Received 1 May 2012
The first 3':5'-cyclic nucleotide-amino acid complex: L-His-cIMP1
In the crystal structure of the L-His-cIMP complex, i.e. L-histidinium inosine 3':5'-cyclic phosphate [systematic name: 5-(2-amino-2-carboxyethyl)-1H-imidazol-3-ium 7-hydroxy-2-oxo-6-(6-oxo-6,9-dihydro-1H-purin-9-yl)-4a,6,7,7a-tetrahydro-4H-1,3,5,25-furo[3,2-d][1,3,25]dioxaphosphinin-2-olate], C6H10N3O2+·C10H10N4O7P-, the Hoogsteen edge of the hypoxanthine (Hyp) base of cIMP and the Hyp face are engaged in specific amino acid-nucleotide (HiscIMP) recognition, i.e. by abutting edge-to-edge and by - stacking, respectively. The Watson-Crick edge of Hyp and the cIMP phosphate group play a role in nonspecific HiscIMP contacts. The interactions between the cIMP anions (anti/C3'-endo/trans-gauche/chair conformers) are realized mainly between riboses and phosphate groups. The results for this L-His-cIMP complex, compared with those for the previously reported solvated L-His-IMP crystal structure, indicate a different nature of amino acid-nucleotide recognition and interactions upon the 3':5'-cyclization of the nucleotide phosphate group.
High-resolution crystal structures of amino acid-nucleotide complexes can serve as model systems to investigate protein-nucleic acid interactions and their manner of recognition. Nucleic acid-binding proteins discriminate between potential binding sites based on their sequence or structure or a combination of both. The forces involved in noncovalent protein-nucleotide/nucleic acid complex formation are relatively weak (hydrogen bonds, hydrophobic interactions, and - stacking between the base and aromatic side chain), but still crucial for the stability of the complex, and the overall affinity results from the sum of many interactions. Electrostatics, hydrophobic interactions and polar contacts involving direct or water-mediated hydrogen bonds are all evident and widespread (Rice & Correll, 2008).
In DNA-protein complexes, histidine has been shown to prefer interactions with guanine (Luscombe et al., 2001), which is a 2-amino derivative of hypoxanthine and thus has the same Hoogsteen edge crucial for protein-DNA recognition. The nucleotide Hoogsteen edge, with its unique functional groups (hydrogen-bond donors and acceptors) exposed in the major groove, is associated with the main specifity of protein-B-DNA complexes. This is distinct from protein-RNA and protein-nucleotide recognition, in which the different edges of a base may play a similar role in protein binding, viz. the Watson-Crick edge, the Hoogsteen edge and the sugar edge (Leontis & Westhof, 2001).
The only examples of coordinating-metal-free complexes of amino acid/oligopeptide-nucleotide deposited so far in the Cambridge Structural Database (CSD, Version 5.33; Allen, 2002) are three isomorphous complexes of IMP (inosine 5'-monophosphate) with L-Ser (CSD refcode ZUWQEN; Mukhopadhyay et al., 1995), L-Glu (QUSMIA; Bhattacharya et al., 2000) and L-Gln (LIRQUY; Bera et al., 1999), two complexes of N7-methylguanosine 5'-monophosphate (m7GMP) with L-Phe and Trp-Glu [DUMJEA10 (Ishida et al., 1988) and SEKXIP10 (Ishida et al., 1991), respectively], and, most recently, isomorphous L-His-IMP·MeOH·H2O (TUVZIU; Slepokura & Petrus, 2010) and L-His-IMP·2.5H2O (Slepokura & Makarewicz, 2012). No crystal data on cyclic nucleotide-amino acid complexes are available to date. The analysis of structural details is difficult in the first four of the above-mentioned structures, mainly due to the lack of at least some of the H atoms and therefore incorrect ionization states. In addition, the crystal structure of the highly hydrated IMP-Ser/Glu/Gln complexes is dominated by IMP-IMP interactions and no direct specific nucleobase-amino acid side-chain interactions are observed.
Therefore, the synthesis and structural analysis of the title L-His-cIMP complex, (I), which reveals both specific and nonspecific interactions of L-histidine (His) with inosine 3':5'-cyclic monophosphate (cIMP), have been undertaken. The conformation and binding mode of cIMP are compared with those found in the solvated L-His-IMP complexes and the other amino acid-IMP complexes deposited in the CSD, as well as with cIMP crystal structures reported to date, viz. cIMP·H2O (BEPRAP; Sundaralingam et al., 1982) and [Cu(cIMP)(phenanthroline)(H2O)2]NO3·2H2O (CATTEW; Sheldrick, 1983).
