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Article

Interference of Sulphonate Buffering Agents with E. coli Hypoxanthine-Guanine Phosphoribosyltransferase Active Site Functioning: A Crystallographic and Enzymological Study

by
Evgeniy A. Zayats
1,*,
Yulia A. Abramchik
1,
Maria A. Kostromina
1,
Vladimir I. Timofeev
2,
Mikhail B. Shevtsov
3,
Alexey V. Mishin
3,
Ilya V. Fateev
1,
Andrey A. Karanov
1,
Alexandra R. Sharafutdinova
1,
Aleksandra O. Arnautova
1,
Irina D. Konstantinova
1,
Valentin I. Borshchevskiy
3 and
Roman S. Esipov
1,*
1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia
2
National Research Centre “Kurchatov Institute”, Moscow 123182, Russia
3
Moscow Center for Advanced Studies, Moscow 123592, Russia
*
Authors to whom correspondence should be addressed.
Crystals 2025, 15(10), 874; https://doi.org/10.3390/cryst15100874
Submission received: 22 August 2025 / Revised: 30 September 2025 / Accepted: 2 October 2025 / Published: 8 October 2025
(This article belongs to the Special Issue Structure and Characterization of Enzymes)
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Versions Notes

Abstract

The investigation of the structure–function relationship in hypoxanthine-guanine phosphoribosyltransferases (HGPRT) is a direction that is relevant for the development of drugs and approaches of enzymatic synthesis of modified nucleosides and nucleotides. This research paper is dedicated to the investigation of binding of sulphonate molecules, such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in the active sites of HGPRT and similar proteins. We report the crystal structure of HGPRT from Escherichia coli (EcoHGPRT) in a complex with HEPES. In the obtained X-ray structure, a HEPES molecule binds to the active site in a position that mimics one of the HGPRT substrates, namely phosphoribosylpyrophosphate (PRPP). Enzymological study has shown that HEPES is an inhibitor of EcoHGPRT, along with two structurally similar molecules, namely MES and PIPES. Comparison of the observed EcoHGPRT/HEPES complex to other reported structures in the context of inhibition study results provides an opportunity to explore the variety of binding modes of HEPES and similar molecules and to discuss the structure–function relationship in this enzyme and similar proteins.

1. Introduction

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, ec:2.4.2.8) is an enzyme involved in nucleic acids exchange metabolism. Using hypoxanthine/guanine and phosphoribosylpyrophosphate (PRPP) as the substrates, HGPRT catalyzes the synthesis of inosine-5-monophosphate/guanosine-5-monophosphate nucleotides (IMP/GMP), with pyrophosphate (PPi) being the second product (Scheme 1A) [1,2,3,4,5,6]. Besides its role in metabolism, HGPRT is highly relevant in several areas of medicinal biotechnology. A multitude of protozoan parasites are purine auxotrophs, thus making HGPRT inhibition a promising strategy for drug development against malaria and other infections [7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25]. Moreover, HGPRT is responsible for the activation of antiviral pyrazine-2-carboxamide derivative prodrugs through conversion into nucleotides in human cells (so-called compounds T-1105 and T-705 “favipiravir”) [26,27,28,29,30,31,32,33]. Therefore, research on HGPRT can be highly relevant for the investigation of the molecular mechanism of such drugs. This is especially relevant, since favipiravir has been broadly used in treatment of SARS-CoV-2 infection during the recent pandemic [34,35,36,37,38]. Moreover, studies on HGPRT include the development of drugs against cancer [39,40,41]. Recombinant phosphoribosyltransferases can also serve as the final enzyme in a multienzymatic cascade, offering production of modified nucleotide pharmaceuticals with exceptional stereo- and regioselectivity in one pot [42,43]. Recombinant HGPRT can also be used in nucleoside synthesis to shift the equilibrium of a transglycosylation reaction towards the formation of the desirable product [44]. While natural biocatalysts are sometimes inefficient with the necessary unnatural substrates, the specificity of HGPRT and other enzymes can be modified through rational enzyme design approaches [45].
The development of the mentioned areas of research on HGPRT requires extensive studies on the relationship between the enzymes structure and function, including investigations using X-ray crystallography. Such studies frequently utilize compounds belonging to the so-called Good’s buffers as the buffering agents, including HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) and MES (2-(N-morpholino)ethanesulfonic acid). The chemical structures of some of these molecules are provided on (Scheme 1B). Various reported structures of phosphoribosyltrapsferase-like (PRT-like) proteins have these molecules in some of their active sites [46,47,48,49]. Nevertheless, the respective papers do not significantly delve into the binding of sulphonate molecules, since they are not the subject of the study. Moreover, while the presence of HEPES/MES in the active sites of HGPRT X-ray structures suggests that it can be an inhibitor of this enzyme, it is unclear how strong the inhibition is if it is really the case. Acosta et al. have observed that the activity of bifunctional enzyme HGPRT/adenylate kinase from Zobellia galactanivoran is lower in MES buffer compared to sodium phosphate buffer [50]. Nevertheless, it is challenging to comprehensively judge the strength of the inhibition of HGPRT by the mentioned compounds, especially relative to each other.
A comparison of the variety of binding modes of HEPES and similar molecules accompanied by an inhibition study using a single enzyme as the model could enrich the understanding of the structure–function relationship in HGPRT. Moreover, investigation of inhibition of HGPRT by Good’s buffer molecules would help to determine whether the presence of the buffering agent molecules could interfere with the results of the study on this enzyme. In this paper, we report the structure of Escherichia coli HGPRT (EcoHGPRT, Uniprot P0A9M3) in complex with a HEPES molecule bound in the enzyme’s active site. We have performed an enzymological study of the inhibition of EcoHGPRT by three similar molecules: HEPES, MES, and PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)). Our results allowed us to perform a comparative analysis of the variety of binding modes of these molecules in the active sites of various PRT-like proteins in the context of the results of our inhibition study.

