Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases

Abstract

dUTPases are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg²⁺-coordination. Our results on transient/steady-state kinetics, ligand-binding and a 1.80 Å-resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg²⁺ accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function.

1. Introduction

Mechanisms responsible for DNA integrity, such as controlled biosynthesis of building block nucleotides and DNA damage repair are of vital importance. The enzyme dUTPase plays a key role in these processes by catalyzing the pyrophosphate hydrolysis of dUTP thereby eliminating the possibility of thymine-replacing uracil incorporation into DNA and providing dUMP as the immediate precursor for de novo TMP biosynthesis [1]. The dUTPase activity resides in different protein families that form monomeric, dimeric or trimeric folds. I n trimeric dUTPases, the homotrimeric oligomerization is essential for adequate enzymatic function [2–6]. These enzymes are indispensable for viability in pro- and eukaryotes alike [7,8] and have been proposed as promising novel targets against cancer cells, Mycobacteria and Plasmodia [1,9–11].

Homotrimeric dUTPases provide an accommodation network for the substrates (dUTP + water) and the phosphate-chain chelating co-factor Mg²⁺ by a rare structural solution. Within the homotrimer, three active sites are formed and conserved sequence motifs (Motifs I through V) from all the three subunits contribute to each active site architecture. The dUTP binding pockets are situated at clefts between two neighboring subunits and are covered by the C-terminal segment of the third subunit. In 3D structures of apoenzymes (1Q5U, 1MQ7 [4,10]) and the recently published enzyme-substrate (dUTPase:Mg:α,β-imido-dUTP) complexes (2HQU, 2PY4 [12,13]), this interesting architecture could be analyzed in detail for human (hDUT) and M. tuberculosis (mtDUT) dUTPases that showed ordering of the full-length C-termini upon substrate binding (c.f. also relevant conclusions based on solution phase experiments [14]). Importantly, these structures represent relevant complexes closely mimicking that of the enzyme-substrate, as i) the α,β-imido-dUTP substrate analogue is also a substrate of dUTPase, it can be hydrolyzed, albeit rather slowly by the enzyme as shown in [15], and ii) the α-phosphorus atom of α,β-imido-dUTP is in the catalytically competent gauche conformation for nucleophilic attack within the enzyme active site, where the divalent metal ion was proposed to facilitate the gauche position (cf [10,16]). Based on these structures, a dual role for the strictly conserved aspartate residue within the dUTPase Motif I (Figure 1A) could be hypothesized. On one hand, this residue may be important in binding of the co-factor Mg²⁺ as its carboxylate group contacts two of the water molecules within the metal co-factor coordination sphere in E. coli, human and mycobacterial dUTPase:Mg:α,β-imido-dUTP complexes [10,12,13,15]. The divalent metal ion cofactor (Mg(II) or Mn(II)) within the active site contributes an approximately twofold catalytic rate enhancement for dUTPases from E. coli [17], Drosophila melanogaster [6] and M. tuberculosis [16] and also increases binding affinity of the triphosphate nucleotide ligand by a factor of around ten. On the other hand, the carboxylate group of the Motif I Asp residue also maintains H-bonded interaction with the terminal amino group of arginine in Motif IV. The dedicated role of the conserved residues in dUTPase function has been analyzed in kinetic and 3D structural details only for the catalytic aspartate within Motif III [15], and kinetic studies were also published for a conserved serine within Motif II [18], but not for other residues. To provide novel insights into the mechanism of this important and unique enzyme, with potential focus on the role of the metal co-factor, we decided to investigate the contribution of Motif I aspartate to structure and function.

Figure 1. Kinetic comparison of wild-type and mutant human and M. tuberculosis dUTPases.

A, Alignment of dUTPase sequences. Bold face, strictly conserved residues in dUTPases, grey background: conserved motifs. The strictly conserved Motif I aspartate is presented in red. B, Proton release kinetics of hDUT^D49N,F158W (grey line) and mtDUT^D28N,H145W (black line) as measured by single turnover stopped-flow (main panel), or by steady-state (inset) for hDUT^F158W (grey solid), hDUT^D49N,F158W (grey striped), mtDUT^H145W (black solid), and mtDUT^D28N,H145W (black striped) pH indicator assay. Measured curves were fiited by single exponentials. C, Single turnover measurements of the hydrolysis step of hDUT^D49N,F158W (open circle) and mtDUT^D28N,H145W (black square) followed by detection of the ³²PPi: γ³²P-dUTP ratio (c.f. Table I). 30 µM enzyme and 20 µM γ³²P-dUTP was used.