The asymmetric unit of (I) consists of a cIMP monoanion and a His cation, as shown in Fig. 1. Analogous to IMP in two isomorphous solvated L-His-IMP complexes, the cIMP nucleotide in (I) adopts a typical anti conformation about the N-glycosidic C1'-N9 bond, although with a CN O4'-C1'-N9-C4 torsion angle of -174.66 (12)°, which is uncommon [compared with -139.2 (5) and -138.5 (3)° observed in IMP in its complexes]. As shown in Fig. 2, the orientation of the Hyp base relative to the ribose ring in IMP and cIMP in their complexes with L-His is the main difference in the structures of these analogous nucleotides, despite the same formal anti glycosyl conformation.
Analogous to IMP in the solvated L-His-IMP complexes, the cIMP nucleotide in (I) is puckered in an envelope C3'-endo (3E) manner, which is confirmed by the pseudorotation parameters (Rao et al., 1981) P = 14.7 (1)° and m = 46.5 (1)°, and the Cremer & Pople (1975) puckering parameters q2 = 0.4561 (15) Å and 2 = 283.22 (17)°. It is worth mentioning that the 3E conformation is one of two typically observed in free IMP and other free and protein-bound nucleotides (Allen, 2002; Moodie & Thornton, 1993), and was also observed in the solvated L-His-IMP complexes, but not in the other previously reported amino acid-nucleotide (both IMP and m7GMP) complexes, in which C2'-endo (2E) puckering is the most common. However, the ribose in the only crystal structures of cIMP reported so far, viz. cIMP·H2O (BEPRAP; Sundaralingam et al., 1982) and [Cu(cIMP)(phenanthroline)(H2O)2]NO3·2H2O (CATTEW; Sheldrick, 1983), is found in a different puckering conformation, i.e. C3'-endo/C4'-exo (3T4). The ribose in protein-bound IMP does not reveal any conformational preference either.
The chair (C) conformation of the six-membered O/P/O/C/C/C 1,3,2-dioxaphosphorinane ring of the cIMP anion is significantly flattened at the P atom. This deformation of the ring towards an envelope (E) is reflected in the values of the dihedral angles between the least-squares plane through the four central atoms of the ring (O3'/O5'/C3'/C5') and the O3'/P1/O5' and C3'/C4'/C5' planes [1 = 44.5 (1)° and 2 = 59.4 (1)°; 2 - 1 = 14.9 (2)°], as well as in the Cremer-Pople puckering parameters: Q = 0.5959 (13) Å, = 172.88 (12)° and = 346.0 (11)°. It has been shown that, in small six-membered cyclic phosphates, the extent of CE deformation is related to protonation or esterification of the cyclic phosphate group (Slepokura & Lis, 2004; Slepokura, 2008; Slepokura & Mitaszewska, 2011). It is convenient to use the values of |2 - 1| (defined as the dihedral angles between the O-P-O and C-C-C planes, respectively) for a quantitative description of ring flattening. In small cyclic phosphates, the |2 - 1| values amount to several degrees for compounds with an ionized phosphate group, and to more than 12° for acidic or esterified molecules. However, this general rule seems not to be true in the cIMP anion of (I) presented here, in which 2 - 1 = 14.9 (2)°, nor in other cIMP anions reported to date (2 - 1 = 18.2 and 14.8° for BEPRAP and CATTEW, respectively), nor in the other 3':5'-cyclic nucleotides deposited in the CSD, in which 2 - 1 ranges from 10.9 to 20.9° (Allen, 2002).
Selected geometric parameters for (I), given in Table 1, indicate the deformation of the phosphate group from the ideal tetrahedral shape, which is seen particularly in the endocyclic O3'-P1-O5' and exocyclic O3-P1-O5 bond angles, which are, respectively, the smallest [103.48 (6)°] and the largest [117.75 (6)°). These values of the endo- and exocyclic O-P-O angles correlate with the respective P-O bond lengths. In (I), as in other ionized six-membered cyclic phosphates (Slepokura, 2008; Slepokura & Mitaszewska, 2011), the P-Oendo bonds are all approximately 1.6 Å, and roughly 0.1 Å longer than the P-Oexo bonds.