2. Materials and Methods

2.1. Enzyme Production, Isolation and Purification

Recombinant EcoHGPRT with a C-terminal 6xHis-tag was produced in E. coli ER2566 strain cells (New England Biolabs, Ipswich, Massachusetts, USA), transformed with a plasmid containing the enzymes gene (based on pET-23d+ Novagen, Darmstadt, Germany). The producer strain culture was cultivated in liquid LB medium (per 1 l: 10 g tryptone, 5 g yeast extract, 10 g NaCl, supplemented with 100 mM ampicillin) until reaching the optical density of OD600 = 0.8. After induction by 0.4 mM IPTG, the cells were further cultivated for 4 h at 37 °C. As a result, the recombinant enzyme was produced in soluble form.
After centrifugation, the cell biomass was dissolved (1:10 w/v) in a buffer containing 50 mM Tris-HCl pH 8.0, 1 mM benzamidine, and 1 mM PMSF. The cell suspension was subjected to ultrasonic disintegration, followed by centrifugation. The supernatant was applied onto a XK16/10 column, packed with 15 mL of Chelating Sepharose Fast Flow sorbent (Cytiva, Marlborough, Massachusetts, USA), pre-equilibrated with Buffer A (50 mM Tris–HCl pH 8.0). The protein was eluted with 5 CV (column volumes) of Buffer C (75 mM imidazole, 50 mM Tris–HCl pH 8.0), followed by 5 CV of Buffer D (250 mM imidazole, 50 mM Tris–HCl pH 8.0). Fractions containing the target recombinant enzyme (SDS PAGE) were pooled and concentrated on the Ultracel 30 kDa membrane (PLTK06210, Millipore, Burlington, Massachusetts, USA) at a concentration of 12 mg/mL. Next, the solution was applied onto a HiLoad 16/60 column packed with Superdex 75 sorbent (GE Healthcare) and pre-equilibrated with Buffer E (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 5% glycerol, 0.04% NaN3). After elution with Buffer E, the fractions containing EcoHGPRT were pooled and concentrated at 15 mg/mL, with a purity of 97.9% (SDS PAGE) on an Ultracel 30 kDa membrane. The aliquots containing recombinant EcoHGPRT were stored at −80 °C. We used SDS PAGE for the determination of protein purity and the Bradford method to determine protein concentration [51,52].