Here, we present kinetic and equilibrium ligand binding results with mutant human and M. tuberculosis dUTPases (hDUT^F158W, mtDUT^H145W, hDUT^D49N,F158W, mtDUT^D28N,H145W). The effect of the mutation on substrate/product binding was determined using the previously engineered active site label tryptophan (hDUT^F158W, mtDUT^H145W) [12–14]. The 3D structure of mtDUT^D28N in complex with Mg:α,β-imido-dUTP was determined by X-ray crystallography at 1.80 Å resolution, allowing detailed insights into the long-range conformational shifts and disorder induced by the Motif I mutation. For a relevant structural comparison, the 3D structure of C-terminally truncated M. tuberculosis dUTPase (mtDUT^T138STOP) was also determined at 1.80 Å resolution. Our results suggest that the perturbations induced by a single Asp/Asn change in Motif I are propagated through a chain of pair-wise interactions finally resulting in increased long-range disorder and drastic decrease of the catalytic efficiency.

2. Materials and Methods

Materials for electrophoresis or chromatography were from Bio-Rad or Amersham Biosciences, phenol red from Merck, molecular biology products were from New England BioLabs or Fermentas. Other materials were from Sigma or Stratagene.

Mutagenesis and protein expression

Site-directed mutagenesis to produce hDUT^D49N,F158W, mtDUT^D28N,H145W, mtDUT^D28N and mtDUT^T138STOP was performed by the QuickChange method (Stratagene) and verified by sequencing of both strands. Proteins were expressed and purified as N-terminally His-tagged constructs as described previously [12,14,19]. It was found that the Trp point mutations had no significant effect on the catalytic properties of dUTPases (cf also [12,14,19]). Stocks at 5–10 mg/ml were flash-frozen in liquid N₂ and stored at −70 °C.

Protein concentration in subunits was determined by UV spectrometry as described [12,13].

Fluorescence Spectra and Intensity Titrations

Fluorescence spectra and intensity titrations were recorded at 20 °C on a Jobin Yvon Spex Fluoromax-3 spectrofluorometer with excitation at 295 nm (slit 1 nm), emission between 320 and 400 nm (slit 5 nm), or at 353 nm as described in [13] using hDUT^F158W, mtDUT^H145W, hDUT^D49N,F158W and mtDUT^D28N,H145W proteins at 3 µM concentration in 20 mM Tris/HCl, 1 mM MgCl₂ and 150 mM NaCl, pH 7.5 buffer and titrated with α,β-imido-dUTP using minute aliquots from a concentrated stock solution.

Continuous spectrophotometric dUTPase assay

A JASCO-V550 spectrophotometer and 10 mm path length 20 °C thermostatted cuvettes were used. Phenol red assay was carried out according to [20,21] in 1 mM Hepes, pH 7.5, buffer containing 100 mM KCl, 40 µM phenol red, and 5 mM MgCl₂. Enzyme concentration was 100 nM for the hDUT^F158W, mtDUT^H145W and 4 µM for the hDUT^D49N,F158W and mtDUT^D28N,H145W enzymes. Substrate dUTP concentration was varied between 0.5–40 µM dUTP for the hDUT^F158W, mtDUT^H145W and 20–40 µM dUTP for the hDUT^D49N,F158W, mtDUT^D28N,H145W enzymes, respectively.

Stopped-flow experiments

Measurements were done using an SX-20 (Applied Photophysics, UK) stopped-flow apparatus, as described [14]. 100 µM phenol red in the above buffer was used and the absorbance was monitored at 559 nm at 20 °C. Time courses were analyzed using the curve fitting software provided with the stopped-flow apparatus or by Origin 7.5 (OriginLab Corp., Northampton, MA). Enzyme concentrations were 12.5 µM and 15 µM for the single Trp mutant and for the double mutant enzymes, respectively. Substrate concentrations were 7.5 (used for the single turnover conditions presented in Fig. 1B), 10 and 50 µM dUTP.

