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Kinetic and crystallographic studies on 2-(-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site

¹ Institute of Organic and Pharmaceutical Chemistry, and ² Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 11635 Athens, Greece³ Department of Organic Chemistry, Faculty of Science, and ⁴ Department of Medical Chemistry, Research Centre for Molecular Medicine, University of Debrecen, Debrecen, Hungary

Reprint requests to: Nikos G. Oikonomakos, Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, 48, Vassileos Constantinou Avenue, 116 35 Athens, Greece; e-mail: ngo{at}eie.gr; fax: +30-210-7273-831.

(RECEIVED November 4, 2004; FINAL REVISION December 7, 2004; ACCEPTED December 10, 2004)

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of -D-glucopyranose, 2-(-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc-1-P) with K_i values of 145.2 (±11.6), 76 (±4.8), and 8.6 (±0.7) µM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb-methyl-oxadiazole complex, 1.92 Å; GPb-benzothiazole, 2.10 Å; GPb-benzimidazole, 1.93 Å). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (~ 32 Å) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.

Keywords: type 2 diabetes; glycogen phosphorylase; -D-glucopyranosyl analogs; inhibition; X-ray crystallography

Abbreviations: GPb, muscle glycogen phosphorylase b • PLP, pyridoxal 5'-phosphate • glucose, -D-glucose • Glc-1-P, -D-glucose 1-phosphate • methyl-oxadiazole, 2-(-D-glucopyranosyl)-5-methyl-1,3,4-oxadiazole • benzothiazole, 2-(-D-glucopyranosyl)-benzothiazole • benzimidazole, 2-(-D-glucopyranosyl)-benzimidazole • r.m.s. deviation, root-mean-square deviation.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041216105.

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Glycogen phosphorylase (GP), because of its central role in glycogen metabolism, has been exploited as a potential target for structure-based design of potent inhibitors, that may be relevant to the control of blood glucose concentrations in type 2 diabetes (Aiston et al. 2001, 2003; Latsis et al. 2002). Several regulatory binding sites, identified in GP, have been used as molecular targets (for review, see Oikonomakos 2002). These are the allosteric site that binds the activator AMP and the inhibitor glucose-6-P, the catalytic site that binds substrates glucose-1-P and glycogen, and the inhibitor glucose, the caffeine binding site, which binds caffeine and related compounds, and the new allosteric inhibitor or indole binding site that binds indole-2 carboxamides and -D-glucopyranosyl ureas. More specifically, the catalytic site has been probed with glucose analog inhibitors, designed on the basis of information derived from the crystal structure of T-state GPb–-D-glucose complex (Martin et al. 1991; Watson et al. 1994, 1995; Bichard et al. 1995; Oikonomakos et al. 1995, 2002a, b; Gregoriou et al. 1998; Somsák et al. 2001, 2003; Chrysina et al. 2003, 2004; Györgydeák et al. 2004). A common feature of these compounds is that upon binding at the catalytic site, they promote the (less active) T-state conformation of the enzyme through stabilization of the closed position of 280s loop (residues 282–287), which blocks access of the substrate to the catalytic site.

We report here on the kinetic and crystallographic study of three new lead compounds, derivatives of -D-glucopy-ranose, 2-(-D-glucopyranosyl)-5-methyl-1,3,4-oxadiazole, 2-(-D-glucopyranosyl)-benzothiazole, and 2-(-D-gluco-pyranosyl)-benzimidazole with GPb. The kinetic experiments reveal that the three compounds are potent competitive inhibitors with respect to the substrate Glc-1-P, whereas the crystallographic results show that each of the compounds binds at the catalytic site and stabilizes the closed position of the 280s loop. Additionally, benzimidazole can occupy the indole binding site and also a novel site that has not been observed previously. Benzimidazole is the first compound to show binding at this novel site.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
The inhibitory efficiencies of methyl-oxadiazole (2), benzothiazole (3), and benzimidazole (4) were tested with GPb, assayed into the direction of glycogen synthesis (Table 1). The three compounds exhibited competition with respect to Glc-1-P, at constant concentrations of glycogen (1% w/v) and AMP (1 mM). Benzimidazole was found to be a better inhibitor (K_i = 8.6 ± 0.7 µM) than benzothiazole (K_i = 76 ± 4.8 µ M) or methyl-oxadiazole (K_i = 145.2 ± 11.6 µM).