2.2. Enzyme Assay

EcoHGPRT activity was determined through detection of the conversion of hypoxanthine into IMP via high-performance liquid chromatography (HPLC). Analytical HPLC was performed on a Hitachi Chromaster system (Pump 5110, Column oven 5310, UV Detector 5410, Chiyoda, Tokyo, Japan), using a Phenomenex Jupiter C18 LC Column, 3 µm, 250 × 4.6 mm, 300 Å (Torrance, California, USA). The following method was used: H2O/0.1% trifluoroacetic acid (TFA), 25 min, flow rate 0.4 mL/min, detection at 254 nm. The reaction mixtures (50 µL) for the determination of IC50 values of HEPES/MES/PIPES included the following: 100 mM Tris-HCl, pH 8.0, 0.5 mM hypoxanthine, 0.5 mM PRPP, 1 mM MgCl2, 0–500 mM HEPES/MES/PIPES. For the determination of EcoHGPRT activity in the presence/absence of HEPES/MES and SO42−/PO43− ions, the reaction mixtures included the following: 100 mM Tris-HCl, pH 8.0, 0.5 mM hypoxanthine, 0.5 mM PRPP, 1 mM MgCl2, 0 or 50 mM HEPES/MES, 0 or 10 mM Na2HPO4/Na2SO4. After addition of EcoHGPRT (0.075 µg), the preheated aliquots were incubated at 37 °C for 5 min, followed by HPLC analysis of conversion of hypoxanthine to IMP. A unit of enzymatic activity of EcoHGPRT was defined as 1 µM of substrate conversion per minute per milligram of enzyme.
The IC50 values were determined through fitting the obtained data to a four-parameter logistic model via nonlinear regression analysis using SciDAVis v2.3.0 software (Equation (1)). The Ki values were calculated using the Cheng–Prusoff equation with a Hill coefficient (Equation (2)) [53]. The KM value used was 0.192 mM for PRPP [54]. V—reaction rate; MIN—minimal reaction rate value; MAX—maximum reaction rate value; S—substrate concentration; C—inhibitor concentration; h—Hill coefficient; Ki—inhibition constant; KM—Michaelis constant.
V = M A X − M I N 1 + ( C I C 50 ) h + M I N
K i = I C 50 1 + S h K M

2.3. Crystallization

In order to obtain crystals of EcoHGPRT, we added 2 μL of the protein solution (15 mg/mL in 50 mM Tris-HCl pH 7.4, 10 mM of MgCl2, 5 mM 3-hydroxypirazine-2-carboxamide, 5% glycerol, 0.04% NaN3) to an equal volume of the precipitant solution (100 mM HEPES pH 6.8, 150 mM sodium citrate). We placed a droplet of the solution on the surface of a siliconized glass plate, which was covering a cuvette containing 1 mL of the precipitant solution. The formation of the crystals could be observed within two weeks. The crystallization conditions were adapted and optimized for the capillary counter diffusion method [55].

2.4. X-Ray Data Processing and Structure Analysis

The diffraction data set was collected at the Elettra (Italy) synchrotron using the XRD2 beamline at a resolution of 2.08 Å. The diffraction data were obtained by the rocking rotation method at a distance of 300 mm between the crystal and detector and a wavelength of 0.9999 Å, with the rocking and rotation angles being 0.1° and 360°, respectively. The set of experimental reflection intensities was integrated using the iMosflm 7.4.0 [56] and scaled using AIMLESS 0.8.2 [57]. We have used the PHASER for phasing [58]. The previously determined structure of EcoHGPRT was used as the starting model while using the molecular replacement method (PDB 8CAG). The iterative model building, model completion, and validation were performed in COOT [59]. Structure refinement was performed using Refmac5 [60].
Biomolecular structures were visualized and analyzed in PyMOL 2.5. The structures of PRT-like proteins were subjected to structural alignment via the SALIGN module of MODELLER 10.7 [61]. The structures with the following PDB ID were subjected to analysis: 9K8M, 1G9T, 1FSG, 4QYI, 1O57, 3HVU, and 1ECG. In structures with PDB ID 1O57 and 1ECG, only the PRT-like domain was taken into account (residues 112–276 and 253–492, respectively). Multiple sequence alignments were carried out using MAFFT 7.490 and rendered via ESPript 3.0 and WebLogo 3 [62,63,64]. Conservation analysis was performed through the alignment of all sequences from reference proteome sources (Uniprot database, keyword KW-1185), annotated as the respective enzyme classification number in the Uniprot database. For PurR, the gene name was used as the search criterion instead of the enzyme classification number. The number of sequences belonging to reference proteomes and annotated as PRT-like proteins (PF00156) was 74,027; among those were the following: HGPRT—10,507, PurF—7794, and PurR—490. For conservation analysis among all PRT-like proteins, the number of sequences was reduced from 74,027 to 3798 through clustering (50% identity and coverage), using MMseqs2 [65].

