Biochemistry 1985, 24, 7827-7833
7827
Crystal Structure of the &,?-Tridentate Manganese Complex of Adenosine 5’-Triphosphate Cocrystallized with 2,2’-Dipyridylamine+ Michael Sabat,t.l Renzo Cini,*vll Tuli Haromy,t and M . Sundaralingam*J Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, and Istituto di Chimica Generale della Universita di Siena, 53100 Siena, Italy I Received June 24. I985
ABSTRACT:
The 1:1:l complex of Mn2+, ATP, and 2,2’-dipyridylamine (DPA) crystallizes as Mn-
(HATP)2-Mn(H20)6*(HDPA)2.1 2 H 2 0 in the orthorhombic space group C222* with unit cell dimensions
a = 10.234 (3) A, b = 22.699 (3) A, and c = 31.351 (4) A. The structure was solved by the multisolution technique and refined by the least-squares method to a final R index of 0.072 using 35 16 intensities. The structure is composed of two A T P molecules sharing a common manganese atom. The metal exhibits a,j3,y coordination to the triphosphate chains of two dyad-related A T P molecules, resulting in a hexacoordinated Mn2+ ion surrounded by six phosphate groups. The metal to oxygen distances are 2.205 (6), 2.156 (4), and 2.144 (5) for the a-, j3-, and y-phosphate groups, respectively. No metal-base interactions are observed. There is a second hexaaqua-coordinated Mn2+ion that is also located on a dyad axis. The hydrated manganese ions sandwich the phosphate-coordinated manganese ions in the crystal with a metal-metal distance of 5.322 A. The ATP molecule is protonated on the N( 1) site of the adenine base and exhibits the anti conformation (x = 66.0°). The ribofuranose ring is in the :Tconformation with pseudorotation parameters P = 179 ( 1 ) O and 7, = 34.1 (6)O. The adenine bases form hydrogen-bonded self-pairs across a crystallographic dyad axis and stack with both DPA molecules to form a column along the dyad. The structure of the metal-ATP complex provides information about the possible metal coordination, conformation, and environment of the nucleoside triphosphate substrate in the enzyme.
E e polyphosphate moiety of nucleotides play a vital role in a wide variety of enzyme-catalyzed reactions. These range from those enzymes directly involved with the replication and transcription of the genetic material to those that function to permit the controlled transfer of energy in metabolism. The actual substrate for most of these enzymes is a complex where the phosphate moiety of the nucleotide is coordinated to a metal ion, usually Mg2+. The metal ion can be coordinated to one, two, or all three phosphate groups of ATP to form a monodentate, bidentate, or tridentate complex, respectively. Although generally difficult to crystallize, a few metal-ATP complexes have previously been reported: NaATP (Kennard et al., 1971; Larson, 1978), ZnATP (Orioli et al., 1981), and CuATP (Sheldrick, 1982). Besides the ATP complexes, the crystal structures of all the various coordination isomers of Co3+complexes with the triphosphate moiety itself have been reported: P-monodentate (Haromy et al., 1983), y-monodentate (equivalent to a-monodentate in the absence of nucleotide) (Haromy et al., 1983), a,y-bidentate (Merritt et al., 1981), P,y-bidentate (Merritt et al., 1981), and a,@,y-tridentate (Merritt & Sundaralingam, 1981). These complexes as well as a number of metal-pyrophosphate complexes have recently been reviewed (Sundaralingam & Haromy, 1985). Trivalent cobalt was used as the metal ligand for these studies because it forms coordination complexes that are inert to spontaneous substitution, thus facilitating their separation, characterization, and crystallization (Cornelius & Cleland, 1978). +Thisresearch was supported by a grant from the National Institutes of Health (GM-17378) and by the College of Agricultural and Life Sciences at the University of Wisconsin. * Correspondence should be addressed to these authors. *Universityof Wisconsin-Madison. 8 Present address: Laboratory of Stereochemistry, National Research Council, 50132 Florence, Italy. 1’ Instituto di Chimica Generale della Universita di Siena.
