Mechanisms of Allosteric Activation and Inhibition of the Deoxyribonucleoside Triphosphate Triphosphohydrolase from Enterococcus faecalis*♦

Background: EF1143 from Enterococcus faecalis is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase. Results: Enzymatic studies and crystal structure of EF1143 bound to dNTPs show that dNTP binding at two adjacent allosteric sites can differentially modulate enzyme catalysis. Conclusion: EF1143 enzyme catalysis is differentially regulated by four dNTPs. Significance: Learning how the enzyme activity is regulated by substrates is fundamental for understanding enzyme catalysis and allostery. EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.

EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.
Enterococcus faecalis is a Gram-positive bacterium found in the intestinal tract of humans. It is a common, life-threatening, hospital-acquired infection, due to frequent mutations that result in the development of antibiotic resistance (17)(18)(19)(20). Understanding the molecular mechanisms of E. faecalis pathogenicity and adaptation to antibiotics is important to prevent disease. Several virulence factors encoded by Enterococcus have been identified (21,22). In this study, we focused on a dNTPase, which controls dNTP pools in the organism.
Biophysical characterization of several dNTPases indicates that they exist as homo-oligomers. For example, crystal structures of the HD domains of human SAMHD1 and bacterial EF1143, a dNTPase from E. faecalis, showed that they are dimer and tetramer, respectively (Protein Data Bank (PDB) IDs 3U1N and 3IRH, respectively) (6,23). Similarly, 067745_AQUAE, a dNTPase from the prokaryotic organism Aquifex aeolicus, is thought to be active as a tetramer (24), whereas Tt-dNTPase from Thermus thermophilus HB8 is a hexamer (25), based on crystal structures (PDB IDs 2HEK and 2DQB, respectively).
The nature of the homo-oligomeric quaternary states of dNTPases suggests allosteric regulation of their enzymatic activity by effector molecules. Indeed, SAMHD1 undergoes dGTP-dependent tetramerization, which is the catalytically active form of the enzyme (26,27), and extensive kinetic characterization of the Tt-dNTPase showed that its enzymatic activity was regulated by both dATP and dCTP (28). The most intriguing feature of the biochemically characterized dNTPases, including SAMHD1, Tt-dNTPase, and EF1143, is that dNTPs are both effectors and substrates. Structural comparison of the apo form of EF1143 with a dGTP effector and dATP substrate-bound form uncovered a few important features of its allosteric regulation and catalytic activity. Still, many mechanistic aspects of its complex regulation remained unclear. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP, bound at two distinctive regulatory sites. The residues at the primary allosteric site are configured to selectively bind only dGTP with high affinity. Detailed biochemical analyses, guided by the crystal structure, suggest that dTTP and dGTP function as negative regulators of the enzyme, whereas dATP and dCTP are positive allosteric effectors, binding to the same promiscuous secondary regulatory site adjacent to the dGTP-binding primary allosteric site. Taken together, these results reveal complex molecular mechanisms of substrate-mediated regulation of EF1143 catalysis.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-Cloning of N-terminally His 6 -tagged E. faecalis HD-domain protein EF1143 was described previously (23). Site-specific mutants of EF1143 were prepared using QuikChange mutagenesis kits (Agilent). Wildtype and mutant EF1143 proteins were expressed in Ros2 (DE3) using auto-induction medium at 18°C for 24 h (29). The recombinant proteins were purified by two-step liquid column chromatography: a 5-ml nickel-nitrilotriacetic acid column (GE Healthcare) and a Hi-Load Superdex200 16/60 column. Purified proteins were stored in a gel filtration column chromatography buffer containing 25 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol, and 0.02% sodium azide.
Protein Crystallization and X-ray Data Collection-The protein stock solution for co-crystallization trials contained 5 mg/ml protein, 1 mM MgCl 2 , 1.8 mM dGTP, 1.8 mM dTTP, 3.6 mM Tris-HCl, and 250 mM NaCl. Crystal screening was done at room temperature in 96-well Corning crystallization plates. Protein stock was mixed with the crystallization conditions in 1:1 ratio. Diffraction quality crystals belonging to the orthogonal space group P2 1 2 1 2 1 were obtained in 0.1 M buffer containing sodium malonate/imidazole/boric acid (at a 2:3:3 molar ratio), pH 5.0, and 25% w/v PEG 1500 in a 2-l sitting drop at 22°C. The crystals were flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K at the LS-CAT (21-ID-F) beamline at the Advanced Photon Source, Argonne National Laboratory. All the data were processed with HKL2000 (30).