3. Results and Discussion

The recombinant EcoHGPRT was produced and purified as described in the materials and methods section. Crystals were obtained through the capillary counter diffusion method, using a solution with 0.15 M sodium citrate and 0.1 M HEPES as the precipitant. A diffraction data set was collected at the Elettra (Italy) synchrotron using the XRD2 beamline, allowing us to solve the structure to a resolution of 2.08 A. The obtained crystals belonged to space group P3121, and contained two independent subunits in the asymmetric unit. The X-ray structure was solved via the molecular replacement method. We were able to observe clear electron density, which was interpreted as a HEPES molecule. The solved X-ray structure was deposited in the Protein Data Bank database (PDB 9K8M), and the statistical parameters for the structure refinement and the experimental data set are provided in Table 1 and Table 2.
In Figure 1, we compare the binding mode of HEPES as observed by us to that of the natural product and substrate reported previously (GMP and PRPP, respectively) [54,66]. Since there is no X-ray structure of EcoHGPRT in a complex with its substrates, we have used the crystal structure of a homologue—HGPRT from Toxoplasma gondii (TgoHGPRT).
As we can observe in Figure 1A, the binding of HEPES occurs mainly through a network of hydrogen bonds formed between its sulphonate group and EcoHGPRT active site. The charged group is located in a pocket formed by a small loop and a part of an α-helix (residues D103–L108). This structural element is responsible for the binding of the 5-phosphate group of the natural substrate and product. Hence, we will later refer to these residues as the “5P-loop”. Besides the sulphonate group, the hydroxyl group of HEPES also participates in the binding of this ligand through a hydrogen bond with Gly44. This residue belongs to another loop, L42–G44. Comparison with the structure of TgoHGPRT in a complex with PRPP suggests that this loop is responsible for the binding of the pyrophosphate group of the natural substrate. Therefore, this loop will later be referred to as the “PP-loop”.
Thus, we have obtained a crystal structure of EcoHGPRT and observed the presence of a HEPES molecule in the active site of the enzyme. The HEPES molecule occupies a position in the active site that is similar to that of the carbohydrate donor substrate, namely PRPP. This result suggests that HEPES could be an inhibitor of the investigated enzyme. In order to verify that HEPES inhibits the investigated enzyme, we have determined the dependency of EcoHGPRT activity on HEPES concentration. The activity of IMP synthesis by EcoHGPRT was measured through defection of the conversion of the nucleobase into the nucleotide product via HPLC. As a result, we have confirmed that HEPES inhibits IMP synthesis by EcoHGPRT with an IC50 value of 187 ± 9 mM.
Next, we have performed a comparative analysis of different positions that HEPES and similar molecules assume in the active site of HGPRT and other proteins with PRT-like fold (Pfam PF00156). Besides the structure reported by us in this article (PDB 9K8M), there are 10 X-ray structures of PRT-like proteins in a complex with a HEPES, MES or PIPES molecule. Four structures represent little interest for our research, since the mentioned ligands are located outside of the active site (or similar structural elements). Among these, three are structures of the complex between EcoHGPRT and phosphonate inhibitors (PDB 5KNS, 5KNT, 5KNU) [67]. The fourth one is the structure of PRT-like protein TT1426 from Thermus thermophilus (PDB 1WD5) [68].
The remaining six structures contain a ligand of interest, located in the active site of the phosphoribosyltransferase (or a similar region of a PRT-like protein that is not an enzyme). Two structures are complexes with a HEPES molecule: HGPRT from Bacillus anthracis (BanHGPRT2, PDB 4QYI) and the purine operon repressor from Bacillus subtilis (BsuPurR, PDB 1O57) [46]. The next two are structures of HGPRT from Trypanosoma cruzi (TcrHGPRT, PDB 1TC1) and Bacillus anthracis (BanHGPRT1, PDB 3HVU) in a complex with MES [47]. It should be noted that Bacillus anthracis has two different HGPRT enzymes, referred to here as BanHGPRT1 and BanHGPRT2. Finally, there are structures of glutamine phosphoribosylpyrophosphate amidotransferase (ec:2.4.2.14) from Escherichia coli in a complex with PIPES (EcoPurF, PDB 1ECF and 1ECG) [48,49]. The comparison of binding modes of HEPES, MES, and PIPES molecules in the active sites of PRT-like proteins is presented in Figure 2 (TcrHGPRT/MES complex is not shown since the ligand position is similar to the one observed in BanHGPRT1).