0006-2960/85/0424-7827$01.50/0
The MgATP complex has long eluted crystallization attempts. Most divalent ions catalyze the hydrolysis of the polyphosphate chain yielding a mixture of metal phosphates and metal nucleoside 5’-monophosphates. Recently, the preparation and crystallizations of the complexes of ATP with Mg2+,Ca2+,S P , Mn2+,Cd+, Cu2+,and Zn2+in the presence of 2,2’-dipyridylamine (DPA) were reported (Cini et al., 1982, 1984). The introduction of DPA apparently minimizes the dephosphorylation reaction by forming a very stable complex facilitated by strong stacking interactions between the adenine bases and the DPA molecules. A preliminary note on the structures of the complexes of ATP with Mg2+,Ca2+,Mn2+, and Co2+ has appeared (Cini et al., 1983), and the refined structures of MgATP and CaATP have recently been published (Cini et al., 1984). The detailed structure of MnATP is now reported. The MnATP crystals are of substantially better quality than the crystals for any of the other isomorphous metal complexes, and this has permitted the most accurate determination of a metal-ATP complex to date. At physiological pH both the N ( 1) site of the adenine base and the metal triphosphate chelate would be expected to be deprotonated. The triphosphate chain is also deprotonated in the crystal while the N ( 1) site of the adenine base is protonated. Thus the structural details of MnATP provide important insights into the geometry and conformation of the enzyme substrate chelate complex. These results are also important for NMR spectroscopy because Mn2+ has been used as a paramagnetic ion to probe the conformation and binding to ATP both in solution and in the enzyme. EXPERIMENTAL PROCEDURES The complex crystallized from a solution containing MnSO,, Na2ATP, and 2,2’-dipyridylamine (DPA) in equimolar ratio. A I-mmol solution of DPA in 95% ethanol was added to an aqueous 1-mmol solution of Na2ATP, which was subsequently added to a 1-mmol solution of MnS04. The mixture was 0 1985 American Chemical Society
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heated at 80 ‘C, and crystals suitable for X-ray analysis were formed upon slow cooling of this mixture (Cini et al., 1984). Intensity data were collected on an Enraf-Nonius CAD-4 diffractometer using Ni-filtered Cu Ka radiation (A = 1.5418 A). Unit cell parameters were refined by a least-squares algorithm using 25 automatically centered reflections. Of a total of 4108 unique reflections measured up to a 28 limit of 150°, 3516 reflections that had F > 2u(F) were used for the structure analysis. A crystal decay correction (maximum of 5%) was applied, based on four reflections that were monitored throughout data collection. An empirical absorption correction (4 curve) was also applied to the data as well as corrections for Lorentz and polarization effects. The structure was solved by the direct methods technique using the program MULTAN (Main et al., 1970). Both manganese atoms were found to lie on a 2-fold rotation axis and were thus given a multiplicity factor of 0.5. Two independent molecules of 2,2’-dipyridylamine were found in the structure. One half of each DPA molecule is related to the other half by rotation about a 2-fold axis. Unlike the pyridyl rings, which are made coincident by the 2-fold rotation, the bridging nitrogen atom is present on only one side of the 2-fold axis in any given molecule. Therefore, the bridging nitrogen atom is statistically disordered with half of the bridge nitrogen atom on one side of the 2-fold axis while the other half is on the other side. Preliminary refinement of the structure was carried out by the least-squares technique using isotropic temperature factors. The DPA molecules could either be represented as complete molecules at half occupancy or one ring of each molecule at full occupancy and the bridge nitrogen atoms at half occupancy. Refinement of the entire DPA molecules at half occupancy showed no improvement in the R index when compared to refinement of only one ring at full occupancy, thus indicating that the overlap of the two DPA rings is nearly perfect. Consequently, to minimize the number of parameters, one ring of each DPA molecule was refined at full occupancy with the bridge nitrogen atoms refined at half occupancy. Another feature of DPA is that the ring nitrogen atoms can either be interior [adjacent to the other ring where the C(1)-N( 1)-C( 1’)-N(2’) torsion angle is cis] or exterior [with the torsion angle trans]. The crystal structure of DPA itself (Johnson & Jacobson, 1973) shows that one ring nitrogen is interior while the other is exterior. The 2-fold symmetry rotation will rotate the interior nitrogen atom N(2’) into position C( 1) and will rotate the exterior nitrogen atom N(6) into position C(3’). Since the nitrogen atoms could also occupy positions 2 and 6’, only positions 4 and 5 of each ring can be regarded as pure carbon atoms while the other four ring atoms may be regarded as statistically consisting of 75% carbon and 25% nitrogen. The hydrogen atoms for the ATP molecule were found in difference Fourier syntheses, the strongest hydrogen density being N ( l ) of the protonated adenine base. Some of the protons of the DPA molecules were also found. To preserve charge neutralization, each DPA molecule must be protonated at one of the ring nitrogen atoms; due to the DPA disorder, it was not possible to locate these protons. The structure contains a total of ten independent water positions, two of which lie on 2-fold axes making a total of nine water molecules per ATP. The hydrogen atom densities for the water molecules were diffuse as expected due to the high temperature factors for the water oxygen atoms (Bq = 11-29 A*). The water hydrogen atoms were fixed on the basis of a reasonable hydrogen-bonding network between the water molecules and the
MnATP molecule. The DPA protons were treated statistically like the ring atoms to which they are bonded. The multiplicity for each DPA proton was derived from the expected proton occupancy of each potential proton site. Refinement of the nonhydrogen atoms with inclusion of the hydrogen atom scattering resulted in a final R index of 0.072 for 3516 reflections. A counting statistics weighting scheme was used with the weight of each reflection proportional to l/(u*F 0.02F:). At the completion of refinement the maximum shift/error ratio was 0.15. The scattering factors for the nonhydrogen atoms were taken from Cromer & Waber (1965) while those for the hydrogen atoms were from Stewart et al. (1965).