View this table: [in this window] [in a new window]   Table 1. Chemical structures of the C-(-D-glucopyranosyl) azoles 2-4, showing the numbering system used and inhibition constants

View larger version (96K): [in this window] [in a new window]   Figure 1. A schematic diagram of the muscle GPb dimeric molecule viewed down the molecular dyad. The positions are shown for the catalytic, allosteric, glycogen storage, the caffeine, the indole site, and a novel binding site for benzimidazole. The catalytic site, marked by benzimidazole, is buried at the center of the subunit and is accessible to the bulk solvent through a 15 Å-long channel. Binding of the competitive inhibitor benz-imidazole promotes the less active T state through stabilization of the closed position of the 280s loop (shown in black). The allosteric site, which binds the activator AMP (indicated in the figure), is situated at the subunit–subunit interface some 30 Å from the catalytic site. The inhibitor site or caffeine binding site, which binds purine compounds such as caffeine and flavopiridol (indicated) is located on the surface of the enzyme some 12 Å from the catalytic site and, in the T state, obstructs the entrance to the catalytic site tunnel. The glycogen storage site (with bound maltopentaose) is on the surface of the molecule some 30 Å from the catalytic site, 40 Å from the allosteric site and 50 Å from the new allosteric inhibitor site. The new allosteric or indole binding site, located inside the central cavity, formed on association of the two subunits, binds indole-2 carboxamide analogs, N-benzoyl-N'--D-glucopyranosyl urea, and benzimidazole (indicated). The novel binding site with bound benzimidazole, also located on the surface of the molecule, is some 31 Å from the catalytic site, 32 Å from the allosteric site, and 32 Å from the indole site.

View this table: [in this window] [in a new window]   Table 2. X-ray crystallographic analysis of 2-(-D-glucopyranosyl) azoles in complex with T-state GPb

View larger version (142K): [in this window] [in a new window]   Figure 2. Diagrams of the Fo - Fc electron density maps, contoured at 3, for the bound methyl-oxadiazole (A) and benzothiazole (B) at the catalytic site, and benzimidazole at the catalytic (C), indole (D), and the novel binding site (E). Electron density maps were calculated using the standard protocol as implemented in CNS v1.1 (Brünger et al. 1998) before incorporating ligand coordinates.

View larger version (67K): [in this window] [in a new window]   Figure 3. (A) Interactions between methyl-oxadiazole and GPb at the catalytic site. (B) Comparison between GPb-methyl-oxadiazole complex (light gray) and GPb--D-glucose complex (dark gray) in the vicinity of the catalytic site. The side chain of Leu136 and water molecules are not shown for clarity reasons.

View this table: [in this window] [in a new window]   Table 3. Hydrogen bond interactions of 2--D-glucopyranosyl) azoles at the catalytic site of GPb

In general, methyl-oxadiazole, on binding to GPb, makes a total of 14 hydrogen bonds and 80 van der Waals interactions (6 nonpolar/nonpolar, 18 polar/polar, and 56 polar/ nonpolar) (Table 4). Methyl-oxadiazole is a good inhibitor (K_i = 145.2 ± 11.6 µM) and binds almost 12 times more tightly than -D-glucose or 50 times more tightly than its parent compound -D-glucose (Martin et al. 1991), possibly because of the additional interactions of the methyl-oxadiazole group with the protein.

View larger version (66K): [in this window] [in a new window]   Figure 4. (A) Interactions between benzothiazole and GPb at the catalytic site. (B) Comparison between the GPb–benzothiazole complex (light gray) and the GPb--D-glucose complex (dark gray) in the vicinity of the catalytic site.

Benzothiazole was a better inhibitor (K_i = 76 ± 4.8 µM) than methyl-oxadiazole (K_i = 145.2 ± 11.6 µM). Benzothiazole, on binding to GPb, makes a total of 13 hydrogen bonds and 79 van der Waals interactions (10 nonpolar/non-polar, 7 polar/polar, and 62 polar/nonpolar), the most important of which are those to the side chains of Leu136 and His341. In the GPb–benzothiazole complex, S1 of thiazole makes seven van der Waals contacts to Asn284, His377, and Thr378. The structural results show that sulphur can be accommodated in this position with essentially no disturbance of the structure.

