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Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP⁺ complex reveals a novel cofactor binding mode

Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA

(RECEIVED December 19, 2006; FINAL REVISION February 25, 2007; ACCEPTED February 27, 2007)

	Abstract

TOP Abstract Introduction Results and Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
Plasmid-encoded bacterial R67 dihydrofolate reductase (DHFR) is a NADPH-dependent enzyme unrelated to chromosomal DHFR in amino acid sequence and structure. R67 DHFR is insensitive to the bacterial drug trimethoprim in contrast to chromosomal DHFR. The crystal structure of Q67H mutant of R67 DHFR bound to NADP⁺ has been determined at 1.15 Å resolution. The cofactor assumes an extended conformation with the nicotinamide ring bound near the center of the active site pore, the ribose and pyrophosphate group (PP_i) extending toward the outer pore. The ribonicotinamide exhibits anti conformation as in chromosomal DHFR complexes. The relative orientation between the PP_i and the nicotinamide ribose differs from that observed in chromosomal DHFR–NADP⁺ complexes. The coenzyme displays symmetrical binding mode with several water-mediated hydrogen bonds with the protein besides ionic, stacking, and van der Waals interactions. The structure provides a molecular basis for the observed stoichiometry and cooperativity in ligand binding. The ternary model based on the present structure and the previous R67 DHFR–folate complex provides insight into the catalytic mechanism and indicates that the relative orientation of the reactants in plasmid DHFR is different from that seen in chromosomal DHFRs.

Keywords: crystal structure; folate metabolism; NADP⁺ ; R67 DHFR; symmetric binding; trimethoprim resistance

	Introduction

TOP Abstract Introduction Results and Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
Dihydrofolate reductase (DHFR, EC 1.5.12.3 [EC] ) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) utilizing the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. THF is required for the synthesis of thymidylate, purine nucleosides, methionine, and other metabolic intermediates. Therefore, inhibition of DHFR results in cessation of precursors necessary for DNA synthesis, leading to cell death. Trimethoprim (TMP) has been used in the treatment of various bacterial infections. Bacterial resistance to TMP is correlated with the production of novel R-plasmid–encoded DHFRs (Fleming et al. 1972). Type II R-plasmid–encoded R67 DHFR is of special interest because it is genetically and structurally unrelated to chromosomal DHFR.

R67 DHFR and chromosomal DHFRs have distinct structural and kinetic features. At physiological pH, the functional R67 DHFR is a homotetramer (M_r 8.5 kDa; 78 amino acid residues per subunit), and at low pH, native tetrameric R67 DHFR dissociates to form inactive dimers (Nichols et al. 1993). By contrast, the chromosomal DHFR is a monomeric enzyme with a core topology that resembles the classic Rossmann-fold usually associated with the binding of NADPH (Bellamacina 1996), whereas R67 DHFR has entirely different molecular architecture (five-stranded -barrel) and belongs to a group of nonclassical dinucleotide binding proteins that have a variety of folds. Structures of the dimeric and terameric forms of R67 DHFR have been determined (Matthews et al. 1986; Narayana et al. 1995). The tetramer displays a large central pore (Fig. 1A) with three mutually perpendicular and intersecting twofold axes passing through the middle of the pore. The K_i (TMP) for R67 DHFR is 0.15 mM versus 20 pM for chromosomal DHFR (Howell 2005).

