Crystal structure of human purine nucleoside phosphorylase complexed with acyclovir

In human, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This work reports the first crystallographic study of human PNP complexed with acyclovir (HsPNP:Acy). Acyclovir is a potent clinically useful inhibitor of replicant herpes simplex virus that also inhibits human PNP but with a relatively lower inhibitory activity (K(i)=90 microM). Analysis of the structural differences among the HsPNP:Acy complex, PNP apoenzyme, and HsPNP:Immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design.     

New catalytic mechanism for human purine nucleoside phosphorylase

Human purine nucleoside phosphorylase has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Recently, several structures of human PNP have been reported, which allowed redefinition of the active site and understanding of the structural basis for inhibition of PNP by acyclovir and immucillin-H. Based on previously solved human PNP structures, we proposed here a new catalytic mechanism for human PNP, which is supported by crystallographic studies and explains previously determined kinetic data.     

Structures of human purine nucleoside phosphorylase complexed with inosine and ddI

Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal structures of human PNP in complex with inosine and 2('),3(')-dideoxyinosine, refined to 2.8A resolution using synchrotron radiation. The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor design.     

Crystal structure of human PNP complexed with guanine

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. More recently, the 3-D structure of human PNP has been refined to 2.3A resolution, which allowed a redefinition of the residues involved in the substrate-binding sites and provided a more reliable model for structure-based design of inhibitors. This work reports crystallographic study of the complex of Human PNP:guanine (HsPNP:Gua) solved at 2.7A resolution using synchrotron radiation. Analysis of the structural differences among the HsPNP:Gua complex, PNP apoenzyme, and HsPNP:immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design.     

Crystal structure of human PNP complexed with hypoxanthine and sulfate ion

Purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme, which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. Human PNP has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Here we report the crystal structure of human PNP in complex with hypoxanthine, refined to 2.6A resolution. The intermolecular interaction between ligand and PNP is discussed.     

Structural basis for inhibition of human PNP by immucillin-H

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation. This work reports on the crystallographic study of the complex of human PNP-immucillin-H (HsPNP-ImmH) solved at 2.6A resolution using synchrotron radiation. Immucillin-H (ImmH) inhibits the growth of malignant T-cell lines in the presence of deoxyguanosine without affecting non-T-cell tumor lines. ImmH inhibits activated normal human T cells after antigenic stimulation in vitro. These biological effects of ImmH suggest that this agent may have utility in the treatment of certain human diseases characterized by abnormal T-cell growth or activation. This is the first structural report of human PNP complexed with immucillin-H. The comparison of the complex HsPNP-ImmH with recent crystallographic structures of human PNP explains the high specificity of immucillin-H for human PNP.     

Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus

The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.     

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors.     

Crystal structure of calf spleen purine nucleoside phosphorylase complexed to a novel purine analogue

The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.     

Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine

In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 Å. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K_i = 200 μM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.     

Remote mutations alter transition-state structure of human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine (2'-deoxy)ribonucleosides to give the corresponding purine base and (2'-deoxy)ribose 1-phosphate as products. Human and bovine PNPs (HsPNP and BtPNP) form distinct transition states despite 87% identity in amino acid sequence. A PNP hybrid was produced by replacing K22 and H104 in HsPNP with the corresponding Glu and Arg residues found in BtPNP. We solved the transition-state structure of E:R-HsPNP (K22E:H104R mutant of HsPNP) using competitive kinetic isotope effects (KIE) and global density functional calculations. An array of PNP transition states was generated from optimized structure candidates with varied C1'-N9, C1'-Ophosphate distances, ribosyl pucker configurations and N7-protonation states. Isotopically labeled [1'-3H], [2'-3H], [1'-14C], [9-15N], [1'-14C, 9-15N] and [5'-3H2]inosines gave intrinsic KIE values of 1.210, 1.075, 1.035, 1.024, 1.065, 1.063 with E:R-HsPNP, respectively. The suite of E:R-HsPNP KIEs match a single structure from the array of PNP transition-state candidates. The transition state of E:R-HsPNP is fully dissociative, N7-protonated hypoxanthine (C1'-N9 distance >or= 3.0 A) with partial participation of phosphate (C1'-Ophosphate distance = 2.26 A), 2'-C-exo-ribosyl ring pucker and the O5'-C5'-C4'-O4' dihedral angle near 60 degrees . The transition state of E:R-HsPNP is altered from the fully dissociative DN*AN character for HsPNP to a late phosphate-associative character. E:R-HsPNP differs from native HsPNP by only two residues over 25 A away from the active site. New interactions caused by the mutations increase the catalytic efficiency of the enzyme for formation of a late transition state with increased participation of the phosphate nucleophile. Dynamic coupling motions from the remote mutations to the catalytic sites are proposed.     

