GMP Synthetase: Allostery, Structure, and Function.

Glutamine amidotransferases (GATs) catalyze the hydrolysis of glutamine and transfer the generated ammonia to diverse metabolites. The two catalytic activities, glutaminolysis and the subsequent amination of the acceptor substrate, happen in two distinct catalytic pockets connected by a channel that facilitates the movement of ammonia. The de novo pathway for the synthesis of guanosine monophosphate (GMP) from xanthosine monophosphate (XMP) is enabled by the GAT GMP synthetase (GMPS). In most available crystal structures of GATs, the ammonia channel is evident in their native state or upon ligand binding, providing molecular details of the conduit. In addition, conformational changes that enable the coordination of the two catalytic chemistries are also informed by the available structures. In contrast, despite the first structure of a GMPS being published in 1996, the understanding of catalysis in the acceptor domain and inter-domain crosstalk became possible only after the structure of a glutamine-bound mutant of Plasmodium falciparum GMPS was determined. In this review, we present the current status of our understanding of the molecular basis of catalysis in GMPS, becoming the first comprehensive assessment of the biochemical function of this intriguing enzyme.

Purine containing carbonucleoside phosphonate analogues as novel chemotype for Plasmodium falciparum Inhibition.

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and ß-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.

Structural and molecular determinants of Candida glabrata metacaspase maturation and activation by calcium.

Metacaspases are caspase-like homologs which undergo a complex maturation process involving multiple intra-chain cleavages resulting in a composite enzyme made of a p10 and a p20 domain. Their proteolytic activity involving a cysteine-histidine catalytic dyad, show peptide bond cleavage specificity in the C-terminal to lysine and arginine, with both maturation- and catalytic processes being calcium-dependent. Here, we present the structure of a metacaspase from the yeast Candida glabrata, CgMCA-I, in complex with a unique calcium along with a structure in which three magnesium ions are bound. We show that the Ca2+ ion interacts with a loop in the vicinity of the catalytic site. The reorganization of this cation binding loop, by bringing together the two catalytic residues, could be one of the main structural determinants triggering metacaspase activation. Enzymatic exploration of CgMCA-I confirmed that the maturation process implies a trans mechanism with sequential cleavages.

Tertiary and Quaternary Structure Organization in GMP Synthetases: Implications for Catalysis.

Glutamine amidotransferases, enzymes that transfer nitrogen from Gln to various cellular metabolites, are modular, with the amidotransferase (GATase) domain hydrolyzing Gln, generating ammonia and the acceptor domain catalyzing the addition of nitrogen onto its cognate substrate. GMP synthetase (GMPS), an enzyme in the de novo purine nucleotide biosynthetic pathway, is a glutamine amidotransferase that catalyzes the synthesis of GMP from XMP. The reaction involves activation of XMP though adenylation by ATP in the ATP pyrophosphatase (ATPPase) active site, followed by channeling and attack of NH3 generated in the GATase pocket. This complex chemistry entails co-ordination of activity across the active sites, allosteric activation of the GATase domain to modulate Gln hydrolysis and channeling of ammonia from the GATase to the acceptor active site. Functional GMPS dimers associate through the dimerization domain. The crystal structure of the Gln-bound complex of Plasmodium falciparum GMPS (PfGMPS) for the first time revealed large-scale domain rotation to be associated with catalysis and leading to the juxtaposition of two otherwise spatially distal cysteinyl (C113/C337) residues. In this manuscript, we report on an unusual structural variation in the crystal structure of the C89A/C113A PfGMPS double mutant, wherein a larger degree of domain rotation has led to the dissociation of the dimeric structure. Furthermore, we report a hitherto overlooked signature motif tightly related to catalysis.

The Candida glabrata glycogen branching enzyme structure reveals unique features of branching enzymes of the Saccharomycetaceae phylum.