Radioactive assay

γ³²P-dUTP was synthesized according to [14]. Radioactive measurements were carried out with the double mutant human and M. tuberculosis enzymes according to [14]. Reaction mixtures containing 30 – 50 µM dUTPase and 20 µM dUTP in 20 mM Tris/HCl, 1 mM MgCl₂ and 150 mM NaCl buffer were incubated at 20 °C and stopped by the addition of 2 M HCl. γ³²P-dUTP and ³²PPi were separated using a 10% charcoal solution also containing 2 M HCl and 0.35 M Na-phosphate. Radioactivity was counted in water using a Wallac 1409 DSA liquid scintillation counter.

Crystallization and crystallography

The mutant proteins mtDUT^D28N and mtDUT^T138STOP were crystallized as described for the wild type enzyme [13]. Complete crystallographic datasets were recorded at the EMBL-beamline X12 of DESY (Hamburg). Data were processed using XDS [22] and XSCALE programs. Structures of the mutant enzyme:α,β-imido-dUTP:Mg²⁺ complexes were solved by molecular replacement (Molrep, [23]) using the protein part of the wild type M. tuberculosis dUTPase structure (PDB ID: 2PY4) [13] as the model. Model building and refinement were carried out using the Coot program [24] and Refmac [25] program of the CCP4 program package. Final models contain the bound substrate analogue Mg²⁺-α,β-imido-dUTP. For the sake of clarity, residues are numbered according to physiological dUTPase sequence, disregarding the recombinant tag, in the figures and throughout the text. Coordinates and structure factor data have been deposited with the Protein Data Bank with accession codes 3H6D and 3I93 (cf. Table I). Figures were produced using Pymol [26].

Table I.

Crystallographic data collection and refinement statistics

	mtDUTD28N PDB ID: 3H6D	mtDUTT138STOP PDB ID: 3I93
Space group	P63	P63
Cell parameters a, c (Å)	55.23, 83.65	54.59, 83.05
Data collection
Wavelength (Å)	0.97861	0.97861
Resolution (Å)	20.0 – 1.80 (1.85-1.80)b	19.44 – 1.80 (1.85 – 1.80)b
Measured reflections	45296 (2517)	49588 (3376)
Unique reflections	13348 (992)	16991 (2023)
Redundancy	3.39 (2.54)	5.52 (4.15)
Completeness (%)	99.0 (99.2)	99.7 (99.2)
<I/σ(I)>	14.58 (2.41)	17.77 (2.85)
Rmeas a	0.069 (0.471)	0.066 (0.825)
Refinement statistics
Resolution (Å)	19.16-1.80	19.44-1.80 (1.80-1.85)
Nonhydrogen atoms	1203	1168
Water molecules	89	141
r.m.s.deviation bond (Å) c	0.015	0.014
r.m.s. deviation angles (°) c	1.741	1.549
Rwork d	0.158	0.166
Rfree d,e	0.207	0.206

Values in parentheses correspond to the highest resolution shell

^aR_meas= Σ_h{(n/(n−1))^0.5 Σ_j|<I_h>−I_hj|} / Σ_hjI_hj with <I_h>=(Σ_jI_hj)/n_j.

^bValues for the highest resolution shell in parentheses.

^cRoot mean square deviation from ideal/target geometries

^dR_work,free=Σ_h||F_obs|−|F_calc||/Σ_h|F_obs|.

^eR_free values are calculated for a randomly selected 5% of the data that was excluded from the refinement

3. Results and Discussion

Kinetic properties of Asp/(28/49)/Asn mutant dUTPases

The enzymatic activity of hDUT^D49N,F158W and mtDUT^D28N,H145W enzymes was measured in several different kinetic setups. The Asp/Asn mutation within Motif I resulted in considerable decrease of steady-state rates for both hDUT and mtDUT (approx thousandfold and fiftyfold decrease, respectively, Figure 1B inset, note logarithmic scale, cf also Table II). To investigate which step of the kinetic cycle is mainly affected by the mutation, we carried out transient kinetic experiments. Time courses of the proton release curves of hDUT^D49N,F158W and mtDUT^D28N,H145W in single turnover conditions also indicated comparable decrease in activity as was observed in the steady-state (Figure 1B, Table II, compare k_cat and k_obs values). In the single turnover experiments, we assured that the active site concentration is higher than that of the substrate, and that substrate binding is not rate-limiting and is quantitative ([enzyme] >> K_d^dUPNPP ≈ K_d^dUTP ≈ K_M).