Structure Determination and Sequence Analysis-The structure of the EF1143 in complex with dGTP and dTTP was solved by molecular replacement using Phaser (31) and chain A from the EF1143-dGTP-dATP complex as a search model (PDB ID 3IRH). The structure was completed using Coot (32) and REFMAC (33). Crystallographic data and the refinement statistics are in Table 1. Graphical presentations were done with PyMOL (41). Sequence homology analysis was performed using BLAST (34) and ClustalW (35).
dNTPase Assays-EF1143 enzyme activities were studied in a reaction buffer containing 20 mM Tris-HCl, pH 7.8, 50 mM NaCl, 2 mM MgCl 2 , 5% glycerol, and varying concentrations of dNTPs, as indicated in the figure legends, with 0.1 M EF1143 and 5 M BSA. The reactions were quenched with a final concentration of 50 mM EDTA after specific time intervals (2, 4, 6, and 8 min or 4, 8, 12, and 32 min). The products (dNs) and substrates (dNTPs) were separated by reversed phase HPLC using a CAPCELL PAK C18 column (Shiseido Fine Chemicals, 4.6 ϫ 75 mm) and were quantified by peak integration of absorbance traces at 260 nm, as described previously (27).
Analysis of Kinetic Data-The kinetic data were fitted with either the Michaelis-Menten equation (Equation 1) or the allosteric sigmoidal equation (Equation 2) where v 0 is the initial velocity, V max is the maximum velocity, K m and K half are ligand concentrations at half-maximal velocity, and h is the Hill coefficient.

RESULTS
The Overall Structure of EF1143 in Complex with dGTP and dTTP-The new crystal structure of EF1143 in complex with dGTP and dTTP (PDB ID 4LRL) demonstrates the same tetrameric architecture as the previously reported EF1143 in complex with dGTP and dATP (PDB ID 3IRH, root mean square deviation between the two structures is 0.67 Å over 1631 C␣) and, likely, represents the biological assembly of EF1143 in solution (23). Each EF1143 monomer consists of a bowl-shaped catalytic HD domain (residues 1-329) and a C-terminal subdomain (residues 330 -456), shown with pale tones in Fig. 1A. Four dGTP molecules in the 4LRL structure were found at the previously identified allosteric sites in the interfaces between the monomers. The detailed description of these dGTP-binding sites can be found elsewhere (23) and is reiterated in Fig. 1B. Guanine base forms five hydrogen-bonding interactions with the side chains of Asn-36, Gln-41, and Arg-44, providing base type specificity. The specificity for dGTP binding at this site was confirmed by kinetic studies, as described in a later section (see Fig. 5). Surprisingly, however, in the 4LRL structure, the electron density of dTTP was observed next to dGTP, indicating the existence of a secondary allosteric site (supplemental Fig. S1). This contrasts with the 3IRH structure, where dATP was found in the catalytic site.