Branching enzymes (BE) are responsible for the formation of branching points at the 1,6 position in glycogen and starch, by catalyzing the cleavage of α-1,4-linkages and the subsequent transfer by introducing α-1,6-linked glucose branched points. BEs are found in the large GH13 family, eukaryotic BEs being mainly classified in the GH13_8 subfamily, GH13_9 grouping almost exclusively prokaryotic enzymes. With the aim of contributing to the understanding of the mode of recognition and action of the enzymes belonging to GH13_8, and to the understanding of features distinguishing these enzymes from those belonging to subfamily 13_9, we solved the crystal structure of the glycogen branching enzyme (GBE) from the yeast Candida glabrata, CgGBE, in ligand-free forms and in complex with a maltotriose. The structures revealed the presence of a domain already observed in Homo sapiens and Oryza sativa BEs that we named α-helical N-terminal domain, in addition to the three conserved domains found in BE. We confirmed by phylogenetic analysis that this α-helical N-terminal domain is always present in the GH13_8 enzymes suggesting that it could actually present a signature for this subfamily. We identified two binding sites in the α-helical N-terminal domain and in the carbohydrate binding module 48 (CBM48), respectively, which show a unique structural organization only present in the Saccharomycotina phylum. Our structural and phylogenetic investigation provides new insight into the structural characterization of GH13_8 GBE revealing that unique structural features only present in the Saccharomycotina phylum thereby conferring original properties to this group of enzymes.

Exploring molecular determinants of polysaccharide lyase family 6-1 enzyme activity.

The polysaccharide lyase family 6 (PL6) represents one of the 41 polysaccharide lyase families classified in the CAZy database with the vast majority of its members being alginate lyases grouped into three subfamilies, PL6_1-3. To decipher the mode of recognition and action of the enzymes belonging to subfamily PL6_1, we solved the crystal structures of Pedsa0632, Patl3640, Pedsa3628 and Pedsa3807, which all show different substrate specificities and mode of action (endo-/exolyase). Thorough exploration of the structures of Pedsa0632 and Patl3640 in complex with their substrates as well as docking experiments confirms that the conserved residues in subsites -1 to +3 of the catalytic site form a common platform that can accommodate various types of alginate in a very similar manner but with a series of original adaptations bringing them their specificities of action. From comparative studies with existing structures of PL6_1 alginate lyases, we observe that in the right-handed parallel ß-helix fold shared by all these enzymes, the substrate-binding site harbors the same overall conserved structures and organization. Despite this apparent similarity, it appears that members of the PL6_1 subfamily specifically accommodate and catalyze the degradation of different alginates suggesting that this common platform is actually a highly adaptable and specific tool.

Structure and catalytic regulation of Plasmodium falciparum IMP specific nucleotidase.

Plasmodium falciparum (Pf) relies solely on the salvage pathway for its purine nucleotide requirements, making this pathway indispensable to the parasite. Purine nucleotide levels are regulated by anabolic processes and by nucleotidases that hydrolyse these metabolites into nucleosides. Certain apicomplexan parasites, including Pf, have an IMP-specific-nucleotidase 1 (ISN1). Here we show, by comprehensive substrate screening, that PfISN1 catalyzes the dephosphorylation of inosine monophosphate (IMP) and is allosterically activated by ATP. Crystal structures of tetrameric PfISN1 reveal complex rearrangements of domain organization tightly associated with catalysis. Immunofluorescence microscopy and expression of GFP-fused protein indicate cytosolic localization of PfISN1 and expression in asexual and gametocyte stages of the parasite. With earlier evidence on isn1 upregulation in female gametocytes, the structures reported in this study may contribute to initiate the design for possible transmission-blocking agents.

Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase.

GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. Our results suggest an arrangement at the interdomain interface, of helices with residues that play roles in ATPPase catalysis as well as domain crosstalk enabling the coupling of ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

Active site coupling in Plasmodium falciparum GMP synthetase is triggered by domain rotation.