Table II.

Quantitative characterization of kinetic and ligand binding characteristics of hDUT and mtDUT enzymes.

	hDUTF158W	hDUTD49N,F158W	mtDUTH145W	mtDUTD28N,H145W
kcat (s−1)	6.3 ± 0.8	0.007 ± 0.002	1.2 ± 0.1	0.024 ± 0.005
STO kobs (s−1)	6.5 ± 0.1a,b	0.017 ± 0.003a	1.2 ± 0.1a,c	0.017 ± 0.002a
STO kH (s−1)	5.5 ± 2.5b	0.0036 ± 0.0009	n.d.	0.0064 ± 0.002
KddUPNPP(µM)	1.9 ± 0.2	8.5 ± 1.2	0.32 ± 0.1	4.7 ± 0.5
KddUMP(µM)	32 ± 2b	38 ± 8	72 ± 17	58 ± 14
ΔFmaxdUMP	0.64 ± 0.03b	0.60 ± 0.01	0.87 ± 0.04	0.79 ± 0.09
ΔFmaxdUTP	0.20 ± 0.06b	0.60 ± 0.01	0.14 ± 0.03	0.88 ± 0.05
ΔFmaxdUPNPP	0.40 ± 0.04b	0.68 ± 0.02	0.21 ± 0.02	0.55 ± 0.02
λmaxapo (nm)	353b	354.4 ± 0.5	354.0 ± 0.1	354.2 ± 0.3
λmaxdUMP (nm)	347b	355.1 ± 0.1	354.2 ± 0.3	353.7 ± 0.2
λmaxdUTP (nm)	339b	354.8 ± 0.1	351.9 ± 2.0	354.8 ± 0.3
λmaxdUPNPP (nm)	343b	354.3 ± 0.8	350.8 ± 0.4	353.1 ± 0.5

k_cat was measured in steady-state proton release assay. STO k_obs stands for the observed rate constant in single turnover stopped-flow kinetic experiments; STO k_H for the rate constant of the hydrolysis step as measured directly by quench flow radioactive assay under single-turnover conditions; K_d for dissociation constant, F for fluorescence emission intensity; ΔF_max^dUMP, ΔF_max^dUTP or ΔF_max^dUPNPP for relative fluorescence intensity difference observed upon saturation of apoenzymes with the ligand indicated in the upper index (dUMP, dUTP or dUPNPP, the latter stands for α,β-imido-dUTP); and λ_max for the fluorescence emission wavelength at which maximal emission is observed for the apoenzymes or for the enzymes saturated with the ligand indicated in the upper index. Data represent mean and standard deviation of 3–5 independent measurements.

^aProton release single turnover

^bData published in [14]

^cFluorescence single turnover

We could directly assess the rate constant of the hydrolysis step (k_H) by measuring the fractional hydrolysis of γ³²P-dUTP in single turnover conditions (Figure 1C, Table II). From previous studies we learned that the hydrolysis of the chemical bond and the proton release occur concomitantly and they are rate-limiting in the enzymatic cycle [14]. In the hDUT^D49N,F158W and mtDUT^D28N,H145W mutants, the rate constants of these kinetic steps are also similar and are in the same order of magnitude range as the k_cat, indicating that the mutation specifically affected the hydrolytic step, but did not change the overall enzymatic mechanism.

The point mutation was expected to perturb accommodation of the metal co-factor within the active site. Interestingly, however, the activity decrease induced by the Motif I Asp/Asn mutation is much more drastic than expected for a metal-free case. In the absence of divalent cations, both human and M. tuberculosis dUTPase retain about 50% of the catalytic rate observed in the presence of Mg²⁺ [12,16]). We also measured k_cat in the absence of Mg(II) for both of hDUT^D49N,F158W and mtDUT^D28N,H145W enzymes and found it to be decreased to about 50 %, i.e. the mutation did not abolish the response of k_cat to the presence of Mg(II).