There are four identical secondary allosteric sites per tetramer, but only two were occupied by dTTP in 4LRL. Several distinct features typify the site (Fig. 1C). First, residues belonging to three different chains (particularly, B, C, and D) form the binding pocket. Second, the site appears to be specific for the deoxyribose moiety, with the 3Ј-hydroxyl group interacting with the main chain atoms of Thr-55 (chain C) and Arg-17 (chain D). The substitution of the deoxyribose with the ribose moiety would result in structural clash between the 2Ј-hydroxyl group and Phe-56 (chain C). Third, the triphosphate moiety, specifically, oxygen atoms of the P ␣ and P ␤ groups, is recognized by His-245 of chain C and Arg-209 of chain B. Interestingly, one of the P ␣ oxygen atoms also has an H-bond with the deoxyribose hydroxyl group of dGTP. Fourth, the guanidinium group of Arg-17 in chain D interacts with the ortho-carbonyl group of thymine. The fact that only one of the three functional groups of the dTTP base is involved in H-bonding suggests the promiscuous nature of the secondary site. This is in stark contrast to the specific dGTP binding mode at the primary allosteric site. The promiscuity of this site may play a key role in regulation of the hydrolase activity, depending on the presence of particular dNTPs as elaborated in later sections. dTTP Binding Alters the Geometry of EF1143 near the Active Site-Superposition of chain B of 3IRH and chain C of 4LRL ( Fig. 2A) reveals a potential structural mechanism for modulation of the dNTPase activity by dTTP binding to the secondary allosteric site. The dTTP-binding site is separated from the catalytic site by a helix-turn-helix fragment comprising ␣13 . . . ␣14. The phenol plane of residue Tyr-243, in the ␣13 . . . ␣14 loop, is in close proximity to the deoxyribose moiety of dATP. The position of this residue would regulate the location of the P␣ group, a nucleophilic attack target of the hydroxide ion stabilized by His-114 (supplemental Fig. S2). The distance between C␥ of Tyr-243 and C1 of the dATP ribose in 3IRH is 3.72 Å, on average, whereas the same positions are 3.57 Å apart, on average, in the substrate-free forms when dATP is modeled in the structure. This suggests, first, that the ␣13 . . . ␣14 helix-turn-helix fragment is mobile and, second, that dATP binding tends to expand the binding site. In contrast to the dATP effect, dTTP binding would change the C␥ . . . C1 distance to an average of 3.32 Å. This 0.40 Å shortening could be directly linked to the dTTP steric contact with residue Gln-241 on the opposite side of ␣13, causing Tyr-243 to shift into the catalytic site. Thus, dTTP binding results in contraction of the catalytic site.
An additional structural change related to dTTP binding at the second allosteric site is the movement of Tyr-199 and the loop that includes residues 195-202 (Fig. 2B). In the absence of ligand, Tyr-199 protrudes into the allosteric site. Thus, in the dTTP-bound state, the 195-202 loop must change its conformation, resulting in an almost 6.9 Å movement of the Tyr-199 C␥ position out of the secondary allosteric site. This new loop conformation is acceptable but more energetically strenuous, as suggested by the Ramachandran plot distribution; in the dTTP-bound state, three residues move from the preferred into the allowed regions (data not shown). This observation suggests that Tyr-199 and the associated loop could play the role of a "spring" that ensures the reversibility of ligand binding to the secondary allosteric site.
Mutational Analysis Combined with Activity Assays Correlates with the Structural Features-Our initial biochemical analysis demonstrated that, in the presence of Mg 2ϩ , the hydrolytic activity of EF1143 depended on the presence of at least two different dNTPs, one of which had to be dGTP (23). In this study, we measured the dATPase activity of EF1143 in the presence of both dATP and dGTP, with a dATP:dGTP ratio of 1250:1. To confirm the importance of several critical residues revealed from the 3IRH and 4LRL structures, several mutants were generated and tested for dATPase activity (Fig. 3). First, analysis of 3IRH, with dATP at the active site, suggested that the catalytic residues comprise a Glu-122-His-129 dyad plus His-  114, which together facilitate the in-line nucleophilic attack of a hydrolytic water molecule at the ␣-phosphate center of the dNTP substrate (supplemental Fig. S2). Negligible catalytic activity of the H114A and H129A mutants supports this hypothesis (Fig. 3). Second, dGTP binding at the primary allosteric site features two distinctive patterns (Fig. 1B): ionic interactions between the triphosphate and basic residues (Lys-14, Arg-326, and Lys-330) and hydrogen bonding between the guanine base and hydrophilic residues (Asn-36, Gln-41 and Arg-44). All these residues (except Arg-44) were mutated, individually, to Ala, and their dATPase activities were compared. With the exception of R326A, all the mutants were significantly defective in dNTP hydrolysis. Finally, residues observed to interact with dTTP in 4LRL, at the second allosteric site, were mutated. Although mutation at Arg-17, Tyr-199, or Gln-241 resulted in significant loss of dATPase activity, R206Q or R209S showed only a moderate decrease in hydrolysis. However, double mutation of Arg-206 and Arg-209 resulted in complete loss of the EF1143 catalytic activity. Taken together, this mutagenesis analysis confirms the importance of residues observed to be involved in ligand binding at the catalytic site and the two allosteric sites.