The L-His cation in its complexes with both IMP and cIMP has the carboxy group deprotonated, and amino atom N11 and imidazole atom N16 (N) protonated. As seen in Fig. 2(a), the His conformation is different. In both cases, the planes defined by the imidazolium ring and by the backbone NH3+-C12-COO- fragment are both approximately perpendicular to the C12-C13-C14 plane, which is reflected in the interplanar angles of 78.4 (1) and 87.6 (1)°, respectively, in (I), compared with 86.8 (4)/87.7 (4)° and 88.4 (2)/88.3 (2)° in two solvated L-His-IMP complexes (Slepokura & Petrus, 2010; Slepokura & Makarewicz, 2012). The location of protonated atoms N11 and N18 (N) on different sides of the His cation in (I) differentiates it from His in complexes with IMP, and plays a role in the HiscIMP and HisIMP interactions, as will be discussed below. As shown in Fig. 2, and confirmed by the pseudotorsion angles N11-C12-C14-C15 of 2.7 (1)° [for His in (I)] and N11-C12-C14-N18 of -11.2 (1) and -10.6 (3)° (for His in IMP complexes), in L-His-cIMP the carboxylate (COO-) group and the protonated N18 atom (N) are located on the same side of the His cation, whereas in the solvated L-His-IMP complexes, protonated atoms N11 (NH3+) and N18 (N) are on the same side of the His cation.
All the differences noted above in the chemical nature and structure of the components of the His-IMP and His-cIMP complexes translate into the nature of the interactions between His cations (HisHis) and between nucleotide anions (cIMPcIMP) and the most interesting and crucial to the amino acid-nucleotide recognition process, i.e. the HiscIMP contacts. As shown in Fig. 3, both HisHis and cIMPcIMP interactions arrange adjacent His cations or cIMP anions into infinite ribbons running down the a axis. The overall architecture of the His ribbon in (I) is similar to that in the solvated L-His-IMP complexes: each His cation is joined to four adjacent cations (related by a direct a-axis translation and a 21 symmetry operation) via a network of bifurcated N-HO hydrogen bonds of a salt-bridge type, which are, however, much more compact than in His-IMP complexes (Fig. 3a and Table 2). As in His-IMP complexes, the imidazolium rings, which are located outside the ribbon, are exposed to specific amino acid-nucleotide interactions (Fig. 3b).
In contrast, the interactions between the cIMP anions in (I) are much less numerous than the IMPIMP contacts in solvated His-IMP complexes. In (I), a direct a-axis translation generates infinite ribbons of cIMP anions via O-HO interactions linking the ribosyl O2'-H2' group with atom O3 of an adjacent cyclic phosphate group, and via C-HO contacts linking the same hydroxy group with an adjacent ribosyl C atom (Fig. 3b). Notably, the hypoxanthine bases in (I) are not involved in the cIMPcIMP contacts, and therefore may be used more efficiently for His-cIMP recognition. In addition, the monoionized cyclic phosphate group in (I) has no H atom, which excludes the possibility of phosphate-phosphate interactions. All these observations result in significant differences in mutual orientation and internucleotide interactions between IMP and its cyclic analogue, cIMP in (I). It is worth mentioning that internucleotide contacts seem to predominate in the crystal structures of previously reported nucleotide-amino acid complexes. They are realised mainly via sugar-phosphate interactions (all but His-IMP) and contacts involving the base: sugar-base, like O2'N3(Hyp) in His-IMP complexes, and base-phosphate, like N1(Hyp)OP in three other IMP complexes. In Phe-m7GMP and solvated His-IMP complexes, additional phosphate-phosphate interactions also exist. Against this background, the internucleotide interactions in (I), with only sugar-phosphate contacts present, appear to be very simple.
In both the His-cIMP, (I), and His-IMP crystal complexes, the nucleotide Hoogsteen edge, commonly used in protein-nucleic acid recognition, is also involved in amino acid-nucleotide (HiscIMP and HisIMP) interactions. As shown in Figs. 1 and 3(b), in (I), the histidine side chain-Hyp base recognition is realised via specific edge-to-edge hydrogen bonds utilizing both Hyp Hoogsteen atoms N7 and O6, i.e. N18-H18O6(Hyp) and C17-H17N7(Hyp) (Table 2), as well as via - stacking of the cation type [centroid-centroid distances of 3.409 (2) and 3.438 (2) Å, respectively, for Cg(His)Cg(Hyp-six-membered) and Cg(His)Cg(Hyp-five-membered) involving the Hyp anion at (x + 1, y, z), with an interplanar spacing of 3.22 (1) Å; Fig. 3b]. In the isomorphous His-IMP complexes, interionic C15-H15O6(Hyp) and N16-H16N7(Hyp) interactions are found. Different HisHoogsteen(Hyp) hydrogen bonds in His-cIMP and His-IMP complexes are accompanied by different conformations of the His cation (discussed above) and thus different orientations of the imidazolium ring relative to the nucleotide base. However, in each case, the orientation is retained such that the interactions of the and positions of the His cation with, respectively, atoms O6(Hyp) and N7(Hyp) of the nucleotide anion are achieved.