Mutation of Motif I Asp to Asn perturbs interaction between the active site label Trp in Motif V with substrate and product

To investigate if the mutation within Motif I perturbed the interaction between the nucleotide ligand and Motif V, we exploited a single Trp reporter previously engineered into the middle of this conserved region. This active site label was successfully used to determine the dissociation constant of complexes formed between α,β-imido-dUTP or dUMP nucleotide ligands and human or mycobacterial dUTPases by differential fluorescence spectroscopy [12–14]. The quantitative data based on these fluorimetric titrations are in very good agreement with previously determined K_M or K_d data for similar complexes [27,28]. Figure 2 A, B reports the titration data obtained with hDUT^F158W, mtDUT^H145W, and hDUT^D49N,F158W, mtDUT^D28N,H145W proteins and α,β-imido-dUTP or dUMP. The data could be nicely fitted with 1:1 stoichiometry and the obtained dissociation constants are included in Table II. We found that the Asp/Asn mutation within Motif I significantly weakened the interaction of the protein with α,β-imido-dUTP (fourfold and 15-fold increment in K_d in hDUT^D49N,F158W and mtDUT^D28N,H145W proteins, respectively), whereas dUMP binding is only slightly affected (Figure 2A,B, Table II). This result indicates that the Motif I mutation perturbed the accommodation of the phosphate chain, although it has no direct contact to this moiety (cf below), which is mostly coordinated by Motif V [4,10,14,29].

Figure 2. Substrate and product binding reported by Trp fluorescence.

A–B, Fluorescence titration of hDUT^F158W, mtDUT^H145W (solid square), and hDUT^D49N,F158W, mtDUT^D28N,H145W (open circle) dUTPases with the substrate analogue α,β-imido-dUTP and the product dUMP (inset). For K_d values, refer to Table II. C–D, Fluorescence quench of the active-site Trp upon ligand binding at saturating concentrations (1mM dUMP, 1mM dUTP, 250µM α,β-imido-dUTP) in hDUT^F158W, mtDUT^H145W (solid) and hDUT^D49N,F158W, mtDUT^D28N,H145W (striped) dUTPases.

In addition, the extent of the fluorescence quench of the Trp active site label upon binding of different nucleotide ligands to the enzyme can also be used to differentiate between binding of the triphosphate substrate or monophosphate product and allows for determining the occupancy of different kinetic states in equilibrium in both human and M. tuberculosis dUTPases [12–14]. Such cognate changes are probably due to specific stacking interactions between the aromatic ring of the Trp residue and the uracil ring as Motif V closes upon the active site [12,13] during the catalytic cycle. In wild type trimeric dUTPases, this stacking interaction is strictly conserved as either Phe (e.g. human) or His (e.g. in M. tuberculosis) is located at this position within the C-terminal Motif V [29].

In steady-state (observed with large excess of dUTP), the large fluorescence quench of hDUT^F158W indicates that the low-fluorescence conformation, reflecting the conformation adopted during catalytic cleavage, is predominant [14]. In both hDUT^D49N,F158W and mtDUT^D28N,H145W mutants, we observed a much smaller quench of the Trp fluorescence upon dUTP or α,β-imido-dUTP binding (Figure 2 C,D). This result probably indicates the impaired ability of the active site to adopt the conformation required for catalytic function. This phenomenon may account for the much decreased catalytic activity of the mutants and is consistent with the increased disorder of the Motif V region in the structure (cf below). On the contrary, the Asp/Asn mutation in Motif I did not perturb the extent of the fluorescent quench upon dUMP binding (Figure 2C,D). This result suggests that product accommodation within the active site may remain intact (cf also dissociation constants in Table II).

Three-dimensional structural analysis reveals far-reaching conformational effects of the single point mutation in Motif I

Kinetic and ligand binding results suggested that the point mutation within Motif I had major perturbing effects on enzyme function. To investigate the structural basis underlying these drastic effects, crystallization of both hDUT^D49N and mtDUT^D28N mutants, in the presence of saturating concentrations of α,β-imido-dUTP and Mg²⁺, was attempted. Unfortunately, the hDUT^D49N crystallization trial did not yield diffracting crystals. However, well-diffracting specimens could be obtained for the complex of mtDUT^D28N protein:α,β-imido-dUTP:Mg complex. Using the model structure of the wild-type enzyme [13], it was straightforward to solve and refine the 3D structure of the mutant complex. The structural details are reported in Figures 3, 4 and in Table I. Motif V residues, with the sole exception of the side chain of Arg140, could not be localized in the density maps due to their disorder.

Figure 3.