As we can see in Figure 2, all of the illustrated molecules are located between the 5P-loop and PP-loop, thus mimicking the position of the natural substrate PRPP. Nevertheless, there is a noteworthy variety of positions of the HEPES molecule. In EcoHGPRRT, the sulphate group of HEPES is located in a pocket formed by the 5P-loop, while the interactions with the PP-loop are limited to a single hydrogen bond through the hydroxyl group. While the interactions between HEPES and the 5P-loop are similar in both EcoHGPRT and BanHGPRT2, interactions with the PP-loop are more complicated in the latter enzymes’ active site. The interaction between the PP-loop of BanHGPRT2 and the hydroxyl group of HEPES is mediated through phosphate and magnesium ions. The interactions between BsuPurR and the ligands is even more different, with the HEPES molecule assuming a reverse position compared to the one observed in EcoHGPRT and BanHGPRT2. The sulphonate group of HEPES binds to the PP-loop of BsuPurR, as opposed to 5P-loop in the above-mentioned enzymes. Meanwhile, a sulphate ion occupies the pocket-formed 5P-loop of BsuPurR, while the hydroxyl group of HEPES is oriented toward the sulphate ion.
It is curious that the X-ray structure reported by us illustrates that HEPES binding can occur without the presence of a negatively charged ion (which was not present in the solution used during protein crystallization). The interaction of HEPES with the PP-loop of EcoHGPRT is limited to a single hydrogen bond, while in the other structures both the 5P-loop and PP-loop interact with a negatively charged chemical group/ion. The binding of PIPES also occurs in a similar matter, with the two symmetrical sulphonate groups interacting with the 5P-loop and PP-loop of EcoPurF. Meanwhile, the examination of the complex between BanHGPRT1 and MES suggests that the interaction only with the 5P-loop is sufficient for ligand binding.
We have determined the IC50 and Ki values for inhibition of EcoHGPRT activity by HEPES, MES, and PIPES. The obtained values are provided in Table 3, while the inhibition curves are shown in Figure S1. The Ki values were estimated assuming a completive inhibition model, while the precise inhibition type was not investigated in this study. According to these results, HEPES and MES are approximately equivalent inhibitors of EcoHGPRT. This fact suggests that the interaction with the PP-loop plays only a limited role in the inhibition of the enzyme by HEPES. One could speculate that HEPES could be a stronger inhibitor in the presence of sulphate or phosphate ions, due to the formation of structures we could observe with BanHGPRT2 and BsuPurR. In order to test this assumption, we have measured EcoHGPRT activity in the presence/absence of 50 mM HEPES/MES and 10 mM PO43−/SO42− ions (Supplementary Materials, Figure S2). The presence of 10 mM PO43−/SO42− ions has caused a decrease in activity comparable to 50 mM HEPES/MES. Combining the presence of the inorganic ion and sulphonate inhibitor has caused a slight further decrease in enzymatic activity. Nevertheless, the effects were similar for both HEPES and MES. The latter molecule is unable to interact with ions like we can observe in the BanHGPRT2 complex with HEPES, since MES does not have a hydroxyethyl group. Therefore, the observed result can be explained by simultaneous inhibition by two molecules, without any synergetic effect caused by possible ion-sulphonate interactions in the active site.
In contrast to the discussion above, we have observed significantly stronger inhibition of EcoHGPRT by PIPES compared to MES and HEPES, with IC50 values being more than twofold lower. There are multiple factors that could explain this result. It could be possible that the PIPES molecule assumes a position similar to the one observed in EcoPurF active site, thus binding to both the 5P-loop and PP-loop with the symmetrical charged groups. In a PIPES molecule, the two sulphonate groups are connected through covalent bonds, unlike the sulphonate—PO43−/SO42− complexes, illustrated in Figure 2B,C. In comparison to a free ion, it would be easier for the covalently bonded sulphonate group to interact with the PP-loop, thus positively contributing to the inhibition strength. Moreover, PIPES is a symmetrical molecule; therefore, it theoretically has two identical positions in the active site that are equivalent for inhibition.
It should be noted that one should be cautious while extrapolating the structure–function relationship between HGPRT and other PRT-like proteins. In Figure 3 we compare the sequences of PP-loop and 5P-loop between the investigated enzymes and illustrate the amino acid residue conservation. As we can see in Figure 3A,B, the sequences of these loops are similar between different HGPRT, and the residues that form these loops are highly conserved. The importance of these conserved residues for catalysis has been previously shown on TgoHGPRT and the human enzyme HsaHGPRT [69,70,71].
The sequences of both the PP-loop and the 5P-loop differ significantly in both PurR and PurF enzymes in comparison to HGPRT. In particular, the PP-loop is longer by one residue in PurR compared to PurF and HGPRT. The binding of sulphate/sulphonate ions by PurR/PurF includes interactions with conserved residues that are different in HGPRT.