The specific nucleotide-amino acid interactions involving the base Hoogsteen edge are crucial in the recognition process. Direct amino acid side-chain-Hyp interactions were not observed in the other IMP-amino acid complexes, except for His-IMP, but they may be analysed in macromolecular protein-IMP complexes [see Slepokura & Petrus (2010) for examples]. However, it is also known that, in protein-IMP complexes, the Watson-Crick edge is also involved in nucleotide-amino acid interactions, e.g. N1(Hyp)amino acid side-chain interactions are commonly observed. As shown in Fig. 4, in (I), each cIMP anion utilizes the base Watson-Crick edge to form nonspecific N-HO and C-HO HiscIMP interactions, linking the Hyp atom N1 to the carboxylate group of the adjacent His cation, and Hyp atom O6 to the C12-H12 group of the same His cation. In this way, two different R22(8) rings (Bernstein et al., 1995) are generated for each Hyp base with the participation of two different His cations. In contrast, the Watson-Crick edge of IMP in His-IMP complexes is involved in solvent-mediated IMPMeOH/H2OIMP contacts, and therefore does not bind His at all.
As seen from a number of contacts involving the cIMP nucleotide anionic cyclic phosphate group in (I), it may be stated that its role in nucleotide-amino acid interactions is much smaller. The phosphate group, as it has no H atom, forms only two hydrogen bonds: one of a salt-bridge type with ammonium atom N11, and the other with imidazolium atom N16 of a different His cation (Fig. 4 and Table 2). As a result of these interactions, each cIMP anion is bound to four different His cations: to two via the nucleotide base and to the other two via the cyclic phosphate group. The fifth His cation is joined by - stacking of the cation type, as shown in Figs. 3(b) and 4. A similar observation is noted in His-IMP complexes. However, despite the same number of surrounding ions, the nature of these interactions is significantly different. In His-IMP complexes, as many as three of the nearest His cations are bound to the IMP anion via the phosphate group (two of them in a monodentate manner and the third in a bidentate manner).
In conclusion, this paper reports the preparation and solid-state structure of, and intermolecular interactions in, the first example of a 3':5'-cyclic nucleotide-amino acid complex, namely L-His-cIMP, (I). By comparison with the analogous L-His-IMP complexes, it has been demonstrated that the 3':5'-cyclization of the nucleotide may reduce the nucleotide-nucleotide interactions and therefore intensify the nucleotide-amino acid interactions. In (I), both the Hoogsteen edge and the face of the Hyp base participate in specific HiscIMP recognition via edge-to-edge interactions and - stacking. The cIMP base Watson-Crick edge is involved in additional nonspecific nucleotide-amino acid contacts, which are not observed in L-His-IMP complexes. In contrast, the role of the phosphate group in nonspecific interactions is reduced compared with His-IMP complexes. This report has also focused on the cIMP conformation, against the background of the available structural information on 3':5'-cyclic nucleotides.
| || Figure 1 |
Views of the His cation and cIMP anion of (I), showing the atom-numbering schemes and the symmetry-independent N-HO and C-HN hydrogen bonds (dashed lines). The intramolecular O2'-H2'O3' contact is also shown. Displacement ellipsoids are drawn at the 50% probability level.
| || Figure 2 |
Comparison of the conformations of (a) the cIMP/IMP anions and (b) the His cations in L-His-cIMP (black; this work) and L-His-IMP·MeOH·H2O (grey) (Slepokura & Petrus, 2010; light orange in the electronic version of the paper). The common reference points are the labelled atoms. In IMP, the relative orientation of the hypoxanthine (Hyp) base and ester atom O5' is stabilized by an intramolecular C8-H8O5' hydrogen bond (dashed line).
| || Figure 3 |
(a) The ribbon of His cations running down the a axis and (b) the specific HiscIMP edge-to-edge hydrogen bonds (thick dashed lines; red in the electronic version of the paper) and - stacking (thin dashed lines; red) with the cIMP ribbon. Intermolecular HisHis and cIMPcIMP interactions are shown as dashed lines (blue). H atoms not involved in hydrogen bonds have been omitted for clarity. [Symmetry codes: (iv) x + 1, y, z; (v) x + , -y + , -z.]
| || Figure 4 |
The complete set of HiscIMP interactions in (I). Specific and nonspecific hydrogen bonds are shown as heavy dashed lines and - stacking is shown as thin dashed lines. H atoms not involved in hydrogen bonds have been omitted for clarity. [Symmetry codes: (ii) x - , -y + , -z + 1; (iii) -x + , -y + 1, z - ; (vi) -x + 2, y + , -z + ; (viii) x + , -y + , -z + 1.]