+
RESULTSAND DISCUSSION Both manganese atoms are located on 2-fold rotation axes. Mn(1) is surrounded by six phosphate groups from two dyad-related ATP molecules, resulting in an unusual environment. Mn(2) is coordinated to six water molecules, and again only three of the water molecules are unique since the other three are dyad-related. As discussed earlier, the DPA molecules also have a pseudo-2-fold axis running through the “center” of each molecule, making only one half of each molecule unique. The final positional parameters for all atoms in the crystal structure are presented in Table I. An ORTEP drawing (Johnson, 1976) showing the atom numbering for the MnATP complex and the DPA molecules is presented in Figure 1. Triphosphate Geometry and Conformation. The triphosphate bond lengths and angles are given in Table I1 together with the corresponding values for the other known metal-ATP and metal-triphosphate coordination complexes. The bridging P-0 bond lengths for MnATP range from 1.57 to 1.64 A while the other P-0 bond lengths range from 1.47 to 1.51 A. Except for the poorly determined NaATP structure, the O(5’)-P( 1) bond length is the shortest of the polyphosphate chain bonds. In MnATP, the O(P23)-P(3) length is longest, a phenomenon also observed for CuATP(A) but not seen for any of the other ATP complexes. The bond angles at P ( l ) and P(2) are significantly less than the ideal tetrahedral angle of 109.47’ for all of the ATP complexes while the bridge oxygen angles are all substantially wider than the tetrahedral angle. The polyphosphate chain of MnATP is in a folded conformation to facilitate the tridentate coordination. The P( 1)-P(3) distance and P( l)-P(2)-P(3) (P-P-P) virtual bond angle serve as measures of the degree to which a triphosphate chain is extended or contracted. These parameters and the principal polyphosphate chain torsion angles for the known metal-ATP and metal-triphosphate complexes are summarized in Table 111. The P-P-P angle for all the tridentate metal complexes ranges from 85’ to 98’. In contrast, monodentate and @,y-bidentatetriphosphate complexes show a more extended polyphosphate chain with P-P-P angles as high as 118’. The a,y-bidentate complex shows a P-P-P angle of 86O, which is within the range observed for the tridentate complexes due to the steric constraint imposed by the a , y coordination. The P( 1)-O( 12)-P(2)-0(23) and O(12)-P(2)-0(23)-P(3) torsion angles are constrained for all tridentate complexes due to the steric requirement that all three phosphate groups be within coordination distances from the metal atom. One of these two torsion angles is positive (51-99’) while the other is negative (-67’ to -1 13’) as seen in Table 111. Thus, the preferred conformation of the triphosphate chain in the tridentate complexes is in the gauche+,gauche- domain. Even though the tridentate complexes are highly restrained due to the triple coordination,
VOL. 2 4 , N O . 26, 1 9 8 5
CRYSTAL STRUCTURE O F MANGANESE ATP
7829
M
/4.905 Mn(2)-6.831-5.239
yp(31f6.475 \ P(2)5.592-Mn(21 /
1
p(Ok 7.771
FIGURE 3:
Interactions and distances between the phosphorus atoms of the triphosphate moiety and the hexaaqua-coordinated Mn(2) ions.
a
‘%
FIGURE 1 : ORTEP drawings of MnATP and the two DPA molecules drawn to the same scale. Nonhydrogen atoms are represented by 50% probability ellipsoids, and hydrogen atoms are represented by spheres of arbitrary size.
d
I.568
bo&,
FIGURE 2: The hexacoordinated Mn( 1) ion showing some important bond lengths for the triphosphate moiety.
nevertheless the chelate rings display a certain latitude of conformation flexibility. Metal Coordination. The Mn(1) atom is coordinated to the triphosphate chains of two different dyad-related ATP molecules (Figure 2). Both the MnATP and MgATP complexes display a,P,y-tridentate metal coordination to the triphosphate chain. The a, p, and y coordination bond lengths of 2.205 (6), 2.156 (4), and 2.144 (5) A, respectively, are
slightly longer than the MgATP coordination distances of 2.10 (2), 2.08 (2), and 2.01 (2) A,respectively. In the MnATP structure, the a coordination distance is significantly longer (0.05 A) than the 0 or y coordination distances. However, in the MgATP complex, it is seen that the a and p coordination bonds are almost equal in length while the y coordination bond is about 0.07 A shorter. The increase of the Mn coordination bond lengths compared to the corresponding Mg distances arises mainly from the difference in the radii of the metal ions. The elongation of the a coordination bond seen for MnATP is much more pronounced in the structure of ZnATP-2,2’bipyridine (Orioli et al., 1981) and CuATP-1,lOpherianthroline (Sheldrick, 1982). In ZnATP the a