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Abstract: Despite being fundamental to multiple biological processes, phosphorus (P) availability in marine environments is often growth-limiting, with generally low surface concentrations. Picocyanobacteria strains encode a putative ABC-type phosphite/phosphate/phosphonate transporter, phnDCE, thought to provide access to an alternative phosphorus pool. This, however, is paradoxical given most picocyanobacterial strains lack known phosphite degradation or carbon-phosphate lyase pathway to utilise alternate phosphorus pools. To understand the function of the PhnDCE transport system and its ecological consequences, we characterised the PhnD1 binding proteins from four distinct marine Synechococcus isolates (CC9311, CC9605, MITS9220, and WH8102). We show the Synechococcus PhnD1 proteins selectively bind phosphorus compounds with a stronger affinity for phosphite than for phosphate or methyl phosphonate. However, based on our comprehensive ligand screening and growth experiments showing Synechococcus strains WH8102 and MITS9220 cannot utilise phosphite or methylphosphonate as a sole phosphorus source, we hypothesise that the picocyanobacterial PhnDCE transporter is a constitutively expressed, medium-affinity phosphate transporter, and the measured affinity of PhnD1 to phosphite or methyl phosphonate is fortuitous. Our MITS9220_PhnD1 structure explains the comparatively lower affinity of picocyanobacterial PhnD1 for phosphate, resulting from a more limited H-bond network. We propose two possible physiological roles for PhnD1. First, it could function in phospholipid recycling, working together with the predicted phospholipase, TesA, and alkaline phosphatase. Second, by having multiple transporters for P (PhnDCE and Pst), picocyanobacteria could balance the need for rapid transport during transient episodes of higher P availability in the environment, with the need for efficient P utilisation in typical phosphate-deplete conditions.

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Abstract: Phosphoenolpyruvate carboxykinase (PEPCK) is a well-characterized enzyme involved in primary glucose metabolism, responsible for catalyzing one of the key steps of gluconeogenesis. It is well demonstrated that PEPCK can efficiently catalyze the reversible interconversion of oxaloacetic acid (OAA) to phosphoenolpyruvate (PEP) in vitro, but the enzyme is typically ascribed a metabolic role that requires preferential catalysis in the direction of PEP synthesis in vivo. Here we present structural and functional data that demonstrate the preferential synthesis of PEP from OAA catalyzed by PEPCK in vivo is facilitated by anion-mediated enzyme inhibition that reduces enzyme activity more significantly in the direction of OAA synthesis than in the direction of PEP synthesis. From our studies we conclude that the specific binding of small, ubiquitous anions like chloride, present in millimolar concentrations under normal cellular conditions allows for metabolic control by restricting PEPCK to function in the direction of PEP synthesis.

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Abstract: Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.

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Abstract: Amino acid transporters play a key role controlling the flow of nutrients across the lysosomal membrane and regulating metabolism in the cell. Mutations in the gene encoding the transporter cystinosin result in cystinosis, an autosomal recessive metabolic disorder characterised by the accumulation of cystine crystals in the lysosome. Cystinosin is a member of the PQ-loop family of solute carrier (SLC) transporters and uses the proton gradient to drive cystine export into the cytoplasm. However, the molecular basis for cystinosin function remains elusive, hampering efforts to develop novel treatments for cystinosis and understand the mechanisms of ion driven transport in the PQ-loop family. To address these questions, we present the crystal structures of cystinosin from Arabidopsis thaliana in both apo and cystine bound states. Using a combination of in vitro and in vivo based assays, we establish a mechanism for cystine recognition and proton coupled transport. Mutational mapping and functional characterisation of human cystinosin further provide a framework for understanding the molecular impact of disease-causing mutations.

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Abstract: The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for β-glucuronidase activity. We identified SN243, a genuine β-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase 3 family member. This glycoside hydrolase family contains only one recently added β-glucuronidase, showing that a functional metagenomic approach can shed light on assignments that are currently ‘unpredictable’ by bioinformatics. Kinetic analyses of SN243 characterized it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous glycoside hydrolase 3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre-volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.