Myelin Basic Protein Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

Crystal Structure of Inhibitor-Bound Bacterial Oligopeptidase B in the Closed State: Similarity and Difference between Protozoan and Bacterial Enzymes.

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the ß-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the ß-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

Rational Mutagenesis in the Lid Domain of Ribokinase from E. coli Results in an Order of Magnitude Increase in Activity towards D-arabinose.

Development of efficient approaches for the production of medically important nucleosides is a highly relevant challenge for biotechnology. In particular, cascade synthesis of arabinosides would allow relatively easy production of various cytostatic and antiviral drugs. However, the biocatalyst necessary for this approach, ribokinase from Escherichia coli (EcoRK), has a very low activity towards D-arabinose, making the synthesis using the state-of-art native enzyme technologically unfeasible. Here, we report the results of our enzyme design project, dedicated to engineering a mutant form of EcoRK with elevated activity towards arabinose. Analysis of the active site structure has allowed us to hypothesize the reasons behind the low EcoRK activity towards arabinose and select feasible mutations. Enzyme assay and kinetic studies have shown that the A98G mutation has caused a large 15-fold increase in kcat and 1.5-fold decrease in KM for arabinose phosphorylation. As a proof of concept, we have performed the cascade synthesis of 2-chloroadenine arabinoside utilizing the A98G mutant with 10-fold lower amount of enzyme compared to the wild type without any loss of synthesis efficiency. Our results are valuable both for the development of new technologies of synthesis of modified nucleosides and providing insight into the structural reasons behind EcoRK substrate specificity.

Unusual Cytochrome c552 from Thioalkalivibrio paradoxus: Solution NMR Structure and Interaction with Thiocyanate Dehydrogenase.

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called "intrinsically disordered" nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.

Computational designing of a novel subunit vaccine for human cytomegalovirus by employing the immunoinformatics framework.

Human cytomegalovirus (HCMV) is a widespread virus that can cause serious and irreversible neurological damage in newborns and even death in children who do not have the access to much-needed medications. While some vaccines and drugs are found to be effective against HCMV, their extended use has given rise to dose-limiting toxicities and the development of drug-resistant mutants among patients. Despite half a century's worth of research, the lack of a licensed HCMV vaccine heightens the need to develop newer antiviral therapies and vaccine candidates with improved effectiveness and reduced side effects. In this study, the immunoinformatics approach was utilized to design a potential polyvalent epitope-based vaccine effective against the four virulent strains of HCMV. The vaccine was constructed using seven CD8+ cytotoxic T lymphocytes epitopes, nine CD4+ helper T lymphocyte epitopes, and twelve linear B-cell lymphocyte epitopes that were predicted to be antigenic, non-allergenic, non-toxic, fully conserved, and non-human homologous. Subsequently, molecular docking study, protein-protein interaction analysis, molecular dynamics simulation (including the root mean square fluctuation (RMSF) and root mean square deviation (RMSD)), and immune simulation study rendered promising results assuring the vaccine to be stable, safe, and effective. Finally, in silico cloning was conducted to develop an efficient mass production strategy of the vaccine. However, further in vitro and in vivo research studies on the proposed vaccine are required to confirm its safety and efficacy.Communicated by Ramaswamy H. Sarma.

Immunoinformatic Design of a Multivalent Peptide Vaccine Against Mucormycosis: Targeting FTR1 Protein of Major Causative Fungi.

Mucormycosis is a potentially fatal illness that arises in immunocompromised people due to diabetic ketoacidosis, neutropenia, organ transplantation, and elevated serum levels of accessible iron. The sudden spread of mucormycosis in COVID-19 patients engendered massive concern worldwide. Comorbidities including diabetes, cancer, steroid-based medications, long-term ventilation, and increased ferritin serum concentration in COVID-19 patients trigger favorable fungi growth that in turn effectuate mucormycosis. The necessity of FTR1 gene-encoded ferrous permease for host iron acquisition by fungi has been found in different studies recently. Thus, targeting the transit component could be a potential solution. Unfortunately, no appropriate antifungal vaccine has been constructed as of yet. To date, mucormycosis has been treated with antiviral therapy and surgical treatment only. Thus, in this study, the FTR1 protein has been targeted to design a convenient and novel epitope-based vaccine with the help of immunoinformatics against four different virulent fungal species. Furthermore, the vaccine was constructed using 8 CTL, 2 HTL, and 1 LBL epitopes that were found to be highly antigenic, non-allergenic, non-toxic, and fully conserved among the fungi under consideration. The vaccine has very reassuring stability due to its high pI value of 9.97, conclusive of a basic range. The vaccine was then subjected to molecular docking, molecular dynamics, and immune simulation studies to confirm the biological environment's safety, efficacy, and stability. The vaccine constructs were found to be safe in addition to being effective. Finally, we used in-silico cloning to develop an effective strategy for vaccine mass production. The designed vaccine will be a potential therapeutic not only to control mucormycosis in COVID-19 patients but also be effective in general mucormycosis events. However, further in vitro, and in vivo testing is needed to confirm the vaccine's safety and efficacy in controlling fungal infections. If successful, this vaccine could provide a low-cost and effective method of preventing the spread of mucormycosis worldwide.