Benzimidazole (4)
Substitution of S1 in benzothiazole by nitrogen (N2 in benzimidazole) resulted in a better inhibitor with a K_i = 8.6 ± 0.7 µM. N2 of the imidazole moiety makes a direct hydrogen bond with O of His377, which shifts by ~ 0.8 Å toward the ligand (dihedral angle [CB-CA-C-O] changes by ~ 21°) (Fig. 5A). This interaction has been observed for the amide nitrogen of -1-N-acetamido-D-glucopyranose (K_i = 32 µM) (Oikonomakos et al. 1995; Watson et al. 1995), spirohydantoin of glucopyranose (K_i = 3.1 µM) (Bichard et al. 1995; Gregoriou et al. 1998; Oikonomakos et al. 2002a) and D-glucopyranosyl formamide analogs (K_i:310–1800 µM) (Chrysina et al. 2003). N1 makes water-mediated interactions with Leu136 N and Asp283 OD1 through Wat311 (Wat233 in the -D-glucose structure), which shifts by ~ 1.0 Å; Wat311, in turn, interacts with Gly135 N and Asp283 OD1 through Wat72, which shifts by ~0.7 Å, and Gly134 N, Gly137 N, Glu88 OE2, and Asn133 N through Wat103 (Wat123 in the -D-glucose structure), which shifts by ~ 0.7 Å (Table 3).

View larger version (68K): [in this window] [in a new window]   Figure 5. (A) Interactions between benzimidazole and GPb at the catalytic site. (B) Comparison between the GPb–benzimidazole complex (light gray) and the GPb–-D-glucose complex (dark gray) in the vicinity of the catalytic site.

Superposition of the three compounds bound at the catalytic site shows that the ligands superimpose well (not shown), except that the replacement of N2 by S changed the geometry of the imidazole ring and imposed a different orientation of the phenyl ring (shifted by ~ 0.8 Å compared to that of benzimidazole). The positions of the glucosyl components of each of the structures are similar, indicating that the glucosyl recognition site does not change substantially.

Benzimidazole, on binding to GPb, also occupies the indole binding site, shown to bind a number of indole-2-carboxamide analogs (Rath et al. 2000a) and N-benzoyl-N'--D-glucopyranosyl-urea (Oikonomakos et al. 2002b). The site is almost buried in the central cavity of the enzyme. Access to the central cavity (Fig. 1) is partially blocked at one end of the twofold axis, which relates the two subunits, by residues Arg33, His34, Arg60, and Asp61 from the cap region (residues 36–47) and 2 helix (residues 47–78) and their symmetry-related equivalents and at the other end by the tower helix residues Asn270, Glu273, and Ser276 of the tower helix (residues 262–276) and their symmetry-related equivalents.

The conformation of the bound benzimidazole at the indole binding site is similar to that described above for the catalytic site, except that the torsion angles O5-C1-C13-N1 and O5-C5-C6-O6 are -116° and -75°, respectively (different from those in the catalytic site -116° and 67°). Benzimidazole on binding at the indole binding site of GPb makes a total of nine hydrogen bonds (Table 5) and exploits 78 van der Waals interactions, 25 of which are interactions between nonpolar groups, and there are in total 20 contacts to the symmetry related subunit (Table 6; Fig. 6A). The benzimidazole group exploits 55 van der Waals contacts, which are dominated by the contacts (26) to Arg60. Arg60 NH1 and NH2 are hydrogen bonded to the hydroxyl O2 group, which in turn, is hydrogen bonded to hydroxyl O3' from the symmetry-related molecule of benzimidazole. There is also a hydrogen bond from N1 to Glu190 O, and Asn187 O (through Wat418) and from N2 to Thr38' O (where the prime refers to residues from the symmetry-related subunit). The glucopyranose moiety makes 24 van der Waals contacts with the protein, of which 16 are to residues from the symmetry-related subunit. The glucopyranose and His57' rings are inclined approximately 80°, and there are extensive contacts between them. There is a hydrogen bond from Lys191 NZ to the hydroxyl O6. In addition, hydroxyl groups O2 and O3 are hydrogen bonded to O3' and O2', respectively, of the glucopyranose from the symmetry-related bound benzimidazole molecule. However, there are no aromatic–aromatic, amino-aromatic, and CH- interactions that are characteristic of the tight binding of the 4-fluorobenzyl moiety of CP320626 (IC₅₀ = 334 ± 10 nM) (Oikonomakos et al. 2000).