Survey of dNTPase Activity with Combinations of dNTPs-
Our discovery of the promiscuous second allosteric site in 4LRL prompted us to speculate that the enzymatic activity of EF1143 is differentially regulated, depending on which dNTP occupies this regulatory site. To gain more insight into this, we first measured the dNTPase activity of EF1143 against each dNTP, in the absence or presence of one, two, or three additional dNTPs (Fig. 4). Consistent with our previous study (23), EF1143 failed to hydrolyze any of the dNTPs when a sole dNTP was present in the reaction mixture (Fig. 4, A-D, column 1 in each). Next, the data confirmed our previous results that, among the four dNTPs, only dGTP could function as an activator of dATP and dCTP hydrolysis (Fig. 4, B  and C, columns 2-4). When the rate of dGTP hydrolysis was compared among reaction mixtures containing two dNTP types, dGTP hydrolysis was clearly enhanced by the presence of dATP or dCTP, but not dTTP, with dATP being the more potent activator (Fig. 4A, columns 2-4). Further, dTTP hydrolysis was virtually nonexistent when any one other dNTP was combined with dTTP in the reaction mixture (Fig. 4D, columns 2-4). This observation is consistent with 4LRL, where dTTP binding at the second allosteric site shrinks the catalytic site. However, dTTP hydrolysis did occur when both dGTP and dATP were present (Fig. 4D, column 5),    suggesting that both dATP and dGTP are co-activators for hydrolysis of dTTP, with dGTP as the primary activator. In contrast, dTTP does not activate or enhance the enzyme's activity against any dNTP. For example, the addition of dTTP to reaction mixtures that contained dGTP and dATP or dCTP had no effect on the hydrolysis of dATP (Fig. 4B, column 2 versus column 7) or dCTP (Fig. 4C, column 2 versus column 6) at the initial concentrations tested. With all four dNTPs present at equal concentrations, the order of dNTP hydrolysis was dGTP Ͼ dCTP Ͼ dATP Ͼ dTTP (Fig. 4, A-D, compare column 8 from each panel). Based on all these comparisons, we conclude that dGTP is a primary activator because appreciable dNTP hydrolysis was not observed in any reaction mixture in the absence of dGTP. This conclusion is consistent with the crystal structures of EF1143, with dGTP at the primary allosteric site (Fig. 1). In addition, dATP is a stronger secondary activator than dCTP because the rates of dGTP and dTTP hydrolysis were higher with dATP in the reaction mixture as compared with those with dCTP.

Determination of Kinetic Constants of dGTP at the Allosteric Site and Catalytic
Site-Analysis of the two crystal structures (3IRH and 4LRL), along with our dNTPase activity survey (Fig.  4), suggests that dGTP binding at the primary allosteric site is a prerequisite for the dATP-and dCTP-dependent activation of EF1143. To determine the binding constant for dGTP at the allosteric site, the rates of dATP and dCTP hydrolysis were determined in the presence of increasing concentrations of dGTP (Fig. 5A). Both sets of data were fitted with the Michaelis-Menten model (Equation 1), suggesting a simple model of dGTP binding at the primary allosteric site. Interestingly, the K m values were similar for both dATP and dCTP (0.17 Ϯ 0.05 and 0.17 Ϯ 0.08 M, respectively). This suggests that dGTP binding at the allosteric site is independent of dATP and dCTP binding at the catalytic site. Further kinetic analyses, with higher dGTP concentrations, suggested that dGTP binding at the catalytic site also follows the Michaelis-Menten model (Equation 1) (Fig. 5B). The rates of dATP and dCTP hydrolysis were moderately constant over the range of dGTP concentrations examined (supplemental Fig. S3). Taken together, the data suggest that dGTP has a strong (ϳ2000-fold) preference for the allosteric site as compared with the catalytic site.