coordination distance of 2.71 (4) A is over 0.6 A longer than either the p or y distances of 2.09 (3) and 2.02 (3) A, respectively. The Zn atom is also coordinated to the y position of a second ATP molecule with a distance of 2.00 (3) A. The other two coordination sites of the Zn atom are directed to the nitrogen atoms of a bipyridine molecule with distances of 2.13 (3) and 2.14 (4) A. Nearly the same coordination scheme was observed in the structure of CuATP where the a coordination distance of 2.878 (9) A is over 0.9 A greater than the p or y distances of 1.942 (9) and 1.925 (8) A, respectively. The coordination distance to the y-phosphate of the second ATP is 2.284 (8) A while the distances to the phenanthroline nitrogen atoms are 1.989 (10) and 2.013 (10) A. Triphosphate Interactions and Environment. In many enzymatic reactions, a second metal ion besides the metal-ATP substrate is involved in substrate binding and enzyme catalysis (Mildvan, 1981; Rosevear et al., 1983). The present structure is of interest because a similar scenario is observed with a second manganese ion also present in the vicinity of the triphosphate chain. The metal-triphosphate dimers are sandwiched between hexaaqua-coordinated Mn(2) atoms (Figure 3). For clarity, one of the triphosphate chains is removed from Figure 3, and two translation-related Mn(2) complexes are shown. Several of the anionic phosphate oxygen atoms engage in hydrogen-bonding interactions to the water molecules coordinated to Mn(2). Both Mn( 1)-Mn(2) distances in Figure 3 are identical (5.322 A) due to symmetry. The Mn(2) to P distances, which differ depending on which Mn(2) atom is used for the measurement, vary from 4.905 to 7.771 A (Figure 3). These distances would be important for the interpretation of NMR and other spectroscopic data. The octahedral coordination angles around the manganese ions (see Table IV) show distortions from the ideal values of 90’ and 180O. The angles about the Mn( 1) coordination bonds show a maximum distortion of 5 O from the ideal octahedral values. The polyphosphate torsion angles are highly con-
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Table I: Fractional Positional Parameters for All Atoms of the MnATP-DPA Complex“ atom X Y z B, multiplicity atom X 2220 (6) 3499 (2) 4283 (2) 3.2 (1) OW(5) 2638 (17) 2166 (8) 2956 (3j 4466 (2j 3.3 i2j OW(6) 4899 (17) 2226 (7) 3.4 (2) 2845 (2) 4875 (2) OW(7) 5000 2347 (7) 2.7 (2) 3346 (3) 5109 (2) OW(8) 3088 (19) 2381 (6) 2.6 (2) 3913 (3) 4957 (2) OW(9) 3653 (22) 2298 (7) 2.6 (1) 4002 (3) 4512 (2) OW(10) 3068 (18) 2332 (7) 3.5 (1) 4519 (2) 4328 (2) H(N1) 215 2485 (7) 3.3 (1) 5292 (2) 4306 (2) H(2) 212 2522 (9) 3.5 (2) 3977 (3) 5628 (2) Hl(N6) 231 2446 (6) 2.8 (1) 3383 (2) 5545 (2) H2(N6) 239 2292 (8) 2.9 (2) 2893 (3) 5847 (2) 258 H(8) 3310 (8) 3.6 (2) 2898 (3) 6199 (2) 234 H(19 4421 (6) 5.3 (2) 2594 (3) 6066 (2) H(2’) 354 2535 (10) 2633 (3) 4.1 (2) 6572 (2) H(02’) 525 2489 (8) 5.8 (2) 2013 (2) 6551 (2) H(39 290 1173 (9) 3.9 (2) 2850 (3) 6497 (2) H(03’) 335 2937 (2) 1065 (6) 3.7 (1) 6038 (1) H(4? 53 4.7 (2) 3432 (4) 6724 (3) Hl(5’) 79 809 (9) 4.3 (2) 3867 (2) 6614 (2) H2(5’) -1 1710 (6) 4533 (1) 1527 (2) 6734 (1) 3.72 (3) H(NlD1) -90 4595 (2) 1197 (7) 7190 (2) 4.4 (1) H(lD1) -93 4844 (2) 2661 (7) 6548 (2) 5.3 (2) H(2D1) -104 4702 (2) 268 (7) 6454 (2) 4.6 (1) H(3D1) -104 5237 (1) -735 (2) 6502 (1) 3.24 (3) H(4D1) -96 3.8 (1) 6947 (1) -1250 (5) 5242 (2) H(5D1) -82 6146 (2) 5.0 (2) -1657 (6) 5186 (3) H(6D1) -88 3.8 (1) 170 (6) 5798 (2) H(NlD2) 568 6421 (1) 6259 (1) H(lD2) 567 6754 (1) 3.31 (3) 853 (2) 7168 (1) 3.7 (1) 1100 (6) 5940 (2) H(2D2) 575 6783 (2) 6.1 (2) -90 (8) 6767 (2) H(3D2) 565 5.1 (2) 6522 (2) 2122 (7) 6405 (3) 553 H(4D2) 7500 2.75 (1) 5280 (1) 0 0.500 554 H(5D2) 5322 (3) 3.8 (3) 0.500 -1076 (12) 5014 (6) H(6D2) 566 5.0 (2) 5242 (3) 4452 (3) -937 (8) Hl(W1) 392 4811 (3) 4416 (3) -987 (8) 5.3 (2) H2(W1) 361 4630 (3) 3882 (4) -1008 (8) 5.0 (2) Hl(W2) 424 4858 (3) 3371 (3) -940 (8) 4.0 (2) H2(W2) 283 5304 (3) 3410 (3) -884 (8) 4.0 (2) Hl(W3) 296 4.9 (2) 5486 (3) 3957 (4) -905 (9) H2(W3) 240 5662 (19) 4803 (12) 5328 (8) 10.7 (8) 0.500 H(W4) 514 5347 (5) 8.9 (4) 5651 (10) 5451 (5) Hl(W5) 304 4964 (4) 5626 (5) 6.8 (3) 5677 (9) H2(W5) 305 4881 (5) 9.9 (5) 5638 (1 1) 6260 (6) Hl(W6) 399 H2(W6) 511 5213 (5) 8.3 (5) 5580 (10) 6593 (6) 5620 (5) 8.8 (4) 5583 (12) 6395 (6) H(W7) 424 5682 (5) 8.6 (4) 5645 (12) 5782 (6) Hl(W8) 325 7500 5924 (2) 9.00 (8) 5000 0.500 H2(W8) 275 6861 (3) 11.3 (3) 4256 (10) 5859 (5) Hl(W9) 294 7701 (5) 19.1 (7) 3731 (11) 5189 (9) H2(W9) 424 3210 (13) 6411 (6) Hl(W10) 214 7665 (3) 14.0 (5) 2409 (4) 5000 13.7 (8) 0.500 H2(W10) 327 . , 7500 “Values are multiplied by lo4 for nonhydrogen atoms and lo3 for hydrogen atoms. ’
.
I
strained in this structure to preserve the nearly octahedral coordination about the hexacoordinated metal atom. In contrast, the coordination around Mn(2) deviates substantially from octahedral symmetry with distortions as high as 20° from the ideal value. Geometry and Conformation of the Nucleoside Moiety. All of the endocyclic adenine ring bond lengths are within 0.01 A of the ideal bond lengths for the protonated adenine base except for the N(7)-C(8) bond length of 1.292 (9) A, which is significantly shorter than the ideal value of 1.316 A (Taylor & Kennard, 1982). The adenine ring bond angles all agree within 1’ with the ideal angles. The glycosyl torsion angle x [0(4’)-C( l’)-N(9)*(8)] (Sundaralingam, 1969) is in the anti domain with a value of 66 (1)O. The ribose ring bond lengths agree to within 0.025 A with those reported for the structure of adenosine (Lai & Marsh, 1972) while the endocyclic ring angles agree to within lo. The ribose ring adopts the C(2’)-endo C(3’)-exo conformation with pseudorotation parameters P = 179 (1)O and 7, = 34.1 (6)O (Altona &
.
I
Y 8853 (6) 4299 (9) 3573 (9) 3026 (6) 4366 (6) 7635 (5) 351 268 454 48 1 412 256 335 279 278 185 255 334 352 504 485 478 386 303 302 399 463 500 533 644 706 667 560 545 616 488 521 663 627 216 845 91 1 446 406 382 345 296 467 440 774 723
z
7218 (4) 6712 (9) 7500 8685 (4) 8888 (6) 7156 (7) 40 1 43 1 408 447 588 568 627 616 685 663 660 703 665 567 539 463 434 472 549 583 566 539 472 458 517 586 598 680 677 785 78 1 793 752 724 722 744 672 695 758 874 839 888 865 723 724
B, multiplicity 15.8 (5) 29.2 (11) 19.4 (12) 0.500 20.3 (6) 22.0 (8) 20.3 (7) 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 0.500 4.0 0.375 4.0 0.375 4.0 0.875 4.0 4.0 4.0 0.875 8.0 0.500 8.0 0.375 8.00.375 8.0 0.875 8.0 8.0 8.0 0.875 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0
Sundaralingam, 1972; Rao et al., 1981). The exocyclic C(3’)-C(4’)-C(5’)-0(5’) torsion angle (+) is gauche+ with a value of 55 (1)O. DPA Molecules. The 2,2’-dipyridylamine molecules are not coordinated to the metal ions. This is in contrast to the structure of Cu(I1)-uridine 5‘-monophosphate-2,2’-dipyridylamine (Fischer & Bau, 1978) where the aromatic base is tightly bonded to the copper ion. The disorder in the DPA molecules causes the observed bond distances and angles to be less accurate. The bond lengths range from 1.31 (1) to 1.40 (1) A for DPA(1) and from 1.29 (2) to 1.47 (2) A for DPA(2). These values can be compared with those for DPA (Johnson & Jacobson, 1973), where the average C-C bond length is 1.37 (1) A, the average ring C-N length is 1.34 (1) A, and the bridge C-N bond lengths are both 1.38 A. Hydrogen Bonding. On the basis of the water positions, all eligible protons appear to have reasonable hydrogen-bonding acceptors. A list of the proposed hydrogen bonds is given in Table V. N ( 1) of the adenine base, which is protonated with
CRYSTAL S T R U C T U R E OF M A N G A N E S E ATP
VOL. 24, NO. 26, 1985
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FIGURE4: Packing diagram of the MnATP-DPA complex viewed down the a axis. Note the DPA and A=A stacking hydrophobic walls along the b axis at c = 0 and 'Iz.