View larger version (75K): [in this window] [in a new window]   Figure 6. (A) Interactions between benzimidazole and GPb at the indole binding site. Residues from subunit 1 are shown in light gray and their symmetry-related equivalents (from subunit 2) are shown in dark gray. (B) Comparison between the GPb–benzimidazole complex (light gray) and the GPb–N-benzoyl-N'--D-glucopyranosyl-urea complex (dark gray) in the vicinity of the indole binding site.

A structural comparison between the GPb–N-benzoyl-N'--D-glucopyranosyl urea and GPb–benzimidazole complex shows that the two complexes have very similar overall structures and do not differ significantly at the interfaces between monomers. The two structures closely resemble each other; the positions of the C, main-chain and side-chain atoms for residues 18–249, 262–312, and 326–829 deviate from their mean positions by 0.297 Å, indicating no significant changes between the complex structures. Figure 6B shows details of the contacts made at the indole binding site for GPb–N-benzoyl-N'--D-glucopyranosyl urea complex and the GPb–benzimidazole complex. The two ligands superimpose quite well except that the glucopyranose and the phenyl moieties of N-benzoyl-N'--D-glucopyranosyl urea are shifted ~ 1.0 Å and ~ 1.5 Å toward the symmetry-related residues Phe53', His57', and Pro188', and the hydrophobic pocket lined by Val64, Thr67, and Pro229, respectively (compared to the position of benzimidazole).

There were also indications in both 2F_o–F_c and F_o–F_c electron density difference maps for binding of a benzimidazole molecule at a novel binding site located at the surface of the molecule ~ 31 Å from the catalytic site, ~ 32 Å from the indole binding site, and ~ 32 Å from the allosteric site (Fig. 1). The electron density was sufficiently well resolved, for the substituent group of benzimidazole and most of the atoms of glucopyranose, to indicate the conformation of the ligand (Fig. 2E). The conformation of the bound benzimidazole at the new site is similar to that described above for the catalytic and the indole binding site, except that the torsion angles O5-C1-C13-N1 and O5-C5-C6-O6 are -154° and –32°, respectively.

The interactions of benzimidazole with GPb involve six hydrogen bonds and 27 van der Waals contacts, 11 of which are interactions between nonpolar groups (Fig. 7A; Tables 5,6). The benzimidazole substituent pocket is formed by residue 202 (of the 8 strand-residues 198–209), residues 219–221 (of the 9 strand-residues 212–223, antiparallel to 8), residues 249 and 252 (of the loop of residues 248–260 between 11, residues 237–247, and tower helix-residues 261–274), residues 271 and 272 (of the tower helix), and residues 290 and 294 (of the 8 helix-residues 289–314). In the native 100 K GPb structure (Gregoriou et al. 1998), the site contains five located water molecules (Wat497, Wat534, Wat747, Wat823, and Wat828), and these are apparently displaced upon binding of benzimidazole. There are hydrogen bonds from N1 and N2 of the imidazole ring to the side-chain atoms Gln219 OE1 and Lys294 NZ, respectively. The glucopyranose moiety makes relatively few van der Waals interactions with the protein, and there are four potential hydrogen bonds from the sugar ring O5 and hydroxyl O2 and O6 groups to Gln219 NE2, Glu290 OE2, and Lys294 NZ.

View larger version (42K): [in this window] [in a new window]   Figure 7. (A) Interactions between benzimidazole and GPb at the new ligand binding site. (B) Comparison between the GPb–benzimidazole complex (light gray) and native GPb (dark gray) in the vicinity of the new site. (C) Comparison between the GPb–benzimidazole complex (light gray) and human liver GPa (dark gray) in the vicinity of the new binding site.