View larger version (24K): [in this window] [in a new window]   Figure 1. Cofactor binding and stoichiometry. (A) The enzyme is comprised of four subunits (1–4) shown in gray and black. The simultaneous binding of two cofactor molecules (CPK model without adenosine 2'-phosphate portion) on the front or rear side of the active site pore results in steric clash, primarily between the ribose groups. (B) The protein portions comprised of residues Val66, His67, Ile68, and Tyr69 from four subunits bordering the central active site pore are shown as black and gray ribbons. The paired cofactor molecules that approach each other from opposite sides (indicated by arrows that align with a twofold symmetry axis) exhibit minimal stacking of the nicotinamide rings. Vertical bars align with a twofold axis, and the symmetry-related His67 side-chains are shown as van der Waals surfaces. The central dot shows another twofold axis passing through the plane of the paper. (C) Schematic diagram representing the possible modes of cofactor binding. Left and right of the vertical bar denote the front and rear portions of the pore as viewed down one of the two short axes of the active site. There are two binding positions in each half of the pore. The sites represented as horizontal lines are labeled A and B for one half of the pore and C and D for the other half of the pore (top panel on the left). The center of the pore is depicted as a vertical bar. The three lower panels on the left show possible binding modes without steric hindrance. The bottom panel on the left shows one bound NADP+ molecule in site A (see text for details). It can also be bound in either site B or C or D (data not shown). The right part of the figure shows different binding modes that are precluded due to stereochemical clashes between the cofactors. It may be noted that binding site A stacks on site D and clashes with site C but is not shown in the figure for clarity (the same applies to site B). The extent of stacking of ligands is larger in the R67 DHFR–folate complex than in the holoenzyme complex.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
Overall structure
The enzyme folding is similar to the previously published structures (Fig. 1A), with NADP⁺ bound in the central active site (Fig. 1A,B). Least-squares fit of C atoms of residues 21–78 in apo R67 DHFR (Narayana 2006) with equivalent atoms in the holoenzyme complex (root-mean-square deviation [RMSD] of 0.30 Å) shows a rigid protein backbone. Steric clashes between the symmetry-related ligands restrict the number of bound cofactors per tetramer (Fig. 1C).

Conformation of the bound cofactor
NADP⁺ binds to R67 DHFR in an extended conformation with the nicotinamide ring near the center of the active site and the remaining portion stretching toward the outer pore. In general, the cofactor in the enzyme-bound form exhibits extended conformation in contrast to the folded architecture in solution. The nicotinamide, ribose, and the pyrophosphate (PP_i) group were seen in a difference Fourier map (Fig. 2A,B,C).

View larger version (38K): [in this window] [in a new window]   Figure 2. Omit difference Fourier map and environment of the bound cofactor. Fo – Fc omit map was computed using protein atoms and solvent molecules, excluding those in the active site pore. (A) The nicotinamide ring is superimposed on the map contoured at 2.5. (B) The density for nicotinamide ribose (1.5) was supportive for an anti conformation about the glycosidic bond. (C) The PPi moiety was seen at 3.0 level (gray). The anomalous difference Fourier map (20–1.15 Å resolution) showed distinct and strong peaks (blue) corresponding to the PN and PA atoms of the cofactor. (D) Schematic diagram depicting direct and water-mediated (single or double) interactions (distances 3.3 Å) between the cofactor and the enzyme. The cofactor atoms are shown as sticks. The atoms belonging to the four subunits are in blue, cyan, green, and red. The dashed lines indicate hydrogen bonds.

The nicotinamide ribose adopts a C2'-endo geometry as in chromosomal DHFRs. Although there is close resemblance with respect to the nicotinamide-ribose moiety between R67 DHFR and the chromosomal DHFRs, the conformations of the rest of the cofactor differ markedly. Major deviations occur at torsion angles O4'-C4'-C5'-O5' and C4'-C5'-O5'-PN. Viewing along the virtual PA···PN bond, the phosphate groups are staggered. The adenosine 2'-phosphate moiety is generally ordered in chromosomal DHFR complexes, unlike R67 DHFR.

Cofactor binding stoichiometry
Two combinations, either sites A and D or sites B and C, are free from steric clashes between the cofactors and are designated as paired sites for ease of description of interligand interactions (Fig. 1C). The structure reveals that each half of the pore can accommodate only one NADP⁺, resulting in a maximum of two NADP⁺ molecules per tetramer (Fig. 1B). Nonetheless, the stoichiometry for NADP⁺ is 1 per tetramer according to ITC studies (Bradrick et al. 1996; Park et al. 1997). To reconcile this observation, we interpret that due to electrostatic repulsion between the positively charged nicotinamide rings occupying the paired sites, the second available site is vacant, leading to one NADP⁺ per tetramer. Assuming NADPH binds in the same orientation as does NADP⁺, we conclude that two NADPH molecules bind per tetramer due to the absence of repulsive forces between the paired nicotinamide rings. This result is in accord with previous ITC studies.