3D structural analysis of mtDUT^D28N and mtDUT^T138STOP proteins in complex with Mg:α,β-imido-dUTP (determined in the present study, PDB ID 3H6D, 3I93), as compared to the wild-type complex (PDB ID 2PY4) [13]). Residues of the β-hairpin Motif III were used for the superimposition. A, Increased flexibility within the mtDUT^D28N mutant complex. Overall structure of mtDUT and mtDUT^D28N in ribbon model, colored according to B factors (from dark blue to green to yellow: low to intermediate to high B factors). The catalytic water and Mg²⁺ are highlighted with red and magenta arrows, respectively. Note increased flexibility in the mutant complex structure around the active site protein atoms, for the bound nucleotide (stick model), especially for the phosphate chain, and metal as well as catalytic water (spheres). The mtDUT^T138STOP complex does not show such increase in disorder of the Mg(II) or nucleotide ligands. B, Location of the catalytic water in mtDUT (yellow carbons, pink water spheres) and mtDUT^D28N (green carbons, dark red water sphere) complexes. The Asp/Asn residue, the ligands α,β-imido-dUTP and Mg(II) (gray sphere) and the Mg(II)-coordinating waters (pink waters) are shown in ball and stick representation using atomic coloring. Note the rotation of the mutated side chain and the altered catalytic water position. The distance of the catalytic water to the α-phosphorus is 3.2 Ǻ in the wild type to be compared with 2.7 Ǻ in the mtDUT^D28N mutant, however, the in-line positioning is unfavorably altered (cf angles 169° vs 156°, in the wild type vs mtDUT^D28N mutant, respectively).

Figure 4.

Interaction maps around the phosphate chain of the substrate analogue Mg:α,β-imido-dUTP as accommodated within the active site of mtDUT (A) and mtDUT^D28N (B). Subunits are color-coded (cyan/dark blue/light blue), waters are in red, Mg²⁺ in magenta. Numbers indicate distances in Å. Note that within the mtDUT^D28N protein:Mg:α,β-imido-dUTP complex, all interactions (except for Arg 140 NH1 and NH2) are lost between Motif V and the substrate, as the arm becomes flexible and cannot be observed in the density data. In addition, the Asp28(Motif I, subunit A):Arg110(Motif IV, subunit A):Ser148(Motif V, subunit B) network of H-bonding is also eliminated in mtDUT^D28N. In the mutant structure distances which correspond to more than 10% shift are highlighted in bold and italic (the proximity of the catalytic water to the α-phosphate oxygen is indicated in italic bold and in red color).

Interestingly, and in contrast to our expectations, the metal ion co-factor Mg²⁺ could still be localized, albeit with increased mobility (Figure 3A, Table III). The three phosphate oxygens of the substrate analogue α,β-imido-dUTP provided the same tri-dentate coordination pattern for the metal as reported previously [10,12,13,15]. The three waters in the hexagonal coordination sphere of the metal, which were well localized in the wild type structure (pdb ID: 2PY4), however, could not be fitted into the electron density map probably due to high mobility (cf Figure 4, Table III).

Table III.

Thermal (B) factor analysis of the wild type (PDB ID:2PY4) and mutant (PDB ID: 3H6D, 3I93) mycobacterial dUTPase enzyme 3D structures

Averaged B-factor (Å2)		Residue # or ligand	mtDUT		mtDUTD28N		mtDUTT138STOP
Residue location	Conserved Motif		Main chain	All	Main chain	All	Main chain	All
Inner channel		M57	15.4	16.3	15.4	16.9	12.5	14.3
		V58	14.1	14.2	14.3	14.2	11.8	11.6
		G59	13.5	13.6	14.0	14.0	13.3	13.3
		L60	13.5	14.6	14.1	15.7	14.1	15.3
		V61	12.2	12.7	14.2	15.1	14.0	14.1
		H62	13.1	13.8	13.7	14.5	14.8	16.0
	IV	I111	13.8	15.8	17.4	19.2	15.7	16.1
	IV	A112	12.7	13.0	15.1	15.2	13.9	14.1
	IV	Q113	13.8	14.9	14.6	19.9	13.9	16.0
		L114	13.9	14.7	14.7	15.3	14.5	15.0
		L115	13.9	14.1	13.8	14.0	12.9	15.6
		V116	14.0	14.4	13.8	14.3	12.6	13.2
		Q117	15.0	15.0	14.5	15.6	13.0	14.2
		R118	14.9	21.0	16.1	29.8	13.4	17.7
Active site	I	D/N28	14.8	15.5	18.7	21.8	14.9	15.7
	II	R64	12.9	13.7	17.5	26.8	15.0	18.8
	II	S65	12.5	13.0	18.8	19.6	15.2	15.4
	II	G66	13.6	13.6	18.7	18.7	15.1	15.1
	III	T81	14.8	15.2	15.9	16.8	15.9	16.9
	III	D83	13.6	14.3	13.8	16.1	15.8	17.1
	IV	R110	14.2	16.3	18.1	31.0	16.3	20.6
	IV	Q113	13.8	14.9	14.6	19.9	14.6	16.8
	V	R140	21.7	19.7	*	42.5	*	*
	V	G146	18.9	18.9	*	*	*	*
	V	S147	20.5	21.0	*	*	*	*
	V	S148	22.5	21.5	*	*	*	*
		dUPNPP	n.a.	14.7	n.a.	34.0	n.a.	15.4
		Mg	n.a.	15.3	n.a.	37.7	n.a.	16.8