Comparison of the GPb–benzimidazole complex with the R state human liver GPa (Rath et al. 2000b) suggests that the ligand at the new binding site is likely to have lower affinity for the R state GPa (Fig. 7C). Superposition of the structure of the liver GPa (subunit A) with the structure of the GPb–benzimidazole complex gave an r.m.s. deviation of 1.146 Å for C atoms, for ordered residues 24–205, 214–249, 270–281, 289–313, 327–553, 564–711, and 787–835. In the R state relative to the T state, one subunit is rotated so as to bring the two subunits closer at the 2' (residues 48'–78')-cap (42–45) interface and causing them to move further apart at the tower/tower' interface of the dimer. In the inactive human liver GPa, the catalytic site is blocked by the 280s loop (residues 280–286) that follows the tower helix (residues 267–274). In the active human liver GPa, the tower helices are shorter by two turns and the 280s loop folds up in an ordered conformation and allows access of the substrate glycogen to the catalytic site and also creates the recognition site for inorganic phosphate substrate (Johnson and Lewis 2001). Thus, changes at the subunit interfaces are closely coupled to changes at the catalytic site. Modeling studies suggest that if GPb–benzimidazole were to be incorporated into the corresponding site of the R-state liver GPa structure, Gln219, Val221, and Leu271 of the tower helix would be required to move to open up the site in order to optimize contacts with benzimidazole. Hence, it would be anticipated that the affinity of benzimidazole for the R state would be less than that of the T state, but there is no experimental evidence for this. On the basis of our observations, we suggest that the binding of benzimidazole at the novel binding site stabilizes the T-state conformation of the enzyme. The physiological significance of this site has yet to be established.

In a crystallographic experiment with 5 mM benzimidazole (2 h soak) (data not shown), electron density maps indicated strong binding of benzimidazole at the catalytic site, but practically no binding at either the indole inhibitor site or the novel binding site. It seems, therefore, that the catalytic site is the primary binding site for benzimidazole binding.

In conclusion, benzimidazole was found to be the most potent inhibitor among the three compounds, with a K_i value of 8.6 µM. Replacement of N2 of benzimidazole by either oxygen (O7) or sulphur (S1) in methyl-oxadiazole or benzothiazole, respectively, disrupted the hydrogen bond formed between N2 and His377 O in the GPb–benzimidazole complex and resulted in less potent inhibitors. The van der Waals interactions with the residues of the catalytic site are reduced from 89 in benzimidazole to 79 in methyl-oxadiazole or benzothiazole. Benzothiazole (K_i = 76 µM) was a better inhibitor than methyl-oxadiazole (K_i = 145.2 µM). A possible explanation for the difference in K_i may lie in the number of the nonpolar/nonpolar contacts of the two ligands with the protein. In the GPb–benzothiazole structure there are 10 nonpolar/nonpolar interactions, while in the GPb–methyl-oxadiazole there are only six nonpolar/nonpolar interactions. In addition, the number of water molecules displaced in the complex with methyl-oxadiazole is reduced to two, compared with three observed in the complex with benzothiazole, possibly leading to an increase in entropy. The ability of the ligands to displace waters, to exploit existing or recruit new waters appears to be important for the variation observed in the potency of the inhibitors studied. The structural results also show that the three compounds interact with the side chains of Asn282, Asp283, and Asp284 holding the 280s loop in its closed T-state conformation, therefore inhibiting access of the substrate to the catalytic site. These observations will be of value in the design of further potent and specific inhibitors of the enzyme. Nevertheless, further investigation is required to explore whether the new (benzimidazole) binding site, located in a surface cavity formed by residues Phe202, Tyr203, Val221, and Phe252, could be exploited as a target for the design of new inhibitors.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
The synthesis of methyl-oxadiazole (2), benzothiazole (3), and benzimidazole (4) was described in Hadady et al. (2004). Rabbit muscle GPb was isolated, purified, recrystallized, and assayed as previously described (Oikonomakos et al. 1995). Kinetic studies were performed in the direction of glycogen synthesis in the presence of constant concentrations of glycogen (1% w/v), AMP (1 mM), and various concentrations of Glc-1-P (3–20 mM) and the inhibitors. The kinetic results are summarized in Table 1.