The difference between D103 in EcoHGPRT and similar residues in PurF and PurR is especially interesting in the context of previously reported research on the role of this Asp residue in catalysis, studied in other HGPRT through site-directed mutagenesis. Mutations D137A in HsaHGPRT and D150A in TgoHGPRT have resulted in a 22-fold and 6-fold reduction in KM towards PRPP, respectively [70,71]. Canyuk et al. suggests that the observed increase in affinity towards PRPP can be explained by the removal of a negative charge in the 5P-loop, which interacts with the negatively charged phosphate group of the substrate [71]. It should also be noted that both D137A and D150A mutations have caused a significant reduction in kcat. Canyuk et al. illustrate that while the D137A increases the enzymes’ affinity towards PRPP, the position that the substrate assumes in the mutant enzymes’ active site is less productive for catalysis.
In contrast to HGPRT, PurR proteins have a highly conservative, positively charged Lys residue in the position of D103 in EcoHGPRT. Meanwhile, PurF has a super conservative Arg residue as an immediate neighbour to the discussed position. Such differences can play a significant role in the affinity of the 5P-loop towards negatively charged ions in PurR and PurF compared to HGPRT.
The results of the sequence conservation analysis illustrate that while the loop conformations are similar in PRT-like proteins, there are significant differences in amino acid residues. These differences can influence the affinity of PP-loop and 5P-loop towards sulphonate molecules. Therefore, despite the observed similarities in the location of sulphonate molecules, extrapolation of the structure–function relationship between these enzymes should be performed with caution. The binding mode of PIPES to EcoPurF is very similar to the one observed for HEPES/MES in the HGPRT active site, taking into account the interaction between the sulphonate ion and the 5P-loop. In contrast, the observed differences in the PP-loop and 5P-loop sequences suggests that the highly different “inverse” position of HEPES observed in the BsaPurR X-ray structure might be specific to this type of enzyme.
To conclude, we have confirmed that HEPES, MES, and PIPES are inhibitors of EcoHGPRT. In the obtained crystal structure, the position of HEPES in the active site of EcoHGPRT mimics that of the natural substrate PRPP. The observed IC50 values are comparable to the common concentrations of buffering agents in X-ray crystallography. Thus, even though the investigated molecules are relatively weak inhibitors, their effects should still be accounted for in research dealing with HGPRT as an enzyme design object or drug development target.
Solving and investigating the X-ray structure of EcoHGPRT in a complex with HEPES has allowed us to compare the ligand position to other reported structures of PRT-like proteins with sulphonate molecules in their active sites. Common features of the observed binding modes of HEPES/MES/PIPES in PRT-like proteins are as follows: (1) localization in a position similar to the natural carbohydrate donor substrate of HGPRT, namely PRPP; (2) interaction of the molecule with the 5P-loop, the PP-loop, or both through charged chemical groups. There are three variants of the binding of sulphonate molecules to PRT-like proteins. The first one is when the sulphonate group interacts only with the 5P-loop, as observed in the EcoHGPRT/HEPES and BanHGPRT1/MES complexes. The second option is an interaction with both the 5P-loop and the PP-loop, while the binding of the molecule to one of these loops is mediated through an inorganic phosphate/sulphate ion. In this regard, the EcoHGPRT structure reported by us in this article is a remarkable example of HEPES binding without the participation of a sulphate/phosphate ion. The third option is the binding of the two sulphonate groups of PIPES to both the 5P-loop and the PP-loop. The fact that PIPES turned out to be the strongest inhibitor among the investigated molecules suggests the positive role of this interaction on ligand binding when the two charged groups are covalently bound.
All of these discussed results concerning the binding of the sulphonate molecules to the active site of HGPRT can be useful for further investigation of the structure–function relationship in this enzyme. The next logical steps for the research direction are enzymological studies aimed at the determination of the precise type of inhibition by investigated compounds. Another interesting direction could be obtaining the X-ray structures of EcoHGPRT in a complex with MES and PIPES, which would allow us to rule out the possible influence of differences in active site structure on sulphonate molecule binding. The understanding of the interactions between HGPRT and sulphonate molecules might be useful for further enzyme or drug-design studies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cryst15100874/s1, Figure S1: Dependence of the enzymatic activity of EcoHGPRT on the concentration of HEPES, MES or PIPES, represented as % relative to reaction without any inhibitors. Each data point represents a single measurement, while the lines show the inhibition curves built using the calculated parameters; Figure S2. Enzymatic activity of EcoHGPRT, represented as % relative to reaction without MES/HEPES (50 mM) or SO42−/PO43− (10 mM). Data is represented as mean and standard derivation of mean (n = 3). * p ≤ 0.05 compared to the control group (without MES/HEPES or SO42−/PO43−) according to the Mann- Whitney test.