Residues are numbered according to the wild type sequence. B-factors > 16 are featured in bold. Residues indicated by * could not be fitted into the electron density map because of high mobility. Note that residues constituting the β-strands that border the inner channel of the protein trimer remain ordered in the mutant enzymes, as well. However, flexibility of residues around the active site increase significantly in the mtDUT^D28N enzyme complex as compared to the wild type and even as compared to the C-terminally truncated mtDUT^T138STOP enzyme complex, indicating considerable weakened interactions between conserved motifs of the catalytic centre. Note also the much increased mobility of the ligand α,β-imido-dUTP (dUPNPP) and the Mg(II) cofactor involved in its coordination in the.Motif I mutant enzyme complex as compared to both wild type and mtDUT^T138STOP. n.a.; not applicable.

The mutated asparagine residue could be nicely resolved in the structure. Its carboxamide moiety shows a ~45° rotation as compared to the carboxylate moiety of the wild-type aspartate residue (that also has the same conformation in human dUTPase) (Figure 3B). This rotation has multiple severe effects. First, it eliminates the possibility of coordinating the two water molecules involved in Mg²⁺ - chelation. In fact, neither these two water molecules, nor the third water also involved in the Mg²⁺ coordination sphere and H-bonded to the two former waters, can be localized in the structure of the mutant complex (Figure 4). These perturbations result in the increased mobility of the Mg²⁺ ion (Figure 3A, Table III). Second, the rotation of the carboxamide moiety also disrupts the interaction between Asn28 (Motif I) and Arg 110 (Motif IV) (Figure 4). The Arg 110 guanidino moiety in the mutant structure shifts away from its interacting partners in the wild-type enzyme, thereby eliminating both contacts with Ser 148 (Motif V of the neighboring subunit) (Figure 4). These contacts in the wild-type enzyme:α,β-imido-dUTP:Mg complex provided an intersubunit linkage facilitating ordering of Motif V upon the active site. Other interactions involving Motif IV Gln 113 within the active site are also weakened (Figure 4). Interestingly, in the mutant structure, the conserved Ser in Motif II (Ser65) (cf [18]) is found in two different conformations (A and B) with equal occupancy: in one of these the terminal oxygen is pointing towards the imido group, as in the wild-type, while in the other one, it is making contact with the α-phosphate group (cf Figure 4) (these two Ser conformers were already seen in other structures as well [16]).

In addition to these observed changes, it was also of interest to investigate the position and coordination of the “catalytic” water molecule, i.e. the water molecule located in-line to the scissile bond at the α-phosphorus atom (cf. [15]). We found that the Asp/Asn mutation perturbs its location so that it is now less suitable for in-line nucleophilic attack (Figure 3B). The angle reflecting in-line attack is decreased from the closely linear 169° in the wild type (also observed in other structures [12,15] to 156° in the mutant. Perhaps even more adverse for in-line nucleophilic attack is that the water gets considerably closer to one oxygen on the α-phosphorus (cf distance of 2.0 Ǻ in red on Figure 4B) and this proximity may well decrease its nucleophile character. In this altered position, the tetrahedral coordination pattern of the water is also disturbed (Figure 4B) as reflected in the increased distances to several of its interacting partners (notably, the distance to Motif IV conserved Gln113 Oε is increased from 2.7 to 3.6 Ǻ). In addition, the altered coordination pattern of the α-phosphate group is also reflected in its increased distance to Motif IV Gln113 and its contact to one conformer of Motif II Ser65 [18] (cf legend to Figure 4).