Cofactor–protein and cofactor–water–protein interactions
The binding position of NADP⁺ can be divided into three regions. Site A, where the pyridine ring and nicotinamide-ribose are bound; site B, where the PP_i is bound; and site C, where the adenosine 2'-phosphate is bound. Figure 2D shows the hydrogen bonding network involving the cofactor with protein and waters.

Site A
The side-chain of His67 is stacked on the B-face of the nicotinamide. Two pairs of symmetry-related side-chains of His67 form flat surfaces at the top and bottom in the center of the active site. His67 side-chains in each pair are inclined by 20°. The stacking interaction of the imidazole ring of His67 over nicotinamide and the concomitant van der Waals interactions between the side-chains in the nearby pair of His67 residues may synergistically contribute in large part to the tight binding of the cofactor compared with the wild-type enzyme.

The carboxamide nitrogen N7 of the nicotinamide donates a hydrogen bond to carbonyl oxygen of Val66. The oxygen O7 forms hydrogen bonds with water molecules Wat145, Wat146, and Wat149. The exocyclic oxygen O2'N has water-mediated interactions with carbonyl oxygen atoms of Val66 and Ile68 (Fig. 2D). The ribose O3'N forms an intramolecular hydrogen bond with O2A and a water-mediated interaction with carbonyl Ile68. The endocyclic oxygen O4' is in van der Waals contact with the side-chain of His67.

Site B
O1N of the phosphate anion interacts with amino acid residues through one or two water molecules. The interacting residues are Lys32, Ser34, Ala36, Tyr46, Thr51, Gly64, Val66, and Ile68. There is an intraligand link between O1N and O5'A via two water molecules, Wat144 and Wat151. The phosphate atom O2N has direct interaction with the hydroxyl group of Tyr69. Further, O2N displays a water-mediated interaction with Ala37:O and Ser65:O. The phosphodiester oxygen O3 forms a direct hydrogen bond, albeit long (3.2 Å), to Lys32:N. A single water molecule (Wat144) bridges O5'A with Lys32:N and Ser34:O. Lys32 is conserved in all R-plasmid encoded variants, suggesting a functional role, and was implicated in binding to phosphate anions of the cofactor by ITC studies (Hicks et al. 2003).

Site C
There were no features corresponding to adenosine 2'-phosphate moiety in the difference Fourier maps, presumably due to a combination of flexibility, static disorder, and partial occupancy owing to symmetric binding. The mobility of this section may be important for its function. For a comparison of NADP⁺ and folate binding environment, see Supplemental materials.

Structure–cooperativity correlation
Previous ITC and solution NOE studies indicate that interligand interactions play a dominant role in binding and catalysis (Bradrick et al. 1996; Li et al. 2001). Two cofactors, one on each half of the tetramer, are bound as described above. Two microscopic K_d (K_d1 = 0.027 µM and K_d2 = 0.62 µM) were determined for the binding of NADPH to Q67H mutant enzyme, suggestive of two distinct binding sites exhibiting negative cooperativity (Park et al. 1997). Wat149 is conserved in all R67 DHFR structures determined to date. In the absence of the nearby NADPH, Wat149 is involved in hydrogen bonding interaction with the backbone amide of Ile68. Wat149 and the nicotinamide ring are mutually exclusive at a given cofactor binding site due to steric clashes. Therefore, binding of the cofactor entails displacement of Wat149 at a specific binding site. However, in the vacant paired site, symmetry-related Wat149 is unperturbed and may serve as an important structural feature for tight cofactor binding through hydrogen bond formation with O7 (Fig. 2D). When the second cofactor binds in the paired binding site, it will displace the symmetry-related water molecule Wat149 that is engaged by O7 of the first cofactor via hydrogen bonding. In addition, the second cofactor does not have access to its symmetry-related Wat149 for hydrogen bonding interaction because it was previously displaced by the first cofactor molecule. In other words, the two symmetry-related water molecules (Wat149) associated with the paired cofactors are displaced upon binding of the two cofactors. We presume that the first cofactor binding site is stronger due in part to the presence of the hydrogen bonding interaction with Wat149 among other interactions, whereas the binding of the second cofactor exhibits negative cooperativity due in part to loss of a favorable hydrogen bond involving Wat149 in both bound ligands. We differentiate two distinct binding sites for the cofactor based on the differential accessibility of a key water molecule Wat149. For details on the structural basis of cooperativity between the cofactor and folate, see Supplemental Materials.