GMP synthetase (GMPS), a key enzyme in the purine biosynthetic pathway performs catalysis through a coordinated process across two catalytic pockets for which the mechanism remains unclear. Crystal structures of Plasmodium falciparum GMPS in conjunction with mutational and enzyme kinetic studies reported here provide evidence that an 85° rotation of the GATase domain is required for ammonia channelling and thus for the catalytic activity of this two-domain enzyme. We suggest that conformational changes in helix 371-375 holding catalytic residues and in loop 376-401 along the rotation trajectory trigger the different steps of catalysis, and establish the central role of Glu374 in allostery and inter-domain crosstalk. These studies reveal the mechanism of domain rotation and inter-domain communication, providing a molecular framework for the function of all single polypeptide GMPSs and form a solid basis for rational drug design targeting this therapeutically important enzyme.

Blocking human enterovirus 71 replication by targeting viral 2A protease.

OBJECTIVES: Human enterovirus 71 (EV-71), a member of the Enterovirus genus, constitutes a major public health issue in the Asia-Pacific region, where it is associated with several severe neurological complications. There is currently no effective vaccine or antiviral against EV-71. The aim of this study was to determine whether the six amino acid peptide LVLQTM, which was previously shown to inhibit human rhinovirus (HRV) 2A protease (2A(pro)) activity in vitro and HRV replication in vivo in mice, could be of more general use against enteroviruses and more particularly against EV-71. METHODS: To investigate whether the LVLQTM peptide was a pseudosubstrate of EV-71 2A(pro), a recombinant luciferase containing the LVLQTM sequence was designed so that recognition of this sequence by 2A(pro) led to luciferase activation. Direct interaction between EV-71 2A(pro) and the LVLQTM peptide was further confirmed by isothermal titration calorimetry. We then tested the effects of the peptide on EV-71 2A(pro) cleavage activity and EV-71 replication in HeLa cells. RESULTS: We showed that the LVLQTM peptide behaved as an effective substrate analogue of EV-71 2A(pro), which binds into the active site of the protease with a dissociation rate constant of 9.6 µM. Moreover, LVLQTM significantly inhibited eIF4G cleavage activity of 2A(pro) as well as EV-71 replication in HeLa cells. CONCLUSIONS: This study demonstrates that the LVLQTM peptide that has previously been shown to inhibit HRV replication is also an effective inhibitor of EV-71 2A(pro) and therefore of EV-71 replication, opening new doors in the development of new antivirals against EV-71.

Ex vivo and in vivo inhibition of human rhinovirus replication by a new pseudosubstrate of viral 2A protease.

Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2A(pro)) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.

Structural basis of X-ray-induced transient photobleaching in a photoactivatable green fluorescent protein.

We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of pi conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photobleaching in FPs, which are of importance in the rational design of photostable variants.

Reverse pH-dependence of chromophore protonation explains the large Stokes shift of the red fluorescent protein mKeima.

The recently developed red fluorescent protein Keima exhibits the largest Stokes shift (180 nm) observed to date. Combining X-ray crystallography with (in crystallo) UV-visible absorption, fluorescence, and Raman spectroscopy, we have investigated molecular determinants of this peculiar property. The results demonstrate a pH-dependent "reverse chromophore protonation" triggered by the key residue Asp157 and which couples to cis/trans isomerization of the chromophore. These data provided guidelines to rationally design a useful Keima variant.

Structural basis for the phototoxicity of the fluorescent protein KillerRed.

The red fluorescent protein KillerRed, engineered from the hydrozoan chromoprotein anm2CP, has been reported to induce strong cytotoxicity through the chromophore assisted light inactivation (CALI) effect. Here, we present the X-ray structures of KillerRed in its native and bleached states. A long water-filled channel is revealed, connecting the methylene bridge of the chromophore to the solvent. This channel facilitates the transit of oxygen and of reactive oxygen species (ROS) formed by reaction with the excited chromophore. The functional roles of key mutations used to produce KillerRed are discussed, strong chromophore distortions in the bleached state are revealed, and mechanisms for ROS production and self protection are proposed. The presence of a partially mature, photo-resistant, green-emitting state is characterized, which accounts for enhanced CALI by "pre-bleached" KillerRed.

Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed beta-propeller domain of the HIV-1 cellular interactor EED.

BACKGROUND: The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA), integrase (IN) and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D) protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies. RESULTS: Our data suggested that the C-terminal domain of EED was folded as a seven-bladed beta-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique alpha-helix in the fourth beta-blade. More importantly, the position of flexible loops and accessible beta-strands on the beta-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the beta-propeller. CONCLUSION: Identification of antibody-, MA-, IN- and EZH2-binding sites at the surface of the EED isoform 3 provided a global picture of the immunogenic and protein-protein interacting regions in the EED C-terminal domain, organized as a seven-bladed beta-propeller protein. Mapping of the HIV-1 MA and IN binding sites on the 3D-model of EED core predicted that EED-bound MA and IN ligands would be in close vicinity at the surface of the beta-propeller, and that the occurrence of a ternary complex MA-EED-IN would be possible.

Structure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering.

Pseudoalteromonas haloplanktis is a psychrophilic Gram-negative bacterium isolated in Antarctica, that lives on organic remains of algae. This bacterium converts the cellulose, highly constitutive of algae, into an immediate nutritive form by biodegrading this biopolymer. To understand the mechanisms of cold adaptation of its enzymatic components, we studied the structural properties of an endoglucanase, Cel5G, by complementary methods, X-ray crystallography and small angle X-ray scattering. Using X-ray crystallography, we determined the structure of the catalytic core module of this family 5 endoglucanase, at 1.4A resolution in its native form and at 1.6A in the cellobiose-bound form. The catalytic module of Cel5G presents the (beta/alpha)(8)-barrel structure typical of clan GH-A of glycoside hydrolase families. The structural comparison of the catalytic core of Cel5G with the mesophilic catalytic core of Cel5A from Erwinia chrysanthemi revealed modifications at the atomic level leading to higher flexibility and thermolability, which might account for the higher activity of Cel5G at low temperatures. Using small angle X-ray scattering we further explored the structure at the entire enzyme level. We analyzed the dimensions, shape, and conformation of Cel5G full length in solution and especially of the linker between the catalytic module and the cellulose-binding module. The results showed that the linker is unstructured, and unusually long and flexible, a peculiarity that distinguishes it from its mesophilic counterpart. Loops formed at the base by disulfide bridges presumably add constraints to stabilize the most extended conformations. These results suggest that the linker plays a major role in cold adaptation of this psychrophilic enzyme, allowing steric optimization of substrate accessibility.

Kinetics study of Bungarus fasciatus venom acetylcholinesterase immobilised on a Langmuir-Blodgett proteo-glycolipidic bilayer.

This study deals with the kinetics properties of an enzyme immobilised in a defined orientation in a biomimetic environment. For this purpose, acetylcholinesterase (AChE) was captured at the surface of a nanostructured proteo-glycolipidic Langmuir-Blodgett film through specific recognition by a noninhibitor monoclonal antibody (IgG) inserted in a neoglycolipid bilayer. Modelling of this molecular assembly provided a plausible interpretation of the functional orientation of the enzyme. The AChE activity being stable for several weeks, the enzyme kinetics were investigated, and fitted perfectly with heterogeneous biocatalytic behaviour representative of cellular enzymatic catalysis. The AChE-IgG-glycolipid nanostructure was directly interfaced with an efficient optical device. Such an association, leading to an intimate contact between the nanostructure and the biochemical signal transducer, gives direct access to the intrinsic AChE behaviour. This study thus demonstrates the potential for direct investigation of the kinetic behaviour of an immobilised enzyme on a lipid bilayer through an efficient transduction system.

Mutational analyses of the core domain of Avian Leukemia and Sarcoma Viruses integrase: critical residues for concerted integration and multimerization.

During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme.

Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro.

Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.

The human polycomb group EED protein interacts with the integrase of human immunodeficiency virus type 1.

Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4(+) cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.