The comparative analysis of the properties and structures of purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27.

Two recombinant purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27 encoded by genes TT_C1070 (TthPNPI) and TT_C0194 (TthPNPII) were purified and characterized. The comparative analysis of their sequences, molecular weight, enzymes specificity and kinetics of the catalyzed reaction were realized. As a result, it was determined that the TthPNPI is specific to guanosine while the TthPNPII to adenosine. According to the results of the size exclusion chromatography and SAXS study both enzymes are hexameric molecules. Based on the sequence alignment with homologous purine nucleoside phosphorylases (PNPs), Asn was identified as a purine base recognizing residue in the active site of TthPNPI and Asp in TthPNPII. The three-dimensional structure of TthPNPII was solved at 2.5 Å resolution by molecular replacement method using crystals grown in microgravity. Position of phosphate in the active site cavity is located. The possible arrangement of adenosine and guanosine in TthPNPII active site cavity is considered using superposition with the structures of homologous trimeric and hexameric PNPs complexed with corresponding substrates. The peculiarities of oligomeric structure of TthPNPII in comparison with homologous PNPs are described. It is shown that two trimeric molecules of TthPNPII in the asymmetric part of the unit cell are connected by three two-fold axis into a hexamer with 32-point symmetry. This type of hexameric structure of PNP is found for the first time. The interface area between the subunits in trimeric molecule and between the trimers in TthPNPII hexamer is described.Communicated by Ramaswamy H. Sarma.

Immunoinformatics analysis to design novel epitope based vaccine candidate targeting the glycoprotein and nucleoprotein of Lassa mammarenavirus (LASMV) using strains from Nigeria.

Lassa mammarenavirus (LASMV) is responsible for a specific type of acute viral hemorrhagic fever known as Lassa fever. Lack of effective treatments and counter-measures against the virus has resulted in a high mortality rate in its endemic regions. Therefore, in this study, a novel epitope-based vaccine has been designed using the methods of immunoinformatics targeting the glycoprotein and nucleoprotein of the virus. After numerous robust analyses, two CTL epitopes, eight HTL epitopes and seven B-cell epitopes were finally selected for constructing the vaccine. All these most promising epitopes were found to be antigenic, non-allergenic, nontoxic and non-human homolog, which made them suitable for designing the subunit vaccine. Furthermore, the selected T-cell epitopes which were found to be fully conserved across different isolates of the virus, were also considered for final vaccine construction. After that, numerous validation experiments, i.e. molecular docking, molecular dynamics simulation and immune simulation were conducted, which predicted that our designed vaccine should be stable within the biological environment and effective in combating the LASMV infection. In the end, codon adaptation and in silico cloning studies were performed to design a recombinant plasmid for producing the vaccine industrially. However, further in vitro and in vivo assessments should be done on the constructed vaccine to finally confirm its safety and efficacy.Communicated by Ramaswamy H. Sarma.

Structure of the microbial carboxypeptidase T complexed with the transition state analog N-sulfamoyl-l-lysine.

Carboxypeptidase T (CPT) from Thermoactinomyces vulgaris (EC 3.4.17.18) has a broad substrate specificity, the mechanism of which remains unclear. It cleaves off arginine residues by 10, and lysine residues by 100 times worse than hydrophobic leucine residues despite the presence of negatively charged Asp260 at the bottom of the primary specificity pocket. To study the relationship between the structure and specificity the 3D structure of CPT in complex with the stable transition state analog N-sulfamoyl-l-lysine (SLys) was determined in which the S-atom imitates the sp3-hybridized carbon in the scissile-bond. Crystals grown in microgravity has the symmetry of space group P6322. The present complex structure was compared with the previously reported complex structure of CPT and N-sulfamoyl-L-arginine (SArg). The location/binding of SLys in the active site of CPT very closely resembled that of SArg, and the positively charged N-atom of SLys was at the same position as the corresponding positively charged N-atom of SArg. The SLys complex is stabilized by the hydrogen bond between the nitrogen atom and OH-group of Thr257. The contact areas of the residues Tyr255, Leu211, and Thr262 with SLys were reduced in comparison with the same of SArg. This difference in bonding of SArg and SLys side chains in the primary specificity pocket induces shifts differences within the catalytic center (especially Tyr255-O20 and S18-Arg129 N1 gap) that may influence the enzyme's catalytic reaction. Therefore, this information may be useful for the design of carboxypeptidases with improved selectivity towards Arg/Lys for biotechnological applications.