Modeling ground state of the catalytic ternary complex
A previous model of the ternary complex (Narayana et al. 1995) is revised in light of the present holoenzyme structure. See Supplemental Material for details about the construction and figure of a catalytic ternary model. Highlights of the model are (1) the A-side of the nicotinamide ring is toward the pore where the pteridine ring resides in the Michaelis complex. This is in agreement with the previous finding that R67 DHFR belongs to the A-stereospecific class of dehydrogenases. (2) Enhanced stacking between pteridine and pyridine rings approximates an endo configuration, and the two rings tilt away from each other as suggested for the transition state complex (Andres et al. 1996). (3) The distances C3 (nic)-C7 (fol) and C5 (nic)-C7 (fol) are 3.64 Å and 4.3 Å, respectively, a trend consistent with the interligand NOE data (Li et al. 2001). Several interligand distances are in agreement with those reported in ILOE studies (see Supplemental Materials). (4) The nicotinamide C4, C5, and C6 atoms lie closest to C5 of folate, and the carboxamide group of the nicotinamide is nearest to C7 and N8 of folate in accord with the ILOE data. (5) The exocyclic NA2 (fol) is within hydrogen bonding distance to O2' of the nicotinamide ribose, as noted in ILOE studies. A key feature derived from the modeling studies is that R67 DHFR exhibits a different binding mode for the reactants compared with its chromosomal counterpart.

	Materials and Methods

TOP Abstract Introduction Results and Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
Protein preparation, crystallization, and data collection
Recombinant Q67H mutant of R67 DHFR was expressed and purified as previously described (Park et al. 1997). The N-terminal 16 residues were cleaved using chymotrypsin, yielding a fully active protein of 62 amino acids. The truncated enzyme bound to the cofactor was crystallized at 4°C, employing the protocol described previously for growing R67 DHFR crystals. X-ray data for a single crystal flash cooled at 100 K were collected at MacCHESS beam line F1. The data were processed using the HKL program (Table 1; Otwinowski and Minor 1997).

A F_o – F_c map was computed using protein and all water molecules except those in the active site. This map revealed features for the nicotinamide ring (2.5) (Fig. 2A), nicotinamide ribose (1.5) (Fig. 2B), and the PP_i group (3.0) (Fig. 2C). An anomalous difference Fourier map using the phases derived from the protein and water atoms yielded two strong peaks corresponding to the PN (5) and PA (3) atoms in the pyrophosphate (Fig. 2C). There was no density corresponding to adenosine 2'-phosphate of NADP⁺. Difference Fourier maps did not reveal residues 17–20. The final model has an R-factor of 19.5% (Table 1). All main-chain (, ) angles are within the allowed or generously allowed regions of the Ramachandran plot.

	Electronic supplemental material

TOP Abstract Introduction Results and Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
The Supplemental material contains a figure showing a stereo view of a ternary model and a table of distances derived from X-ray/model/ILOE studies. Details related to comparison of NADP⁺ and folate binding environment, molecular basis for cooperativity between cofactor and folate binding, and construction of a catalytic ternary model are presented.

	Footnotes

 
Supplemental material: see www.proteinscience.org

Reprint requests to: Narendra Narayana, Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA; e-mail: nxn17{at}case.edu; fax: (216) 368-8740.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062740907.

	Acknowledgments

TOP Abstract Introduction Results and Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
We thank Elizabeth Howell and Michael Jackson for providing us with constructs and samples for the initial experiments. We thank Umadevi for protein purification. We thank the staff at MacCHESS beam line (Cornell University) for assistance with the data collection. Atomic coordinates have been deposited in the Protein Data Bank under accession code 2P4T.

	References

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This Article Abstract Full Text (PDF) Supplemental Research Data All Versions of this Article: ps.062740907v1 16/6/1063    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Divya, N. Articles by Narayana, N. Search for Related Content PubMed PubMed Citation Articles by Divya, N. Articles by Narayana, N. Social Bookmarking                   What's this?