Author Contributions

Conceptualization, E.A.Z., I.D.K., V.I.B. and R.S.E.; methodology, E.A.Z., Y.A.A., M.A.K. and I.V.F.; software, E.A.Z., V.I.T. and M.B.S.; validation, M.A.K. and I.V.F.; formal analysis, I.V.F., A.V.M. and A.O.A.; investigation, E.A.Z., Y.A.A., M.A.K., V.I.T., M.B.S., I.V.F., A.A.K., A.R.S. and A.O.A.; resources, Y.A.A., M.A.K. and I.V.F.; data curation, I.D.K. and R.S.E.; writing—original draft preparation, E.A.Z., V.I.T. and I.V.F.; writing—review and editing, E.A.Z., V.I.B. and R.S.E.; visualization, E.A.Z.; supervision, V.I.B. and R.S.E.; project administration, V.I.B. and R.S.E.; funding acquisition, V.I.B. and R.S.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Russian Science Foundation (Project No. 24-14-00458), including the works on genetic engineering, recombinant protein production, purification, and crystallization, enzymological studies. The X-ray data collection and analysis was supported by the Ministry of Science and Higher Education of the Russian Federation (agreement # 075-03-2025-662, project FSMG-2024-0012)).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors.

Acknowledgments

We gratefully acknowledge Elettra and XRD2 beamline for providing beamtime and support under proposal 20225306.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CVColoumn volume
HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HGPRThypoxanthine-guanine phosphorybosyltransferase
MES2-(N-morpholino)ethanesulfonic acid
PIPESPiperazine-N,N′-bis(2-ethanesulfonic acid)
PRPPPhosphoribosylpyrophosphate
PRT-likePhosphoribosyltrapsferase-like
9dG9-deazaguanine

References

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Crystals 15 00874 sch001
Scheme 1. (A) Reaction, catalyzed by hypoxanthine-guanine phosphorybosyltransferase. (B) Chemical structures of HEPES, MES, and PIPES. Red highlights the differences between these molecules.
Scheme 1. (A) Reaction, catalyzed by hypoxanthine-guanine phosphorybosyltransferase. (B) Chemical structures of HEPES, MES, and PIPES. Red highlights the differences between these molecules.
Crystals 15 00874 sch001
Crystals 15 00874 g001
Figure 1. Crystal structures of HGPRT in complex with various ligands. (A) EcoHGPRT in complex with HEPES, PDB 9K8M; (B) EcoHGPRT in complex with GMP, PDB 1G9T; (C) TgoHGPRT in complex with PRPP, two magnesium ions and the nucleobase substrate analogue 9-deazoguanine (9dG), PDB 1FSG.
Figure 1. Crystal structures of HGPRT in complex with various ligands. (A) EcoHGPRT in complex with HEPES, PDB 9K8M; (B) EcoHGPRT in complex with GMP, PDB 1G9T; (C) TgoHGPRT in complex with PRPP, two magnesium ions and the nucleobase substrate analogue 9-deazoguanine (9dG), PDB 1FSG.
Crystals 15 00874 g001
Crystals 15 00874 g002
Figure 2. Comparison of the positions of HEPES/MES/PIPES in the active sites of various PRT-like proteins; (A) EcoHGPRT in complex with HEPES (PDB 9K8M); (B) BanHGPRT2 in complex with HEPES, PO43− and Mg2+ (PDB 4QYI); (C) BsuPurR in complex with HEPES and SO42− (PDB 1O57); (D) BanHGPRT1/MES (PDB 3HVU); (E) EcoPurF in complex with PIPES (PDB 1ECG); (F) TgoHGPRT in complex with 9-deazaguanine (9dG), PRPP and Mg2+ (PDB 1FSG). Some residues are hidden (EcoHGPRT 69–78, BanHGPRT2 70–80, BsuPurR 175–185, TgoHGPRT 115–125).
Figure 2. Comparison of the positions of HEPES/MES/PIPES in the active sites of various PRT-like proteins; (A) EcoHGPRT in complex with HEPES (PDB 9K8M); (B) BanHGPRT2 in complex with HEPES, PO43− and Mg2+ (PDB 4QYI); (C) BsuPurR in complex with HEPES and SO42− (PDB 1O57); (D) BanHGPRT1/MES (PDB 3HVU); (E) EcoPurF in complex with PIPES (PDB 1ECG); (F) TgoHGPRT in complex with 9-deazaguanine (9dG), PRPP and Mg2+ (PDB 1FSG). Some residues are hidden (EcoHGPRT 69–78, BanHGPRT2 70–80, BsuPurR 175–185, TgoHGPRT 115–125).
Crystals 15 00874 g002
Crystals 15 00874 g003
Figure 3. Multiple sequence alignment of investigated PRT-like proteins (A) and WebLogo illustration of PP-loop and 5P-loop residue conservation among HGPRT (B), PurR (C), PurF, (D) and all PRT-like enzymes (E). Y-axis represents probability of residue variant (%). X-axis—examined residues, with numbers corresponding to the protein indicated in brackets.
Figure 3. Multiple sequence alignment of investigated PRT-like proteins (A) and WebLogo illustration of PP-loop and 5P-loop residue conservation among HGPRT (B), PurR (C), PurF, (D) and all PRT-like enzymes (E). Y-axis represents probability of residue variant (%). X-axis—examined residues, with numbers corresponding to the protein indicated in brackets.
Crystals 15 00874 g003
Table 1. X-ray data collection and processing statistics.
Table 1. X-ray data collection and processing statistics.
ParameterValue
Diffraction SourceELETTRA BEAMLINE XRD2
Wavelength (Å)0.9999
Temperature (K)100
DetectorDECTRIS EIGER R 1M
Crystal to detector distance (mm)300
Total rotation range (°)360
Rotation angle per image (°)0.1
Exposure time per image (s)0.1
Space groupP3121
a, b, c (Å)85.22, 85.22, 168.28
α β, γ (°)90, 90, 120
Mosaicity (°)2.7
Resolution range (Å)2.08–29.94 (2.08–2.14)
Total no. of reflections167,079 (10,069)
No. of unique reflections40,751 (2877)
Completeness (%)99.67 (96.5)
Redundancy4.1 (3.5)
<I/σ(I)> from merged data8 (1.9)
Rmeas0.13 (0.43)
Table 2. Structure refinement statistics.
Table 2. Structure refinement statistics.
ParameterValue
PDB ID9K8M
Resolution range (Å)29.94–2.08 (2.14–2.08)
Completeness (%)99.7 (96.5)
No. of reflections, working set38,642 (2877)
No. of reflections, test set2109 (151)
Final Rcryst0.205 (0.289)
Final Rfree0.227 (0.321)
No. of non-H atoms2720
Protein2651
Ions0
Ligands30
Waters39
R.m.s. deviations from ideality
Bonds (Å)0.013
Angles (°)1.908
Average B factors for all atoms (Å2)46.99
   for protein atoms49.99
   for ligands atoms67.09
   for waters44.38
Ramachandran plot
Favoured regions (%)96
Table 3. The parameters describing inhibition of EcoHGPRT by HEPES, MES, and PIPES.1
Table 3. The parameters describing inhibition of EcoHGPRT by HEPES, MES, and PIPES.1
MoleculeIC50, mMHill CoefficientKi, mM
HEPES187 ± 91.52 ± 0.1366.4 ± 3.2
MES171 ± 31.78 ± 0.0567.9 ± 1.2
PIPES77.1 ± 7.01.30 ± 0.1424.7 ± 2.2
1 Ki values were estimated assuming a completive inhibition model (precise inhibition type was not investigated in the study).
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Zayats, E.A.; Abramchik, Y.A.; Kostromina, M.A.; Timofeev, V.I.; Shevtsov, M.B.; Mishin, A.V.; Fateev, I.V.; Karanov, A.A.; Sharafutdinova, A.R.; Arnautova, A.O.; et al. Interference of Sulphonate Buffering Agents with E. coli Hypoxanthine-Guanine Phosphoribosyltransferase Active Site Functioning: A Crystallographic and Enzymological Study. Crystals 2025, 15, 874. https://doi.org/10.3390/cryst15100874

