Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7''s closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7''s cold adaptation rather than changes in dynamics.     

High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP

Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain–containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80–2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1–2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.     

The enzymatic nature of an anonymous protein sequence cannot reliably be inferred from superfamily level structural information alone

As the largest fraction of any proteome does not carry out enzymatic functions, and in order to leverage 3D structural data for the annotation of increasingly higher volumes of sequence data, we wanted to assess the strength of the link between coarse grained structural data (i.e., homologous superfamily level) and the enzymatic versus non-enzymatic nature of protein sequences. To probe this relationship, we took advantage of 41 phylogenetically diverse (encompassing 11 distinct phyla) genomes recently sequenced within the GEBA initiative, for which we integrated structural information, as defined by CATH, with enzyme level information, as defined by Enzyme Commission (EC) numbers. This analysis revealed that only a very small fraction (about 1%) of domain sequences occurring in the analyzed genomes was found to be associated with homologous superfamilies strongly indicative of enzymatic function. Resorting to less stringent criteria to define enzyme versus non-enzyme biased structural classes or excluding highly prevalent folds from the analysis had only modest effect on this proportion. Thus, the low genomic coverage by structurally anchored protein domains strongly associated to catalytic activities indicates that, on its own, the power of coarse grained structural information to infer the general property of being an enzyme is rather limited.     

Structural characterization of the homotropic cooperative binding of azamulin to human cytochrome P450 3A5

Cytochrome P450 3A4 and 3A5 catalyze the metabolic clearance of a large portion of therapeutic drugs. Azamulin is used as a selective inhibitor for 3A4 and 3A5 to define their roles in metabolism of new chemical entities during drug development. In contrast to 3A4, 3A5 exhibits homotropic cooperativity for the sequential binding of two azamulin molecules at concentrations used for inhibition. To define the underlying sites and mechanisms for cooperativity, an X-ray crystal structure of 3A5 was determined with two azamulin molecules in the active site that are stacked in an antiparallel orientation. One azamulin resides proximal to the heme in a pose similar to the 3A4–azamulin complex. Comparison to the 3A5 apo structure indicates that the distal azamulin in 3A5 ternary complex causes a significant induced fit that excludes water from the hydrophobic surfaces of binding cavity and the distal azamulin, which is augmented by the stacking interaction with the proximal azamulin. Homotropic cooperativity was not observed for the binding of related pleuromutilin antibiotics, tiamulin, retapamulin, and lefamulin, to 3A5, which are larger and unlikely to bind in the distal site in a stacked orientation. Formation of the 3A5 complex with two azamulin molecules may prevent time-dependent inhibition that is seen for 3A4 by restricting alternate product formation and/or access of reactive intermediates to vulnerable protein sites. These results also contribute to a better understanding of sites for cooperative binding and the differential structural plasticity of 3A5 and 3A4 that contribute to differential substrate and inhibitor binding.     

Inhibition by purine nucleotides of the release of reactive oxygen species from muscle mitochondria: indication for a function of uncoupling proteins as superoxide anion transporters

Release of reactive oxygen species (ROS), measured as the sum of hydrogen peroxide (H₂O₂) and superoxide anion radical (), from respiring rat heart and skeletal muscle mitochondria was significantly decreased by millimolar concentrations of GTP or GDP. Attempts to differentiate between the two forms of ROS showed that the release of rather than that of H₂O₂ was affected. Meanwhile, intramitochondrial ROS accumulation, measured by inactivation of aconitase, increased. These results suggest that guanine nucleotides inhibit the release of from mitochondria. As these nucleotides are known inhibitors of uncoupling proteins (UCPs), it is proposed that UCPs may function as carriers of , thus enabling its removal from the matrix compartment.     

Molecular functions of the iron-regulated metastasis suppressor,NDRG1, and its potential as a molecular target for cancer therapy

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.