an N(1)-H(N1) distance of 0.86 A, is engaged in a very strong hydrogen bond to 03(P3) of a symmetry-related molecule with an N-0 distance of 2.535 (9) A. Hl(N6) is hydrogen-bonded to 02(P1) of the same symmetry-related molecule with an Ne-0 distance of 3.122 (8) A. H2(N6) is also engaged in a hydrogen-bonding interaction to N(7) of the same molecule with an Ne-N distance of 2.925 (7) A. The ribose O(2') atom is hydrogen-bonded to a water molecule of hydration E2.84 (2) A], and O(3') is hydrogen-bonded to the terminal phosphate group of a symmetry-related ATP molecule [2.64 (1) 211, The bridge nitrogen atom of DPA( 1) is engaged in a hydrogen bond to 02(P2) while the bridge nitrogen atom of DPA(2) is hydrogen-bonded to OW(9) with distances of 2.68 (2) and 2.74 (3) A, respectively. The water molecules are primarily hydrogen-bonded to each other and to the triphosphate chain. As seen in Figure 2, the hexaaqua Mn group hydrogen bonds to the a- and y-phosphate groups of ATP while a translation-related Mn group contributes the hydrogen bond to the P-phosphate of the same ATP molecule. The 18 unique water protons engage in hydrogen bonds varying from the extremely short OW(9)-OW(6) distance of 2.40 (3) A to the rather long OW(lO)-OW(4) distance of 3.36 (2) A. The special-position water molecules OW(4) and OW(7) have only one proton since the other proton is related to the first by a 2-fold rotation and each proton is engaged in exactly the same hydrogen-bonding interaction to different symmetryrelated molecules. Crystal Packing. A packing diagram of the MnATP-DPA complex is presented in Figure 4. The adenine base is engaged in a stacking arrangement with the DPA molecules. Two dyad-related adenine bases form a base pair involving N(6)-N(7) hydrogen bonds. These A=A self-pairs sandwich both DPA molecules. The A=A to DPA(1) base-stacking distance is 3.38 (4) A, and A=A to DPA(2) stacking distance is 3.37 (4) A, and the DPA(1) to DPA(2) distance is 3.48 (1) A, which when added together yield the length of the unit cell a axis. The base stacking of DPA( 1) and DPA(2) over the A=A pairs is shown in Figure 5 . The water molecules all tend to cluster in the vicinity of the hydrophilic polyphosphate chains to form water channels between the hydrophobic walls generated by the A=A and DPA stacked columns.
CONCLUSIONS The nature of metal binding to ATP has been investigated by nuclear magnetic resonance techniques both in solution and at the enzyme active site. The structures of MgATP and MnATP appear to differ in solution on the basis of several
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Table 111: Conformational Parameters for Various Triphosphate-Metal Complexes distances tridentate complexes MnATP MgATP CaATP NaATP(A) NaATP( B) ZnATP(A) ZnATP(B) CuATP(A) &ATP( B) ff,p,-f-Co-PPP monodentate complexes p-co-PPP 7-Co-PPP bidentate complexes ff,y-Co-PPP P,r-CG-PPP
(A)
torsion angles (deg) P(1)-O(l2)0(12)-P(2)P(2)-0(23) 0(23)-P(3)
angle (deg), P-P-P
P( 1)-P(2)
P(2)-P(3)
2.90 2.90 2.90 2.97 2.97 2.91 2.90 2.91 2.90 2.90
2.94 2.91 2.92 3.02 3.00 2.96 2.94 2.92 2.93 2.90
3.98 3.94 4.03 4.53 4.27 4.30 4.01 4.24 4.08 4.21
86 85 87 98 91 94 87 93 89 93
64 67 60 -116 51 -106 95 -109 99 -9 5
-100 -101 -97 83 -113 86 -67 77 -7 5 53
2.92 2.94
2.89 2.95
4.97 4.64
118 104
-74 -100
-145 -66
2.94 2.87
2.91 2.91
3.99 4.91
86 116
3 81
-106 115
Table IV: Metal Coordination Bond Angles in MnATP‘ angle angle (deal (deal 87.0 OW ( 1)-Mn( 2)-0 W (2) 89.7 89.1 OW( l)-Mn(2)-OW(3) 87.7 87.1 OW(2)-Mn(2)-OW(3) 80.2 90.6 0W( 1)-Mn(2)-OW( 1)* 171.7 89.7 OW(l)-Mn(2)-OW(2)* 83.9 176.8 OW(l)-Mn(2)-OW(3)* 96.5 175.3 0W(2)-Mn(2)-OW (2) * 81.0 96.2 OWI2l-Mn(2l-OW(3)* 159.4 ‘Since the metal atoms are on dyad symmetry axes, only eight of the fifteen possible angles about each metal atom are independent. Those atoms marked with an asterisk refer to the dyad-related atom.
~(1)-~(3)
3.34(4,8 DPA(I)
DPA (2)
A=P.