Motif V residues (except for the side chain of Arg140) of the mtDUT^D28N complex structure could not be localized in the electron density maps due to their high flexibility. Although it was reported in other dUTPases that Motif V is frequently lacking from the density maps [29,30], recently reviewed in [1], the M. tuberculosis enzyme is an exception in this respect when crystallized in complex with α,β-imido-dUTP in P6₃ space group (PDB IDs 1SIX, 2PY4, 3HZA [10,13] and submitted for publication). Perhaps due to the tight packing of this specific space group, the C-terminus is clearly visible in all these structures. Therefore, in the presently determined mutant structure, isostructural with the wild type structure, lack of well-defined density for Motif V residues can be interpreted as resulting from the mutation. This increased flexibility is also in agreement with the results of the fluorescence spectral titrations (Figure 2), which indicated an altered interaction pattern between the C-terminal Motif V and the triphosphate substrate. Although visible in the density maps, the phosphate chain moiety, the catalytic water, the Mg²⁺ ion and clusters of protein atoms around the active site also show significantly increased mobility reported by the increased B factors (Figure 3A, Table III). This overall disordered character may contribute to the observed decrease in catalytic efficiency. Summarizing the 3D structural observations, the single point mutation within Motf I induced multiple conformational shifts not restricted to short-range interactions. Intersubunit interactions also suffered significant perturbations, finally resulting in highly increased conformational freedom of moieties involved in catalytic function.

To further ascertain that the increased disorder within the active site residues and the nucleotide and Mg(II) ligands are due to the perturbation caused by the Motif I mutation, we also determined the 3D structure of the mtDUT^T138STOP: α,β-imido-dUTP:Mg complex. In this case, the C-terminal segment is truncated. (Kinetic and ligand binding properties of this mutant will be published elsewhere in a systematic study focused on Motif V mutations in dUTPases). We observed that this truncation did not perturb accommodation of the nucleotide ligand within the remaining segments of the active site, and no significant change in disorder parameters can be observed as compared to the wild type complex structure. This observation indicates that the disorder presented in the Motif I mutant structure faithfully reflects the fact that this point mutation disrupted numerous intertwined interactions among protein atoms of conserved residues from different subunits.

4. Conclusions

In homooligomeric enzymes, substrate accommodation in each of the multiple active sites may involve residues from several different subunits, although such intersubunit active sites are usually formed within the joint surface of no more than two subunits. Higher-order (trimeric/tetrameric) involvement in each of the multiple substrate binding pockets are less frequently reported (c.f. [31]). Conserved sequence motifs of human and mycobacterial dUTPase homotrimers provide such architecture by solidifying intersubunit contacts around the bound substrate nucleotide. Localization of the otherwise quite flexible C-terminus (including Motif V) upon the active site in a catalytically competent conformation may be a major advantage of this intertwined network. Disruption of the motif-interaction network at one single location in Motif I of one subunit has far-reaching conformational effects within Motif IV of the same subunit and Motif V of the neighboring subunit. The catalytic rate of the Motif I mutant enzyme is significantly decreased due to increased long-range disorder around the active site and perturbation of the coordination sphere of the water molecule involved in nucleophilic attack. Present results reveal that despite earlier suggestions, the strictly conserved Motif I aspartate is dispensable for accommodation of the metal co-factor. Instead, it plays an unexpected major role in the motif-interaction network, crucial for catalytic function.

Abbreviations

M. tuberculosis

Mycobacterium tuberculosis

dUTP

2’-deoxyuridine triphosphate

dUTPase

dUTP pyrophosphatase

dUPNPP

α,β-imido-dUTP

hDUT

human dUTPase

mtDUT

Mycobacterium tuberculosis dUTPase

hDUT^D49N,F158W

Asp49Asn/Phe158Trp double mutant construct of human dUTPase

mtDUT^D28N, mtDUT^T138STOP, and mtDUT^D28N,H145W, Asp28Asn

Thr138STOP and Asp28Asn/His145Trp mutant constructs of Mycobacterium tuberculosis dUTPase