dATP and dCTP Allosterically Activate the dNTPase-To further understand the activation features of EF1143 by dATP and dCTP (Fig. 4), dNTPase assays were performed with increasing concentrations of dATP or dCTP in a reaction mixture containing a fixed concentration of dGTP, 16 M, at which the primary allosteric site of the enzyme is fully occupied by dGTP (K d of dGTP at the primary allosteric site is 0.17 M). When the rates of dA and dC accumulation were plotted as a function of dATP and dCTP concentrations, respectively, the data were best fitted with sigmoidal curves (Equation 2) ( 5C), indicating allostery. The rate of dC accumulation was more sensitive to dCTP concentration than the rate of dA accumulation was to dATP, resulting in a higher Hill coefficient value and V max for dCTP than dATP. When the rate of dG accumulation was plotted, dATP was far superior to dCTP in allosteric activation of dGTP hydrolysis (Fig. 5D). These data support the observation that dATP is a more potent activator of dGTP hydrolysis than dCTP, as seen when all dNTPs were present at 100 M (Fig. 4A, columns 2 and 3). Further, at 100 M, the rate of dA accumulation is slightly higher than that of dC accumulation (Fig. 5C), which also agrees with the data presented in Fig.  4. Taken together, the results show that dATP and dCTP allosterically activate the dNTPase with a different cooperativity for substrates. Our structural modeling suggests that both dATP and dCTP might bind at the secondary allosteric site, which is occupied by dTTP in 4LRL (see the section below).
dTTP and dGTP Inhibits dNTPase Activity at High Concentration-Comparison of crystal structures 3IRH and 4LRL (Fig. 2) suggests that dTTP binding at the secondary allosteric site could negatively influence EF1143 catalytic activity (Fig. 4). To further investigate this possibility, we measured the rate of dT accumulation in increasing concentrations of dTTP with a fixed concentration of dGTP and dATP or dCTP (Fig.  6A). Note that hydrolysis of dTTP can only be observed in the presence of both dGTP and dATP or dCTP (Fig. 4D). The rate of dTTP hydrolysis increased with increasing concentrations of dTTP, up to 400 M, and then gradually declined at higher concentrations. Strikingly, no conversion of dTTP was observed at 1600 M dTTP with dGTP and dCTP in the reaction mixture. Similar results were observed when the rates of dG, dA, and dC conversion were monitored with increasing concentrations of dTTP (Fig. 6, B and C).
The 4LRL structure provides mechanistic insight into the inhibition mechanism by dTTP. The ligand interacts with residue Gln-241 of helix ␣13, shifting Tyr-243 toward the catalytic binding site by ϳ0.4 Å (Fig. 2A). This movement could either prevent substrate binding or disrupt correct positioning of the dNTP for nucleophilic attack. Modeling of dGTP ligand binding at the second allosteric site (Fig. 7) suggests that it could exert a similar effect on helix ␣13/Gln-241, inhibiting enzyme catalysis. Consistent with this notion, at high dGTP concentrations (greater than 200 M), the dNTPase was inhibited (Fig. 6,  D and E). The dCTP molecule should be able to bind to the secondary allosteric site in a mode similar to that of dTTP. However, the deoxycytidine, in particular the amino group in para-position, would clash with Gln-241 if dCTP binds exactly in the same conformation as observed for dTTP in 4LRL. A similar observation could be made for dATP in either anticonformation or syn-conformation (Fig. 7). Despite these caveats, the binding pocket appears to be large enough to allow cytosine/adenine plane rotation, for example, toward the Arg-236 residue that would prevent collision with Gln-241. The reduced, but still significant activity of the Q241A mutant in the dGTP/dATP mixture (Fig. 3) may be interpreted to support this hypothesis. Therefore, the steric difference exerted by the amino group in dCTP/dATP and the carbonyl group in the analogous position of dTTP/dGTP may differently affect the position of helix ␣13 and residue Tyr-243 in the catalytic site, thus, switching the enzyme between the inhibited and the activated states. Taken alto- gether, the kinetic data support a modulatory role of the secondary allosteric site upon binding to different dNTPs.

DISCUSSION
The proper balance of cellular dNTP pools is critical for maintaining genome stability. The central role of ribonucleoside diphosphate reductases in this has been well documented (4). A recent study of SAMHD1, a mammalian dNTPase, suggested its essential role in balancing dNTP pools in concert with the ribonucleoside diphosphate reductase (5). We and others elucidated the structural basis of dGTP-dependent SAMHD1 tetramerization and allosteric activation (36,37). In this study, we present a crystal structure of EF1143 in complex with dGTP and dTTP (4LRL) and provide insight into an elegant allosteric mechanism of dNTP-mediated inhibition of enzyme catalysis.
Comparison of 4LRL with the apo-EF1143 (2O6I) and dGTP/ dATP bound forms (3IRH), along with extensive enzyme kinetic studies, presents a qualitative model for EF1143 allosteric regulation (Fig. 8). dGTP binding at the primary allosteric site is necessary for hydrolysis of the canonical dNTP substrates. Analysis of the N36A and Q41A mutants confirmed the critical importance of the guanine base-recognizing residues (Fig. 3). The enzyme kinetic analysis indicated high specificity of the primary allosteric site for dGTP (Fig. 5A). Taken together, binding of dGTP could be considered an enzyme preactivation step. Then, the enzyme can be further activated or inhibited by dNTPs binding to the secondary allosteric site, with different affinities. If the site is occupied by dATP or dCTP, the enzyme becomes catalytically active (Fig. 5, C and D). With dGTP or dTTP bound at the site, EF1143 is negatively regulated (Fig. 6). The physiological concentrations of dNTPs in prokaryotes are in the range of 100 -300 M and fluctuate several folds during bacteriophage T4 infection (38 -40). At these concentrations, the primary allosteric site should be fully occupied with dGTP and substrate hydrolysis at the catalytic site could be modulated by different dNTPs binding at the secondary allosteric sites.
To assess whether the proposed mechanism of EF1143 regulation by dNTPs could serve as a general model for homotetrameric dNTPases, we compared the amino acid sequences of EF1143 with 43 of its closest homologs, mostly from Enterococcus and Lactobacillus species (at least 97% coverage and 65% identity, supplemental Fig. S4). Among the closest sequence homologs, most of the critical residues in the catalytic and two allosteric sites are 100% conserved (supplemental Fig. S4, red  boxes). We also carried out a structure-assisted comparison of EF1143 and SAMHD1 (supplemental Fig. S5). The human SAMHD1 dNTPase (sequence identity 36% over 274 residues) is a structural homolog of EF1143. For the conservation analysis, we superimposed SAMHD1 chain A from 4BZB (dGTPbound tetrameric form) to chain C of EF1143 tetramer (supplemental Fig. S5A). Overall, all the dNTP-binding sites between EF1143 and SAMHD1 are highly conserved. At the dGTP-specific primary allosteric site, two critical residues for EF1143 preactivation, Arg-44 and Arg-326, have Arg-145 and Arg-451 as their counterparts in SAMHD1 (supplemental Fig. S5B). Of note, SAMHD1 R145A mutation resulted in less than 1% of wild-type dNTPase activity (6). Interestingly, hydrogen-bonding interactions between the base of deoxyribonucleoside triphosphate and surrounding residues in both enzymes suggest a promiscuity of the secondary allosteric site to other dNTPs (Fig. 1C) (36,37). The catalytic residues Glu-122-His-129-His-114 in EF1143 correspond to Asp-218-His-233-His-210 in SAMHD1 (supplemental Fig. S5C). Thus, the hydrolysis in both enzymes may proceed in a similar manner, through the nucleophilic attack of the activated water species (hydroxide ion) on the P␣ center.
Despite the structural similarities between EF1143 and SAMHD1, several important differences exist. Although both apo-bound and dGTP-bound EF1143 are tetramers in solution (23), SAMHD1 undergoes tetramerization upon dGTP binding (26,27). Further, different dNTP binding at the secondary allosteric site of EF1143 can result in either active or inhibited forms, whereas no such regulation has been reported for SAMHD1.
A complete understanding of EF1143 regulation by dNTPs requires further work. Although our hypothesis that the switch between the active and inhibited modes could be regulated by binding of different ligands to the promiscuous secondary allosteric site agrees with the biochemical data, our suggestion is primarily based on modeling into the rigid protein structure. To prove that dGTP, dATP, and dCTP indeed bind to the secondary allosteric site, more crystallization experiments with different dNTP mixtures are needed. The issue also could be approached theoretically, using flexible molecular docking, molecular dynamics, or Monte-Carlo simulations, using our structures as a starting point. Furthermore, in 3IRH, only two out of four catalytic sites were occupied by dATP, and only two out of four secondary regulatory sites in 4LRL have dTTP. This might be an artifact of crystal packing but could also suggest communication between monomers in the tetrameric assembly. Finally, the function of the prominent and highly conserved C terminus of EF1143 remains unknown. These three structural aspects are currently under investigation.