First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine.

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states-closed and open-in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Structure-based inhibitors targeting the alpha-helical domain of the Spiroplasma melliferum histone-like HU protein.

Here we report bisphenol derivatives of fluorene (BDFs) as a new type of chemical probes targeting a histone-like HU protein, a global regulator of bacterial nucleoids, via its dimerization interface perturbation. BDFs were identified by virtual screening and molecular docking that targeted the core of DNA-binding ß-saddle-like domain of the HU protein from Spiroplasma melliferum. However, NMR spectroscopy, complemented with molecular dynamics and site-directed mutagenesis, indicated that the actual site of the inhibitors' intervention consists of residues from the α-helical domain of one monomer and the side portion of the DNA-binding domain of another monomer. BDFs inhibited DNA-binding properties of HU proteins from mycoplasmas S. melliferum, Mycoplasma gallicepticum and Escherichia coli with half-maximum inhibitory concentrations in the range between 5 and 10 µM. In addition, BDFs demonstrated antimicrobial activity against mycoplasma species, but not against E. coli, which is consistent with the compensatory role of other nucleoid-associated proteins in the higher bacteria. Further evaluation of antimicrobial effects of BDFs against various bacteria and viruses will reveal their pharmacological potential, and the allosteric inhibition mode reported here, which avoids direct competition for the binding site with DNA, should be considered in the development of small molecule inhibitors of nucleoid-associated proteins as well as other types of DNA-binding multimeric proteins.

Dodecamers derived from the crystal structure were found in the pre-crystallization solution of the transaminase from the thermophilic bacterium Thermobaculum terrenum by small-angle X-ray scattering.

The pre-crystallization solution of the transaminase from Thermobaculum terrenum (TaTT) has been studied by small-angle X-ray scattering (SAXS). Regular changes in the oligomeric composition of the protein were observed after the addition of the precipitant. Comparison of the observed oligomers with the crystal structure of TaTT (PDB ID 6GKR) shows that dodecamers may act as building blocks in the growth of transaminase single crystals. Correlating of these results to the similar X-ray studies of other proteins suggests that SAXS may be a valuable tool for searching optimum crystallization conditions. AbbreviationSAXSsmall-angle X-ray scatteringTatransaminaseTaTTtransaminase from Thermobaculum terrenumPLPpyridoxal-5'-phosphateR-PEAR-(þ)-1-phenylethylamineBCATbranched-chain amino acid aminotransferaseDAATD-aminoacid aminotransferaseR-TAR-amine:pyruvate transaminaseCommunicated by Ramaswamy H. Sarma.

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme.

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl2 and CuCl2) as precipitants and the three-dimensional structures were determined at 1.35 Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu+2, Ni+2 and Na+1 cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.Communicated by Ramaswamy H. Sarma.

Molecular dynamics complemented by site-directed mutagenesis reveals significant difference between the interdomain salt bridge networks stabilizing oligopeptidases B from bacteria and protozoa in their active conformations.

Oligopeptidases B (OpdBs) are trypsin-like peptidases from protozoa and bacteria that belong to the prolyl oligopeptidase (POP) family. All POPs consist of C-terminal catalytic domain and N-terminal ß-propeller domain and exist in two major conformations: closed (active), where the domains and residues of the catalytic triad are positioned close to each other, and open (non-active), where two domains and residues of the catalytic triad are separated. The interdomain interface, particularly, one of its salt bridges (SB1), plays a role in the transition between these two conformations. However, due to double amino acid substitution (E/R and R/Q), this functionally important SB1 is absent in γ-proteobacterial OpdBs including peptidase from Serratia proteamaculans (PSP). In this study, molecular dynamics was used to analyze inter- and intradomain interactions stabilizing PSP in the closed conformation, in which catalytic H652 is located close to other residues of the catalytic triad. The 3D models of either wild-type PSP or of mutant PSPs carrying activating mutations E125A and D649A in complexes with peptide-substrates were subjected to the analysis. The mechanism that regulates transition of H652 from active to non-active conformation upon domain separation in PSP and other γ-proteobacterial OpdB was proposed. The complex network of polar interactions within H652-loop/C-terminal α-helix and between these areas and ß-propeller domain, established in silico, was in a good agreement with both previously published results on the effects of single-residue mutations and new data on the effects of the activating mutations on each other and on the low active mutant PSP-K655A.Communicated by Ramaswamy H. Sarma.