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found K _d = 0.12 ± 0.02 μ M for Gua and 0.16 ± 0.01 μ M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were K _d = 0.15 ± 0.01 μ M for Gua (pH 9.0) and 0.25 ± 0.02 μ M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N(7) position.     

Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir

Consumption has been a scourge of mankind since ancient times. This illness has charged a high price to human lives. Many efforts have been made to defeat Mycobacterium tuberculosis (Mt). The M. tuberculosis purine nucleoside phosphorylase (MtPNP) is considered an interesting target to pursuit new potential inhibitors, inasmuch it belongs to the purine salvage pathway and its activity might be involved in the mycobacterial latency process. Here we present the MtPNP crystallographic structure associated with acyclovir and phosphate (MtPNP:ACY:PO₄) at 2.10 Å resolution. Molecular dynamics simulations were carried out in order to dissect MtPNP:ACY:PO₄ structural features, and the influence of the ligand in the binding pocket stability. Our results revealed that the ligand leads to active site lost of stability, in agreement with experimental results, which demonstrate a considerable inhibitory activity against MtPNP (K_i = 150 nM). Furthermore, we observed that some residues which are important in the proper ligand’s anchor into the human homologous enzyme do not present the same importance to MtPNP. Therewithal, these findings contribute to the search of new specific inhibitors for MtPNP, since peculiarities between the mycobacterial and human enzyme binding sites have been identified, making a structural-based drug design feasible.     

Topographical distribution of purine nucleoside phosphorylase in the human neuraxis

The activity of purine nucleoside phosphorylase was determined at various levels of the human neuraxis in 5 brains and 2 spinal cords, using the method of Lewis and Glantz. The determination is based on the decrease in optical density of guanosine at 252 nm and 40 degrees C, with conversion of this compound to guanine and ribose-1-phosphate by phosphorolysis. Our studies show a fairly uniform distribution of the enzyme in the human CNS, with an average value of 209 mumol of guanosine transformed/min/g of wet tissue. The lowest values are found in the spinal cord and cerebellar grey matter, and highest amounts in the occipital grey and white substance.     

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.     

Crystal structure of human alpha-lactalbumin at 1.7 A resolution

The three-dimensional X-ray structure of human alpha-lactalbumin, an important component of milk, has been determined at 1.7 A (0.17 nm) resolution by the method of molecular replacement, using the refined structure of baboon alpha-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P2(1)2(1)2. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0.209 for the 11,373 observed reflections (F greater than or equal to 2 sigma (F)) between 8 and 1.7 A resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0.013 A for covalent bond distances and 2.9 degrees for bond angles. Superposition of the human and baboon alpha-lactalbumin structures yields a root-mean-square difference of 0.67 A for the 123 structurally equivalent C alpha atoms. The C terminus is flexible in the human alpha-lactalbumin molecule. The striking structural resemblance between alpha-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins.     

Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.

The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy. X-ray data to 2.3 A were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 A structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules. The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 A. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2 degrees. This value should be compared with observed values of 10 degrees for the oxidized enzyme-substrate complex and 19 degrees for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found.     

Arsenate and phosphate as nucleophiles at the transition states of human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2'-deoxy)ribonucleosides, generating (2-deoxy)ribose 1-phosphate and the purine base. Transition-state models for inosine cleavage have been proposed with bovine, human, and malarial PNPs using arsenate as the nucleophile, since kinetic isotope effects (KIEs) are obscured on phosphorolysis due to high commitment factors. The Phe200Gly mutant of human PNP has low forward and reverse commitment factors in the phosphorolytic reaction, permitting the measurement of competitive intrinsic KIEs on both arsenolysis and phosphorolysis of inosine. The intrinsic 1'-(14)C, 1'-(3)H, 2'-(2)H, 9-(15)N, and 5'-(3)H(2) KIEs for inosine were measured for arsenolysis and phosphorolysis. Except for the remote 5'-(3)H(2), and some slight difference between the 2'-(2)H KIEs, all isotope effects originating in the reaction coordinate are the same within experimental error. Hence, arsenolysis and phosphorolysis proceed through closely related transition states. Although electrostatically similar, the volume of arsenate is greater than phosphate and supports a steric influence to explain the differences in the 5'-(3)H(2) KIEs. Density functional theory calculations provide quantitative models of the transition states for Phe200Gly human PNP-catalyzed arsenolysis and phosphorolysis, selected upon matching calculated and experimental KIEs. The models confirm the striking resemblance between the transition states for the two reactions.