Table V: List of Proposed Hydrogen-Bonding Interactions in MnATP translation distance hydrogen bond sym” x y z (A) N ( l)-H(N1)*-03(P3) 2 0 1 1 2.535 (9) N(6)-H1 (N6)*-02(Pl) 2 0 1 1 3.122 (8) N(6)-H2(N6)-.N(7) 2 0 1 1 2.925 (7) 0(2’)-H(02’)-OW(8) 4 1 0 1 2.84 (2) 0(3’)-H(03’)*-02(P3) 5 0 -1 0 2.64 (1) Nl(Dl)-H(NlD1)*-02(P2) 1 0 0 0 2.68 (1) Nl(D2)-H(NlD2)*-0W(9) 4 1 0 1 2.74 (3) OW(l)-Hl(W1).-02(Pl) 1 0 0 0 2.99 (1) OW ( 1)-H2( W 1)-*03(P3) 1 0 0 0 2.73 (1) OW(2)-Hl(W2)-OW(6) 0 0 3.07 (3) 1 0 OW(2)-H2(W2)*-01(P2) 4 0 0 1 2.77 (1) OW(3)-Hl(W3)-*0(3’) 8 0 0 1 2.90 (1) OW(3)-H2(W3)***01(P3) 1 0 0 0 2.87 (1) OW(4)-H(W4)-02(P3) 5 0 -1 0 2.68 (1) OW(5)-Hl(W5).-0W(lO) 1 0 0 0 2.81 (2) OW(5)-H2(W5)-01 (Pl) 1 0 0 0 2.78 (2) 0W (6)-H 1(W6)*-02(P 1) 1 0 0 0 2.65 (2) OW(6)-H2(W6)*-OW(7) 0 0 2.97 (3) 1 0 OW (7)-H( W7)-0 W( 5) 8 0 -1 1 2.91 (2) OW(8)-H 1(W8)-OW(9) 1 0 0 0 3.16 (2) OW(8)-H2(W8)**.OW(lO) 8 0 -1 1 3.02 (3) OW(9)-Hl(W9)-*02(P2) 4 0 0 1 2.77 (2) OW (9)-H2( W9)***0 W (6) 4 1 0 1 2.40 (3) 5 -1 OW(lO)-Hl(WlO)-.OW(4) 0 0 3.36 (2) OW(lO)-H2(WlO).*.OW(3) 1 0 0 0 3.21 (2) ‘Symmetry codes: (1) x , y, z; (2) x , -y, -z; (3) -x, -y, ‘ I 2+ z; (4) --x, Y , ‘ 1 2 - 2; (5) ‘ 1 2 + X, ‘ 1 2 + Y , Z; (6) ‘ 1 2 + X, ‘ 1 2 - Y , -2; (7) ‘I2 X , ‘ 1 2 - Y , ‘1, + Z ; (8) ‘1, - X, ‘1, + V , ’ I , - Z.
NMR investigations. Cohn & Hughes (1 962) have shown that Mg2+ does not affect proton resonances in the adenine ring while 31Presonances for the 8- and y-phosphate groups implicate these phosphates as the position of Mg2+ binding.
e
-
-1
-
DPf(l)
FIGURE 5 : Stacking
-
*
3 48(118
- =
-
-t
3 37(4)8
I+
3.38(4)8
of the adenine-adenine self-pairs with both
DPA( 1) and DPA(2).
Happe & Morales (1966) provided further evidence for the absence of adenine ring interaction by showing that Mg2+had no effect on the nitrogen resonances in ATP. However, in the case of the Mn2+ion, Sternlicht et al. (1968) concluded that the Mn2+ion is equally bound to the a,0, and y positions as well as to the N(7) atom of the adenine ring. In contrast, Heller et al. (1970) conclude that metal ion binding to ring positions in MnATP is negligible. Wee et al. (1 974) proposed that this ambiguity for the MnATP system can be resolved by a model where, in addition to the a,&y-tridentate coordination to the polyphosphate chain, the Mn2+ion is indirectly bound to both N(6) and N(7) of two different ATP molecules via two coordinated water molecules. Granot & Fiat (1977) also observed indirect adenine base interactions for paramagnetic metal ions and showed that protonation of the base at N( 1) enhances the metal-base interaction by bringing the base closer to the triphosphate chain. Therefore, Mg does not appear to bind to the adenine base in solution while Mn appears to bind indirectly to the base N(6) and N(7) sites. The crystal structures of the known metal-ATP complexes can be classified into two types of dimers. In the first type, two metal ions are encapsulated by two ATP molecules as is observed in the structures of NaATP, ZnATP, and CuATP. In this group an approximate 2-fold rotation axis relating the two ATP molecules lies between the two metal ions. Since the symmetry is approximate, the two molecules can exhibit different conformations. In the second group, which includes
CRYSTAL STRUCTURE OF MANGANESE ATP
MnATP and its isomorphous counterparts, there is only one metal atom bound by two ATP molecules, and these ATP molecules are related by an exact 2-fold rotation axis passing through the metal ion. The first represents the formation of a true metal-ATP dimer while in the second case a pseudodimer is formed since both ATP molecules share the same metal ion, which is not involved in base binding. Although dimer formation does not normally occur in solution or at the enzyme active site, solution studies have suggested that the addition of an aromatic amine favors dimer formation (Martin & Mariam, 1980). The adenine base and the aromatic amine independently display affinity for metal ions; however, when together, the stacking interaction appears to predominate. Therefore, the metal ion tends to associate with the polyphosphate chains. The structure of MnATP demonstrates an extreme example of this effect with all six metal coordination sites occupied by phosphate oxygen atoms. Because of the importance of the stacking energy, the stacking option is preferred over metal coordination by the nucleic acid base. Although the crystal structures of all known ATP complexes exhibit dimer (or pseudodimer) formation in the solid state, the metal-ATP complex in solution probably does not favor dimer formation in the absence of aromatic amines. Therefore, in solution, the tridentate metal-coordinated ATP molecule will be expected to have three water molecules occupying the remaining metal coordination sites instead of the second ATP molecule. Upon enzyme binding, these water molecules either interact with or are replaced by enzyme functional groups in the active site. In addition, the triphosphate chain and the nucleoside moiety will have the potential to form several hydrogen bonds to the enzyme spacially similar to that observed for the ATP molecule in the crystal lattice.