An explanation for the narrow carbohydrate substrate specificity of adenine phosphoribosyltransferase from Thermus thermophilus from the model of the enzyme, substrate, and magnesium cation cofactor complex.

Communicated by Ramaswamy H. Sarma.

The nature of the ligand's side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex.

The carboxypeptidase T (CPT) from Thermoactinomyces vulgaris has an active site structure and 3D organization similar to pancreatic carboxypeptidases A and B (CPA and CPB), but differs in broader substrate specificity. The crystal structures of CPT complexes with the transition state analogs N-sulfamoyl-L-leucine and N-sulfamoyl-L-glutamate (SLeu and SGlu) were determined and compared with previously determined structures of CPT complexes with N-sulfamoyl-L-arginine and N-sulfamoyl-L-phenylalanine (SArg and SPhe). The conformations of residues Tyr255 and Glu270, the distances between these residues and the corresponding ligand groups, and the Zn-S gap between the zinc ion and the sulfur atom in the ligand's sulfamoyl group that simulates a distance between the zinc ion and the tetrahedral sp3-hybridized carbon atom of the converted peptide bond, vary depending on the nature of the side chain in the substrate's C-terminus. The increasing affinity of CPT with the transition state analogs in the order SGlu, SArg, SPhe, SLeu correlates well with a decreasing Zn-S gap in these complexes and the increasing efficiency of CPT-catalyzed hydrolysis of the corresponding tripeptide substrates (ZAAL > ZAAF > ZAAR > ZAAE). Thus, the side chain of the ligand that interacts with the primary specificity pocket of CPT, determines the geometry of the transition complex, the relative orientation of the bond to be cleaved by the catalytic groups of the active site and the catalytic properties of the enzyme. In the case of CPB, the relative orientation of the catalytic amino acid residues, as well as the distance between Glu270 and SArg/SPhe, is much less dependent on the nature of the corresponding side chain of the substrate. The influence of the nature of the substrate side chain on the structural organization of the transition state determines catalytic activity and broad substrate specificity of the carboxypeptidase T.

Pre-crystallization phase formation of thermolysin hexamers in solution close to crystallization conditions.

Communicated by Ramaswamy H. Sarma.

Functional characterization of PLP fold type IV transaminase with a mixed type of activity from Haliangium ochraceum.

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases and (R)-amine:pyruvate transaminases. Recently, transaminases with a mixed type of activity were identified and characterized. Here, we report biochemical and structural characterization of a transaminase from myxobacterium Haliangium ochraceum (Hoch3033), which is active towards keto analogs of branched-chain amino acids (specific substrates for BCATs) and (R)-(+)-α-methylbenzylamine (specific substrate for (R)-amine:pyruvate transaminases). The enzyme is characterized by an alkaline pH optimum (pH 10.0-10.5) and a tolerance to high salt concentrations (up to 2 M NaCl). The structure of Hoch3033 was determined at 2.35 Šresolution. The overall fold of the enzyme was similar to those of known enzymes of PLP fold type IV. The mixed type of activity of Hoch3033 was implemented within the BCAT-like active site. However, in the active site of Hoch3033, we observed substitutions of specificity-determining residues that are important for substrate binding in canonical BCATs. We suggest that these changes result in the loss of activity towards α-ketoglutarate and increase the affinity towards (R)-(+)-α-methylbenzylamine. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further research to understand the structural basis of substrate specificity in these enzymes.

Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1' subsite from pancreatic carboxypeptidase B.

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1' subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1' subsite was crystallized and its three-dimensional structure was determined at 1.29 Šresolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1' subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1' subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1' subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.

Crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with acyclovir.

Escherichia coli purine nucleoside phosphorylase (PNP), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family I hexameric PNPs. Owing to their key role in the purine salvage pathway, PNPs are attractive targets for drug design against some pathogens. Acyclovir (ACV) is an acyclic derivative of the PNP substrate guanosine and is used as an antiviral drug for the treatment of some human viral infections. The crystalline complex of E. coli PNP with acyclovir was prepared by co-crystallization in microgravity using counter-diffusion through a gel layer in a capillary. The structure of the E. coli PNP-ACV complex was solved at 2.32 Šresolution using the molecular-replacement method. The ACV molecule is observed in two conformations and sulfate ions were located in both the nucleoside-binding and phosphate-binding pockets of the enzyme. A comparison with the complexes of other hexameric and trimeric PNPs with ACV shows the similarity in acyclovir binding by these enzymes.