A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated Protein Kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a compact conformation in which it engages two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, the latter being a non-canonical Pin1-interacting element. The structural information, combined with the results of extensive binding studies and in vivo experiments suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.

Supramolecular Ionic Liquid Gels for Enzyme Entrapment.

Reported herein is an entrapment method for enzyme immobilization that does not require the formation of new covalent bonds. Ionic liquid supramolecular gels are formed containing enzymes that can be shaped into gel beads and act as recyclable immobilized biocatalysts. The gel was formed from two components, a hydrophobic phosphonium ionic liquid and a low molecular weight gelator derived from the amino acid phenylalanine. Gel-entrapped lipase from Aneurinibacillus thermoaerophilus was recycled for 10 runs over 3 days without loss of activity and retained activity for at least 150 days. The procedure does not form covalent bonds upon gel formation, which is supramolecular, and no bonds are formed between the enzyme and the solid support.

Designing Materials for Aqueous Catalysis: Ionic Liquid Gel and Silica Sphere Entrapped Iron-TAML Catalysts for Oxidative Degradation of Dyes.

Materials have been developed that encapsulate a homogeneous catalyst and enable it to operate as a heterogeneous catalyst in water. A hydrophobic ionic liquid within the material was used to dissolve Fe-TAML and keep it from leaching into the aqueous phase. One-pot processes were used to entrap Fe-TAML in basic ionic liquid gels, and ionic liquid gel spheres structured via a modified Stöber synthesis forming SiO2 particles of uniform size. Catalytic activity was demonstrated via the oxidative degradation of dyes. Fe-TAML entrapped in a basic ionic liquid gel exhibited consistent activity in five recycles. This discovery of heterogenized H2O2 activators prepared by sol-gel and Stöber processes opens new possibilities for the creation of engineered catalytic materials for water purification.

Differential reactivity of closely related zinc(II)-binding metallothioneins from the plant Arabidopsis thaliana.

The dynamics of metal binding to and transfer from metalloproteins involved in metal homeostasis are important for understanding cellular distribution of metal ions. The dicotyledonous plant Arabidopsis thaliana has two type 4 seed-specific metallothionein homologues, MT4a and MT4b, with likely roles in zinc(II) homeostasis. These two metallothioneins are 84% identical, with full conservation of all metal-binding cysteine and histidine residues. Yet, differences in their spatial and temporal expression patterns suggested divergence in their biological roles. To investigate whether biological functions are reflected in molecular properties, we compare aspects of zinc(II)-binding dynamics of full-length MT4a and MT4b, namely the pH dependence of zinc(II) binding and protein folding, and zinc(II) transfer to the chelator EDTA. UV-Vis and NMR spectroscopies as well as native electrospray ionisation mass spectrometry consistently showed that transfer from Zn6MT4a is considerably faster than from Zn6MT4b, with pseudo-first-order rate constants for the fastest observed step of k obs = 2.8 × 10-4 s-1 (MT4b) and k obs = 7.5 × 10-4 s-1 (MT4a) (5 µM protein, 500 µM EDTA, 25 mM Tris buffer, pH 7.33, 298 K). 2D heteronuclear NMR experiments allowed locating the most labile zinc(II) ions in domain II for both proteins. 3D homology models suggest that reactivity of this domain is governed by the local environment around the mononuclear Cys2His2 site that is unique to type 4 MTs. Non-conservative amino acid substitutions in this region affect local electrostatics as well as whole-domain dynamics, with both effects rendering zinc(II) ions bound to MT4a more reactive in metal transfer reactions. Therefore, domain II of MT4a is well suited to rapidly release its bound zinc(II) ions, in broad agreement with a previously suggested role of MT4a in zinc(II) transport and delivery to other proteins.

Reconstruction of diaminopimelic acid biosynthesis allows characterisation of Mycobacterium tuberculosis N-succinyl-L,L-diaminopimelic acid desuccinylase.

With the increased incidence of tuberculosis (TB) caused by Mycobacterium tuberculosis there is an urgent need for new and better anti-tubercular drugs. N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a key enzyme in the succinylase pathway for the biosynthesis of meso-diaminopimelic acid (meso-DAP) and L-lysine. DapE is a zinc containing metallohydrolase which hydrolyses N-succinyl L,L diaminopimelic acid (L,L-NSDAP) to L,L-diaminopimelic acid (L,L-DAP) and succinate. M. tuberculosis DapE (MtDapE) was cloned, over-expressed and purified as an N-terminal hexahistidine ((His)6) tagged fusion containing one zinc ion per DapE monomer. We redesigned the DAP synthetic pathway to generate L,L-NSDAP and other L,L-NSDAP derivatives and have characterised MtDapE with these substrates. In contrast to its other Gram negative homologues, the MtDapE was insensitive to inhibition by L-captopril which we show is consistent with novel mycobacterial alterations in the binding site of this drug.

Diversity and distribution of plant metallothioneins: a review of structure, properties and functions.

More than 30 years have passed since the discovery of the first plant metallothionein in wheat embryos, from which the emergence of a uniquely diverse metallothionein family with a fascinating array of structural nuances and molecular properties has been witnessed. Metallothioneins are not only constitutively expressed, but the production of different types of plant metallothionein is also stimulated by a myriad of endogenous and exogenous agents in both a temporally and spatially regulated manner. This ubiquitous, yet discrete expression of metallothioneins not only signifies their importance for plant survival and development, but also suggests a functional divergence for the individual plant metallothionein subfamilies. Understanding why one type of plant metallothionein has more advantageous structural and metal binding attributes over another for a given biological process is a crucial piece in the puzzle of assigning physiological functions to these proteins. In this review, we discuss how in vivo and in vitro studies have advanced our understanding of the structure-property-function relationship for the plant metallothionein family. In particular, we highlight the progress that has been made for the Type 4 plant metallothioneins.