SUPPLEMENTARY MATERIAL AVAILABLE A table giving the anisotropic temperature factors for nonhydrogen atoms and a table listing all observed and calculated structure factors (1 3 pages). Ordering information is given on any current masthead page. REFERENCES Altona, C., & Sundaralingam, M. (1972) J . Am. Chem. SOC. 94, 8205-8212. Cini, R., Cinquantini, A,, Burla, M. C., Nunzi, A., Polidori, G., & Zanazzi, P. F. (1982) Chim. Znd. (Milan) 64, 826. Chi, R., Sabat, M., Sundaralingam, M., Burla, M. C., Nunzi, A., Polidori, G., & Zanazzi, P. F. (1983) J . Biomol. Struct. Dyn. 1, 633-637. Chi, R., Burla, M. C., Nunzi, A., Polidori, G. P., & Zanazzi, P. F. (1984) J . Chem. SOC.,Dalton Trans., 2467-2476. Cornelius, R. D., & Cleland, W. W. (1978) Biochemistry 17, 3 279-3 28 6. Cromer, D. T., & Waber, J. T. (1965) Acta Crystallogr. 18, 1 04- 1 09.
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Fischer, R. E., & Bau, R. (1978) Inorg. Chem. 17, 27-34. Granot, J., & Fiat, D. (1977) J . Am. Chem. SOC.99, 70-79. Haromy, T. P., Gilletti, P. F., Cornelius, R. D., & Sundaralingam, M. (1984) J . A m . Chem. SOC.106, 2812-2818. Heller, M. J., Jones, A. J., & Tu, A. T. (1 970) Biochemistry 9 , 4981-4986. Johnson, C. K. (1976) Oak Ridge Natl. Lab., [Rep.] ORNL (US.)ORNL-5138. Johnson, J. E., & Jacobson, R. A. (1973) Acta Crystallogr., Sect. B Struct. Crystallogr. Cryst. Chem. 829, 1669-1674. Kennard, O., Isaacs, N. W., Motherwell, W. D. S.,Coppola, J. C., Wampler, D. L., Larson, A. C., & Watson, D. G. (1971) Proc. R . SOC.London, A 325, 401-436. Lai, T. F., & Marsh, R. E. (1972) Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. B28, 1982-1989. Larson, A. C. (1978) Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. B34, 3601-3604. Main, P., Germain, G., & Woolfson, M. M. (1970) MULTAN. A System of Computer Programs for the Automatic Solution of Noncentrosymmetric Crystal Structures, Universities of York, England, and Louvain, Belgium. Martin, R. B., & Miriam, Y. H. (1980) Met. Ions Biol. Syst. 8 , 57-124. Merritt, E. A,, & Sundaralingam, M. (1980) Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. B36, 2576-2584. Merritt, E. A,, & Sundaralingam, M. (1981) Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 8 3 7 , 1505-1 509. Merritt, E. A., Sundaralingam, M., & Cornelius, R. D. (1981) Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. B37, 657-659. Mildvan, A. S. (1 98 1) Philos. Trans. R . SOC.London, B 293, 65-74. Orioli, P., Chi, R., Donati, D., & Mangani, S. (1981) J. Am. Chem. SOC.103,4446-4452. Rao, S . T., Westhof, E., & Sundaralingam, M. (1981) Acta Crystallogr., Sect. A : Cryst. Phys., Diffr., Theor. Gen. Crystallogr. A37, 421-425. Schneider, S., & Collin, R. L. (1973) Znorg. Chem. 12, 21 36-21 39. Sheldrick, W. S. (1982) 2.Naturforsch., B: Anorg. Chem., Org. Chem., Biochem., Biophys., Biol. 37B, 863-871. Sternlicht, H., Jones, D. E., & Kustin, K. (1968) J . Am. Chem. SOC.90, 7110-7118. Stewart, R. F., Davidson, E. R., & Simpson, W. T. (1965) J . Chem. Phys. 42, 3175-3187. Sundaralingam, M. (1 969) Biopolymers 7, 821-860. Sundaralingam, M., & Haromy, T. P. (1985) Stereochem. Enzym. React. Proc. Steenbock Symp., 15th, 1985. Taylor, R., & Kennard, 0. (1982) J . Am. Chem. SOC.104, 3209-32 12. Wee, V., Feldman, I., Rose, P., & Gross, S . (1974) J . Am. Chem. SOC.96, 103-1 12.
