Experimental details for the ITC assay. The enthalpy change (ΔH) associated with the reaction of rkbPAP with p-NPP was determined by injecting 10 µL of p-NPP (20 mM) into the reaction cell (200 µL) containing 0.95 µM of rkbPAP, and by allowing the reaction to proceed to completion.The reaction is exothermic as indicated by the negative value of the heat pulse (dQ/dT); the value of ΔH can be calculated by integrating the area under the curve in Fig. S2a (minus the endothermic heat associated with the dilution) divided by the amount of p-NPP hydrolyzed in the cell. The value for the endothermic heat generated by the dilution was obtained by injecting 10 µL of p-NPP (20 mM) into the reaction cell containing only buffer: ΔH = 3089 ± 52 µcal/µmol (i.e. 12.9 ± 0.22 kJ/mol), in good agreement with the corresponding ΔH value determined by enzymatic assays (13.2 ± 0.18 kJ/mol) [1]. In this study, we focused on measuring initial catalytic rates in order to minimize the effect of the reaction product and competitive inhibitor phosphate [2][3][4][5][6].Different concentrations of p-NPP were prepared and 8 µL of 0.24 µM rkbPAP were injected into the substrate solutions. Fig. S2b shows a typical calorimetric trace of a reaction. The initial rates determined in µcal/s, when divided by ΔH (µcal/µmol), are transformed into µmol/s, representing the amount of p-NPP hydrolyzed per second in the cell. These rates were subsequently plotted against the p-NPP concentration and fitted to the Michaelis-Menten equation (Eq. 1; Fig. S2c), resulting in a kcat value (i.e. Vmax/[PAP]) of ~190 s -1 and a Michaelis constant Km of ~4 mM.

Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K i values in the 30-60 lM range. Ligand efficiency values are 0.39-0.44 kcal ⁄ mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.Key words: X-ray, crystallography, drug design, fragment screening, purple acid phosphatase, osteoporosis Purple acid phosphatases (PAP) are metalloenzymes that hydrolyse phosphate esters and anhydrides, generally at low pH values (1). The distinctive purple colour of these enzymes is due to a metal to ligand charge transfer from a tyrosine phenolate to a chromophoric Fe(III) (2,3). The cornerstone of the active site of PAP is the presence of two metal ions; Fe(III) is always present in the chromophoric site, while the second site can be occupied by a redox active Fe(II ⁄ III) in mammals (4,5) or a Zn(II) or Mn(II) in plants (6,7). Mammalian PAP is a 35 kDa monomeric protein also known as tartrate-resistant acid phosphatase (TRAP or TRAcP). In contrast, plant PAP is a 110 kDa homodimer, with each subunit consisting of two domains, an N-terminal one whose function is unknown and a catalytic C-terminal domain that strongly resembles the overall structure of the mammalian enzyme (1). Crystal structures of human (8), pig (9), rat (10,11) and plant PAPs (12,13) have been determined and show that the amino acid ligands of the metal ions are completely conserved across plant and animal PAPs, but there are some differences in the identities of the residues that line the active site.A number of biological roles for PAP have been proposed. These include (i) the transport of iron from the mother to the developing foetus during gestation (14); (ii) bone resorption in osteoclasts (evidence for this activity is derived from the effect of PAP on osteoclasts that were cultured on cortical bone slices (15) and from t...

