Catherine H. Botting

Polymeric Chains of SUMO-2 and SUMO-3 Are Conjugated to Protein Substrates by SAE1/SAE2 and Ubc9

Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence ψKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detectedin vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.

Size selective protein adsorption on thiol-functionalised SBA-15 mesoporous molecular sieve

The mesoporous silica SBA-15, functionalised with propylthiol groups during synthesis and rendered porous (mean pore diameter 51 A) by extraction of surfactant template molecules, shows strong and size selective adsorption of proteins, selectively excluding those with molecular weights of ca. 40000 u and above. A model for the adsorption process is proposed, in which reversible physisorption is followed by irreversible chemisorption. Adsorption of proteins on an unfunctionalised SBA-15 from which the template has been removed by calcination, (mean pore diameter 56 A) shows shape selective and reversible adsorption of proteins with molecular weights of ca. 43000 u and below.

Creatine kinase is physically associated with the cardiac ATP-sensitive k+ channel in vivo

Cardiac sarcolemmal ATP‐sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal KATP channel protein complex. The inside‐out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate KATP channels activity. Following immunoprecipitation of guinea‐pig cardiac membrane fraction with the anti‐SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at ~48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti‐SUR2A immunoprecipitate and the cross reactivity between an anti‐CK antibody and the anti‐SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal KATP channel protein complex.

Ribosomal proteins are targets for the NEDD8 pathway

Identification of the molecular targets for post‐translational modifications is an important step for explaining the regulated pathways. The ubiquitin‐like molecule NEDD8 is implicated in the regulation of cell proliferation, viability and development. By combining proteomics and in vivo NEDDylation assays, we identified a subset of ribosomal proteins as novel targets for the NEDD8 pathway. We further show that the lack of NEDDylation in cells causes ribosomal protein instability. Our studies identify a novel and specific role of the NEDD8 pathway in protecting a subset of ribosomal proteins from destabilization.

The 3D structure of a periplasm-spanning platform required for assembly of group 1 capsular polysaccharides in Escherichia coli

Capsular polysaccharides (CPSs) are essential virulence determinants of many pathogenic bacteria. Escherichia coli group 1 CPSs provide paradigms for widespread surface polysaccharide assembly systems in Gram-negative bacteria. In these systems, complex carbohydrate polymers must be exported across the periplasm and outer membrane to the cell surface. Group 1 CPS export requires oligomers of the outer membrane protein, Wza, for translocation across the outer membrane. Assembly also depends on Wzc, an inner membrane tyrosine autokinase known to regulate export and synthesis of group 1 CPS. Here, we provide a structural view of a complex comprising Wzc and Wza that spans the periplasm, connecting the inner and outer membranes. Examination of transmembrane sections of the complex suggests that the periplasm is compressed at the site of complex formation. An important feature of CPS production is the coupling of steps involved in biosynthesis and export. We propose that the Wza–Wzc complex provides the structural and regulatory core of a larger macromolecular machine. We suggest a mechanism by which CPS may move from the periplasm through the outer membrane.

The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain

Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of nonactivated substrates has wide biotechnology applications.

Polymer precursors from catalytic reactions of natural oils

Dimethyl 1,19-nonadecanedioate is produced from the methoxycarbonylation of commercial olive, rapeseed or sunflower oils in the presence of a catalyst derived from [Pd2(dba)3], bis(ditertiarybutylphosphinomethyl)benzene (BDTBPMB) and methanesulphonic acid (MSA). The diester is then hydrogenated to 1,19-nonadecanediol using Ru/1,1,1-tris-(diphenylphosphinemethyl)ethane (triphos). 1,19-Nonadecadienoic acid is hydrogenated to short chain oligoesters, which can themselves be hydrogenated to 1,19-nonadecanol by hydrogenation in the presence of water.

Miswiring the brain: Δ9‐tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin‐2 degradation pathway

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ9‐tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor‐driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC‐sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin‐2, a microtubule‐binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis‐exposed fetuses, defining SCG10 as the first cannabis‐driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c‐Jun N‐terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.

Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

BACKGROUND Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained. RESULTS We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.

Structural analysis of leader peptide binding enables leader-free cyanobactin processing

Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways and many such products have potential as therapeutics. The ATP dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate suggesting recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whilst catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity; it appears to be a conserved strategy in other peptide modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.