Native T-state GPb crystals, grown in the tetragonal lattice, spacegroup P4₃2₁2 (Oikonomakos et al. 1985), were soaked with 100 mM methyl-oxadiazole (for 1 h and 40 min) or 56 mM benzothiazole in 18% DMSO (for 2 h) or 100 mM benzimidazole (for 2 h), prior to data collection. Diffraction data were collected from single crystals in room temperature using synchrotron radiation source at Daresbury Laboratory (beamline PX-9.6, = 0.87 Å) and EMBL-Hamburg outstation (beamline X13, = 0.8015 Å). Data for the 5 mM benzimidazole soak (2 h) (not shown) were collected at a resolution of 2.26 Å on an Image Plate RAXIS IV mounted on a Rigaku Ru-H3RHB generator with a belt drive rotating anode ( = 1.5418 Å). Data reduction and integration followed by scaling and merging of the intensities obtained were performed with Denzo and Scalepack, respectively, as implemented in HKL suite (Otwinowski and Minor 1997).

The crystal structure of GPb--D-glucose was used as a starting model for crystallographic refinement against the experimental data by applying a standard protocol as implemented by CNS (Brünger et al. 1998), involving rigid body refinement followed by positional and individual B-factor refinement (with bulk solvent correction). 2F_o–F_c and F_o–F_c electron density maps calculated were visualized using the program for molecular graphics "O" (Jones et al. 1991). Ligand models, constructed and minimized using the program SYBYL (Tripos Associates Inc.), were fitted to the electron density maps after adjustment of the torsion angles of the computed models. Alternate cycles of manual rebuilding with "O" and refinement with CNS improved the quality of the models.

The stereochemistry of the protein residues was validated by PROCHECK (Laskowski et al. 1993; CCP4, 1994). Hydrogen bonds and van der Waals interactions were calculated with the program CONTACT as implemented in CCP4 (1994) applying a distance cutoff of 3.3 Å and 4.0 Å, respectively. The program calculates the angle O. . .H. . .N (where the hydrogen position is unambiguous) and the angle source . . . oxygen-bonded carbon atom. Suitable values are 120° and 90°. A Luzatti plot (Luzatti 1952) suggests an average positional error for all structures of approximately 0.22–0.25 Å. GPb complex structures were superimposed over well-defined residues using LSQKAB (CCP4 1994). Comparisons of the water molecules in the complex structures were made taking into consideration their equivalent positions. The schematic representation of the crystal structures presented in all figures were prepared with the programs MolScript (Kraulis 1991) and BobScript (Esnouf 1997) and rendered with Raster3D (Merritt and Bacon 1997). The coordinates of the new structures have been deposited with the RCSB Protein Data Bank (http://www.rcsb.org/pdb) with codes 1XL0 [PDB] (methyl-oxadiazole complex), 1XL1 (benzothiazole complex), and 1XKX (benzimidazole complex).

	Acknowledgments

 
This work was supported by the Greek General Secretariat for Research and Technology (GSRT) through the programs PENED-204/2001 (to M.N.K.), ENTER-EP6/2001 (to E.D.C.), the Joint Research and Technology project between Greece and Hungary (2004–2006) (to N.G.O and L.S.), the Hungarian Scientific Research Fund (OTKA T37210 [GenBank] to P.G., T46081 [GenBank] to L.S.), the Hungarian Ministry of Health (ETT 030/2003 to P.G., T.D., and L.S.), the SRS Daresbury Laboratory through IHPP HPRI-CT-1999-00012, and the EMBL-Hamburg outstation under FP6 "Structuring the European Research Area Programme" contract no. RII3/CT/2004/5060008. Zs.H. thanks CMS Chemicals (Oxford, UK) for a stipend supporting her Ph.D. studies with L.S. in Debrecen. We also acknowledge the assistance of the staff at SRS and EMBL-Hamburg outstation for providing excellent facilities for X-ray data collection.

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