Purple acid phosphatases( PAPs) are members of the large family of metallohydrolases, ag roup of enzymes that perform aw ide range of biological functions, while employing ah ighly conserved catalytic mechanism. PAPs are found in plants,a nimals and fungi;i nh umans they play an important role in bone turnovera nd are thus of interest for developingt reatments for osteoporosis. The majority of metallohydrolases use am etalbound hydroxide to initiate catalysis, which leads to the formation of ap roposed five-coordinate oxyphosphorane species in the transition state. In this work, we crystallized PAPf rom red kidney beans (rkbPAP) in the presence of both adenosine and vanadate. The in crystallo-formed vanadate analogue of ADP provides detailed insight into the binding mode of aP AP substrate, captured in as tructure that mimics the putative fivecoordinate transition state. Our observations not only provide unprecedented insightinto the mechanism of metallohydrolases, but might also guide the structure-based design of inhibitors for application in the treatment of severalhuman illnesses.Metallohydrolases form al arge familyo fm ostly bimetallic enzymes that use metal ionst oactivate both the nucleophile and scissile bond of various phosphate or amide substrates. [1][2][3][4] Examples include the antibiotic-degrading metallo-b-lactamases, pesticide-decontaminating organophosphate hydrolases, and purple acid phosphatases (PAPs). [5][6][7] PAPs, the only known metallohydrolases that require ah eterovalent Fe III M II center (with M = Fe, Zn or Mn) for catalytic activity, have been characterized from various mammals, plants and fungi. [2,5] Mostp lant PAPs use either Zn II or Mn II ,w hereas their mammalianc ounterparts employ ar edox-activei ron ( Figure 1). [8,9] The reported substrates for PAPs include adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate( ADP), phosphotyrosine and pyrophosphate. [5,10] The synthetic substrate para-nitrophenyl phosphate (pNPP) is frequently used for functionalstudies. [2,5] The biological functions of PAPs are diverse and dependent on the organism. For mammals, experiments with transgenic mice have demonstrated that PAPp lays an important role in bone metabolism. [11,12] In plants, PAPs playamajor role in the acquisition of phosphorus, especially if there is al imited supplyo fp hosphate. [13,14] Due to their diverse roles, PAPs have attracted attention either as targetsf or new treatments of osteoporosis or for applicationsi na griculture to aid nutrient uptake by crops. [11,15] PAPs operate optimally in the pH range between5 .0 and 6.5, and are characterized by ad istinct purple color due to ac harge-transfer transition between an active-site tyrosine ligand and an Fe III ion. [16,17] The crystal structures of several mammalian and plant PAPs have demonstrated that, despite the difference in the metal ion selectivity and the limited degree of sequence similarity,t heir actives ite geometries are highly conserved (Figure1). [18][19][20][21][22][23] Consequently,t he proposed mod...

Emerging viruses impose global threats to animal and human populations and may bear novel genes with limited homology to known sequences, necessitating the development of novel approaches to infer and test protein functions. This challenge is dramatically evident in tilapia lake virus (TiLV), an emerging orthomyxo-like virus that threatens the global tilapia aquaculture and food security of millions of people. The majority of TiLV proteins have no homology to known sequences, impeding functionality assessments. Using a novel bioinformatics approach, we predicted that TiLV’s Protein 4 encodes the nucleoprotein - a factor essential for viral RNA replication. Multiple methodologies revealed the expected properties of orthomyxoviral nucleoproteins. A modified yeast three-hybrid assay detected Protein 4-RNA interactions, which were independent of the RNA sequence, and identified specific positively charged residues involved. Protein 4-RNA interactions were uncovered by R-DeeP and XRNAX methodologies. Immunoelectron microscopy found that multiple Protein 4 copies localized along enriched ribonucleoproteins. TiLV RNA from cells and virions co-immunoprecipitated with Protein 4. Immunofluorescence microscopy detected Protein 4 in the cytoplasm and nuclei, and nuclear Protein 4 increased upon CRM1 inhibition, suggesting CRM1-dependent nuclear export of TiLV RNA. Together, these data reveal TiLV’s nucleoprotein and highlight the ability to infer protein functionality, including novel RNA-binding proteins, in emerging pathogens. These are important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens. Importance Tilapia is an important source of dietary protein, especially in developing countries. Massive losses of tilapia were identified worldwide, risking the food security of millions of people. Tilapia lake virus (TiLV) is an emerging pathogen responsible for these disease outbreaks. TiLV’s genome encodes ten major proteins, nine of which show no homology to other known viral or cellular proteins, hindering functionality assessment of these proteins. Here we describe a novel bioinformatics approach to infer the functionality of TiLV proteins, which predicted Protein 4 as the nucleoprotein - a factor essential for viral RNA replication. We provided experimental support for this prediction by applying multiple molecular, biochemical, and imaging approaches. Overall, we illustrate a strategy for functional analyses in viral discovery. The strategy is important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens.