 161-164 (1971). ( 1 1 ) M. Blumenstein and M. A. Raftery, Biochemistry, 12, 3585-3590 (1973). (12) A. P. Zens, P. T.Fogle, T. A. Bryson, R. B. Dunlap, R. R. Fisher, and P. D. Ellis, J . Am. Chem. Soc., 98, 3760-3764 (1976). (13) T. J. Williams, A. P. Zens, J. C. Wisowaty, R. F. Fisher, R. B. Dunlap, T. A. Bryson, and R. P. D. Ellis, Arch. Biochem. Biophys., 172, 490-501 (1976). (14) J. R.Barrio, J. A. Secrist 111, and N. J. Leonard, Proc. Narl. Acad. Sci. U.S.A., 69, 2039-2042 (1972). (15) B. A. Gruber and N. J. Leonard, Proc. Natl. Acad. Sci. U.S.A.,72, 3966-3969 (1975).
0002-7863/81/1503-907$01,00/0
A number of dehydrogenases with and without bound NAD’ have been crystallized and subjected to X-ray analyses.’ These studies invariably demonstrated NAD’ to exist in an “extended” form, with sugar and pyrophosphate moieties bound by amino acid side chains while the adenosine heterocycle is inserted into a hydrophobic pocket of the protein. In all these complexes, NAD’ adopts a conformation not conforming with the generally accepted “standard, low energy” structures of nucleotides,I6-’* probably because aqueous environment is replaced by protein surface and protein-nucleotide hydrogen bonds and salt bridges do occur. On this background it was of interest to look at the detailed geometry of NAD’ itself. Published attempts to crystallize this coenzyme date back to 1957,’9but since then no specimens suitable for X-ray analysis could be obtained. In this paper we describe the crystal structure of the lithium dihydrate complex of NAD’. Some of the results of this analysis have already been reported.20p21
Experimental Section As outlined previously,20the lithium dihydrate complex of NAD’ was crystallized in the form of thin needles from about 40% methanolic, aqueous solution adjusted to slightly acidic pH. For X-ray studies, a crystal of dimensions 0.025 X 0.025 X 0.2 mm was mounted together with some mother liquor in a quartz capillary. The crystals belong to the orthorhombicsystem, space group P212121,and cell dimensions are a = 10.073 (3) A, b = 15.839 (4) A, c = 17.821 (4) A, and Z = 4. The X-ray and chemical analyses showed that the crystals contain two molecules of water per Li+.NAD’, yielding a molecular formula C21H26N7Ol4P2Li-2H20, M,= 705.4 per asymmetric unit. The crystal density could not be measured and was calculated to p c = 1.648 g/cm3. The numbering scheme adopted for NAD’ is displayed in Figure 1. Intensity data were collected on a STOE four-circle diffractometer by using Ni-filtered Cu Ka radiation and a 20/0 scan mode. Owing to the rapid decrease of intensities with increasing glancing angle 0, only a limited data set corresponding to a resolution of 1.09 A (e,, = 45O) could be measured. Each reflection was scanned in 2 min with 20-s stationary background counts on both sides of the scan. The data were corrected for Lorentz-polarization factors but not for absorption, and the (16) M. Sundaralingam, Ann. N . Y . Acad. Sci., 255, 1-42 (1975). (17) W. Saenger, Angew. Chem., Int. Ed. Engl., 12, 591-601 (1973). (18) B. Pullman and A. Saran, Prog. Nucl. Acid. Res. Mol. Biol. 18, 216-236 (1976). (19) K. Wallenfels and W. Christian, Methods Enzymol., 3, 882-884 (1957).
0 1981 American Chemical Society
908 J. Am. Chem. Soc., Vol. 103, No. 4, 1981
Reddy et al. A
0 Li
cc ON
A
0 0
OP pseudo diad
K
Figure 1. Chemical structure and numbering scheme in NAD’. The pseudodiad passing through the pyrophosphate oxygen relates both ribosephosphate moieties. 0’ Li
oc
R
ON
Figure 3. The NAD+ dimer linked by a Li’ cation. Antiparallel stack formation is between adenine and nicotinamide residues. A hydrogen bond formed between N(6)A and 0(1), of symmetry-relatedmolecules is indicated by stippled line.
and further geometrical details are listed in Tables 11-V. structure factors have been deposited in the microfilm.
Figure 2. The structure of NAD+ as found in the Li+ complex-Li’ coordination indicated by solid lines.
standard deviations of the structure amplitudes were computed on the basis of counter statistics.22 A total of 338 reflections below the 3a threshold were considered unobserved out of a total of 1365. The structure was solved by Patterson and direct methods employing the multisohtion tangent formula approach MULTAN.~) Because of the limited data set, normalized structure factors E,, below the usually accepted cutoff 1.5 had to be used. With 450 E‘s greater than 1.01 and seven starting reflections consisting of three origin defining reflections, one a( 1) relationship, and three additional reflections, phase sets were computed of which the most consistent had an R factor = 50.4%, absolute figure of merit = 1.42, and combined figure of merit = 2.1 1. An E map computed with this phase set showed maxima which could be identified as atoms of the pyrophosphate group and of the adenine heterocycle. The peaks assigned to phosphorus atoms also showed up in corresponding positions in a sharpened Patterson map computed with (E2 - 1) data. Four cycles of structure factor and difference Fourier calculations allowed us to locate all the nonhydrogen atoms of the NAD’ molecule including the two water molecules. Isotropic refinement using full-matrix least-squares techniques converged after four cycles at R = 0.14, and a difference Fourier map computed at this stage revealed the position of the lithium cation. Refinement was continued with anisotropic temperature factors for the phosphorous and oxygen atoms. The hydrogen atom positions could not be verified from difference Fourier maps, but positions for hydrogens attached to carbon atoms were calculated from the stereochemistry of the C, N, and 0 skeleton and these were included in structure factor calculations during subsequent refinements. The final R value is 0.10 for the 1027 observed data. Results and Discussion In Figures 2 and 3, the structure of the Li+.NAD+ complex is described. Table I gives the final positional and thermal parameters of nonhydrogen atoms. The bond distances and angles (Figure 4) are in general agreement with comparable structures, (22) G. H . Stout and L. H. Jensen, “X-ray Structure Determination”, Macmillan, New York, 1968. (23) P. Main, M. M. Woolfson, and G . Germain, MULTAN, a multisolution tangent formula refinement program, Universities of York and Louvain, 1971.
The
Molecular Conformation and Geometry. General Description of the Structure of NAD’ Prior to advancing to the details of the structure of the Li’. NAD+.2Hz0 complex, let us look at the overall structure of the NAD’ molecule. NAD’ is composed of two 5’-nucleotides, adenylic acid, and nicotinamide-5’-ribonucleotide (Figure 1). These two nucleotides are not linked in the usual head-to-tail orientation via a 3’,5’-internucleotide phophodiester bond but in a head-to-head arrangement via a pyrophosphate group. In other words, while a dinucleotide can be augmented on both ends to yield a longer oligonucleotide, NAD’ represents a complete molecule in itself; it cannot grow further. From a structural point of view, NAD’ could be diad symmetrical (neglecting the difference in heterocycles) with the diad passing through the pyrophosphate oxygen OPP. If the charge distribution is concerned, however, the molecule becomes unsymmetrical. In the neutral state at physiological pH, the pyrophosphate group bears two negative charges distributed over the four free oxygen atoms. One positive charge is located on the nicotinamide residue; i.e., in total one negative charge remains and has to be balanced, in this case by one Li’ cation. In the Li’.NAD’.2Hz0 crystal structure, the NAD’ molecules are arranged head-to-tail (Figure 5) and associated with Li+ cations located between them. Each NAD’ faces two Li’ and vice versa, resulting in a “polymeric” coordination scheme. The apparent diad symmetry of NAD+ is destroyed by this coordination because one Li+ is liganded to N(7) of adenine and to one free oxygen atom of the PN phosphate group while the second Li+ bridges two free oxygen atoms of the pyrophosphate, thus forming a sixmembered ring. This Li+ complexation does not allow a “folded” conformation of NAD+ with intramolecular stacking of the two heterocycles, but Li’eNAD’ rather assumes an “extended” form, similar to but distinctly different from the NAD’ structures complexed to dehydrogenases. Preferred Nucleotide Conformations. From a great number of 5’-nucleotide structures studied thus far, the most preferred and energetically favored conformational parameters can be summarized with a few statements.I6*” The five-membered ribose ring is puckered C(2’)-endo or C(3’)-endo, the heterocycle is in anti orientation about the glycosidic C( 1’)-N bond, and the conformation about the exocyclic C(4’)-C(5’) bond (torsion angle q , C(3’)-C(4’)-C(5’)-0(5’)) is (+)-gauche, locating O(5’) “above” the ribose. [All torsion angles A-B-C-D in this paper refer to the standard nomenclature; Le., they are 0’ if the bonds A-B and C-D are cis planar and counted positive if the far bond C-D rotates clockwise with respect to the near bond A-B.] Finally, the phosphate group is trans to C(4’) and the conformation
Structure of the Lithium Salt of NAD'
J. Am. Chem. SOC.,Vol. 103, No. 4, 1981 909
Table I (a) Fractional Atomic Coordinates of Anisotropically Refined Atoms" atom
X
0 ( 1 ' ) ~ -0.0358 (13) o(2')~ 0.1699 (14) 0(3')A -0.1117 (13) O(~')A 0.0340 (14) 0 ( 1 ) ~ -0.0355 (12) o(2)~ 0.1300 (14) PA 0.0759 (6) 0.1951 (13) OPP PN 0.2375 (6) 0.4277 (13) 0(1')~ 0.4228 (15) o(2')~ 0.3473 (16) 0(3')~ 0.2851 (13) 0(5')~ 0(1)~ 0.3483 (14) 0.1161 (14) o(2)~ 0.0721 (15) 0(7)~ 0.2957 (14) W(1) 0.6104 (17) W(2)
B,, Y B33 z B,, B,, 7 (12) 15 (6) 0.1730 (7) 95 (21) 28 (8) 0.6141 (7) 0.5802 (9) 0.3345 (7) 130 (23) 62 (10) 15 (6) 23 (14) 11 (11) 28 (7) 0.3462 (7) 67 (21) 31 (8) 0.6000 (8) -9 (12) 31 (7) 110 (25) 22 (8) 0.1914 (7) 0.7937 (7) -11 (11) 17 (6) 0.0865 (7) 31 (19) 38 (8) 0.8937 (8) -7 (13) 3 (6) 0.1886 (7) 91 (23) 38 (9) 0.9414 (8) 0.8716 (4) 0.1403 (3) 43 (9) 22 (3) 12 (2) 7 (5) 17 (13) 30 (7) 0.0972 (7) 36 (20) 45 (9) 0.8314 (8) -4 (5) 24 (3) 9 (3) 0.8287 (3) 0.0073 (4) 5 3 (9) -15 (12) 36 (7) -0.0884 (7) 18 (7) 94 (22) 1.0434 (7) -38 (12) 34 (8) -0.0313 (8) 94 (24) 22 (8) 1.2299 (8) -16 (13) 29 (7) 0.0882 (7) 28 (8) 181 (27) 1.1331 (8) 18 (6) -15 (10) -0.0061 (7) 42 (21) 44 (8) 0.9239 (8) 10 (12) 22 (6) 0.0003 (8) 121 (24) 21 (8) 0.7713 (7) 25 (6) -44 (12) -0.0387 (7) 59 (22) 40 (9) 0.8196 (9) -18 (14) 60 (9) 105 (26) -0.2756 (8) 56 (10) 1.2839 (9) 75 (23) 64 (9) 6(13) 37 (10) 0.3935 (9) 0.4482 (9) 57 (19) 120 (17) 119 (14) 76 (27) 0.1301 (11) 0.1662 (12) (b) Fractional Atomic Coordinates of Isotropically Refined Atomsb
B13
B23
0 (9) -7 (11) -12 (10) 9 (11) -10 (9) 9 (9) 0 (4) -23 (10) 10 (5) 22 (11) 46 (12) -10 (12) 5 (10) -2 (12) 4 (10) 0 (13) 6 (12) -67 (17)
6 (6) 32 (7) 8 (6) 19 (7) -5 (6) -7 (6) 0 (3) -7 (7) -3 (3) -2 (6) -18 (6) 0 (7) 6 (7) 12 (6) 0 (7) 28 (8) 25 (8) -48 (14)
atom X Y z B,, atom X Y z Bll 0.2129 (11) 15 (5) c(1')N 0.3626 (25) 1.1179 (16) -0.1138 (13) 48 (7) c(1')A 0.0611 (20) 0.5701 (12) 0.2814 (12) 25 (6) c ( 2 ' ) ~ 0.3376 (24) 1.1771 (15) -0.0448 (13) 35 (6) c ( 2 ' ) ~ 0.1042 (22) 0.6235 (14) 0.3007 (12) 31 (6) c(3')N 0.3208 (22) 1.1070 (13) 0.0157 (11) 23 (6) c ( 3 ' ) ~ -0.0302 (22) 0.6597 (14) 0.2243 (11) 16 (5) c ( 4 ' ) ~ 0.4352 (24) 1.0416 (15) -0.0024 (13) 38 (6) c ( 4 ' ) ~ -0.1037 (20) 0.6744 (12) 0.1911 (12) 34 (6) c ( 5 ' ) ~ 0.4158 (23) 0.9532 (14) 0.0174 (12) 29 (6) c ( 5 ' ) ~ -0.1032 (24) 0.7594 (14) 0.0655 (11) 32 (6) N ( ~ ) N 0.2394 (19) 1.0929 (11) -0.1612 (10) 31 (5) N(~)A 0.3551 (18) 0.3551 (11) 0.1201 (13) 34 (6) c ( 2 ) ~ 0.1907 (22) 1.0137 (15) -0.1597 (12) 34 (6) c ( 2 ) ~ 0.2693 (25) 0.3372 (14) c(3)~ 0.0823 (23) 0.9953 (14) -0.2029 (12) 24 (5) 0.1595 (10) 41 (5) N(~)A 0.2012 (19) 0.3995 (12) 0.1366 (14) 35 (6) c ( 4 ) ~ 0.0161 (25) 1.0571 (16) -0.2470 (14) 45 (7) c ( 4 ) ~ 0.2260 (25) 0.4818 (16) 0.0801 (11) 13 (5) c ( 5 ) ~ 0.0754 (24) 1.1366 (15) -0.2424 (13) 29 (6) c ( 5 ) ~ 0.3204 (20) 0.5012 (13) 0.0392 (13) 29 (6) c ( 6 ) ~ 0.1870 (24) 1.1588 (14) -0.2034 (13) 32 (6) c ( 6 ) ~ 0.3894 (23) 0.4335 (15) 31 (5) c ( 7 ) ~ 0.0217 (27) 1.2148 (17) -0.2879 (15) 59 (8) N(~)A 0.4766 (17) 0.4432 (11) -0.0146 (10) 0.0690 (9) 19 (4) N ( ~ ) N -0.0616 (19) 1.1978 (11) -0.3453 (10) 37 (5) N(~)N 0.3211 (17) 0.5844 (11) 0.1172 (13) 35 (6) Li+ 0.4437 (48) 0.6662 (28) 0.0041 (27) 46 (1 1) c ( 8 ) ~ 0.2359 (25) 0.6164 (14) N(~)A 0.1692 (19) 0.5586 (12) 0.1608 (10) 35 (5) a Temperature factors (X 10.000) in the form T = exp[-(B,,h2 + B,,k' + B3J2 + 2B,,hk + 2BI3hZ+ 2B,,kl)]. Standard deviations of last digits are given in parentheses; they were obtained from the least-squares correlation matrix. b Temperature factors (X 10) in the form B = 8 Z z (A'), with i' the mean square amplitude of atomic vibration. Standard deviations as in footnote a. Table 11. Torsion Angles (Deg) in Ribose Moieties" Table 111 Torsion Angles in Pyrophosphate Chain" nicotinamide C(4')A-C(S')A-0(5')A-PA 163 (1) torsion angle adenosine ribose C(5')Aa(5')A-PA-OPP -125 (1) -0(1)A -8 ( 2 ) XCN, O(l')-C(l')-N(9)-C(8) 52 -0(2)A 123 (1) -N( 1)-C(6) 15 PN-Opp-PA-0(5 ')A 138 (1) 0 ( 1 ')-C(l')-N(9)-C(4) -121 -0(1)A 17 (1) -165 -113 (1) -N(l)-C(2) -0(2)A C(4')-0( 1')-C( 1')-C(2') - 24 7 PAaPP-PN-0(5 'IN 72 (1) 37 -31 0(1')-C( 1')-C(2')