AMA Style

Zayats EA, Abramchik YA, Kostromina MA, Timofeev VI, Shevtsov MB, Mishin AV, Fateev IV, Karanov AA, Sharafutdinova AR, Arnautova AO, et al. Interference of Sulphonate Buffering Agents with E. coli Hypoxanthine-Guanine Phosphoribosyltransferase Active Site Functioning: A Crystallographic and Enzymological Study. Crystals. 2025; 15(10):874. https://doi.org/10.3390/cryst15100874

Chicago/Turabian Style

Zayats, Evgeniy A., Yulia A. Abramchik, Maria A. Kostromina, Vladimir I. Timofeev, Mikhail B. Shevtsov, Alexey V. Mishin, Ilya V. Fateev, Andrey A. Karanov, Alexandra R. Sharafutdinova, Aleksandra O. Arnautova, and et al. 2025. "Interference of Sulphonate Buffering Agents with E. coli Hypoxanthine-Guanine Phosphoribosyltransferase Active Site Functioning: A Crystallographic and Enzymological Study" Crystals 15, no. 10: 874. https://doi.org/10.3390/cryst15100874

APA Style

Zayats, E. A., Abramchik, Y. A., Kostromina, M. A., Timofeev, V. I., Shevtsov, M. B., Mishin, A. V., Fateev, I. V., Karanov, A. A., Sharafutdinova, A. R., Arnautova, A. O., Konstantinova, I. D., Borshchevskiy, V. I., & Esipov, R. S. (2025). Interference of Sulphonate Buffering Agents with E. coli Hypoxanthine-Guanine Phosphoribosyltransferase Active Site Functioning: A Crystallographic and Enzymological Study. Crystals, 15(10), 874. https://doi.org/10.3390/cryst15100874

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