An MeOH-water solution (1:1 v/v) containing a 1:1 molar ratio of cyclic inosine 5'-phosphate sodium salt, Na(cIMP) (10 mg; Sigma) and L-histidine (L-His, 4.4 mg; ROTH) was evaporated slowly at room temperature for several days to give colourless columns of (I).
All H atoms were found in difference Fourier maps. In the final refinement cycles, they were positioned geometrically and treated as riding atoms, with C-H = 0.95-1.00 Å, N-H = 0.88-0.91 Å and O-H = 0.84 Å, and with Uiso(H) = 1.2Ueq(C,Nsp2) or 1.5Ueq(O,Nsp3).
Data collection: CrysAlis CCD (Oxford Diffraction, 2009); cell refinement: CrysAlis RED (Oxford Diffraction, 2009); data reduction: CrysAlis RED; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: DIAMOND (Brandenburg, 2005); software used to prepare material for publication: SHELXL97.
Supplementary data for this paper are available from the IUCr electronic archives (Reference: QS3016 ). Services for accessing these data are described at the back of the journal.
Financial support from the Faculty of Chemistry, University of Wroclaw (grant No. 1494/M/WCH/11), is gratefully acknowledged.
Allen, F. H. (2002). Acta Cryst. B58, 380-388.
Bera, A. K., Mukhopadhyay, B. P., Ghosh, S., Bhattacharya, S., Chakraborty, S. & Banerjee, A. (1999). J. Chem. Crystallogr. 29, 531-540.
Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555-1573.
Bhattacharya, S., Bera, A. K., Ghosh, S., Chakraborty, S., Mukhopadhyay, B. P., Pal, A. & Banerjee, A. (2000). J. Chem. Crystallogr. 30, 655-663.
Brandenburg, K. (2005). DIAMOND. Crystal Impact GbR, Bonn, Germany.
Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354-1358.
Flack, H. D. (1983). Acta Cryst. A39, 876-881.
Ishida, T., Doi, M. & Inoue, M. (1988). Nucleic Acids Res. 16, 6175-6190.
Ishida, T., Iyo, H., Ueda, H., Doi, M., Inoue, M., Nishimura, S. & Kitamura, K. (1991). J. Chem. Soc. Perkin Trans. 1, pp. 1847-1853.
Leontis, N. B. & Westhof, E. (2001). RNA, 7, 499-512.
Luscombe, N. M., Laskowski, R. A. & Thornton, J. M. (2001). Nucleic Acids Res. 29, 2860-2874.
Moodie, S. L. & Thornton, J. M. (1993). Nucleic Acids Res. 21, 1369-1380.
Mukhopadhyay, B. P., Ghosh, S. & Banerjee, A. (1995). J. Chem. Crystallogr. 25, 477-485.
Oxford Diffraction (2009). CrysAlis CCD and CrysAlis RED in Xcalibur PX software. Oxford Diffraction Ltd, Yarnton, Oxfordshire, England.
Rao, S. T., Westhof, E. & Sundaralingam, M. (1981). Acta Cryst. A37, 421-425.
Rice, P. A. & Correll, C. C. (2008). In Protein-Nucleic Acid Interactions: Structural Biology. Cambridge: RSC Publishing.
Sheldrick, W. S. (1983). Z. Naturforsch. Teil B, 38, 982-987.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122.
Slepokura, K. (2008). Carbohydr. Res. 343, 113-131.
Slepokura, K. & Lis, T. (2004). Acta Cryst. C60, o315-o317.
Slepokura, K. & Makarewicz, E. (2012). Private communication (deposition number 878282). CCDC, Union Road, Cambridge, England.
Slepokura, K. & Mitaszewska, I. (2011). Acta Cryst. C67, o161-o165.
Slepokura, K. & Petrus, R. (2010). Acta Cryst. C66, o265-o269.
Sundaralingam, M., Haromy, T. P. & Prusiner, P. (1982). Acta Cryst. B38, 1536-1540.