Open Access

Show Abstract ▼

Abstract: Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Å resolution that along with description of the enantiospecificity of the enzyme affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a Km for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologs in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.

Show Abstract ▼

Abstract: Dendrites are the most common microstructural features in the cast metals, significantly affecting the structure integrity and mechanical properties of the castings. In this study, the in situ synchrotron X-ray radiographic and tomographic imaging techniques were combined to evaluate the critical fracture stress of the growing dendrite tip during the solidification of an Al-15 wt% Cu alloy under an external electromagnetic force. Two dendritic 3D models have been proposed to simulate the dendrite 3D morphologic characteristics and thus revealed that the critical fracture stresses of the Al dendrites at temperatures close to its melting point were in the range of 0.5 kPa–0.05 MPa. The present results demonstrate the feasibility of measuring the high-temperature mechanical properties of the metallic dendrites.

Show Abstract ▼

Abstract: Bipolar disorder is a major psychiatric disorder associated with cognitive impairment and a high suicide rate. Frontline therapy for this condition includes lithium (Li+)-containing treatments that can exert severe side effects. One target of Li+ is inositol monophosphatase-1 (IMPase1); inhibition of IMPase1 through small-molecule compounds may provide an alternative treatment for bipolar disorder. One such compound is the anti-inflammatory drug ebselen, which is well tolerated and safe; however, ebselen’s exact mechanism of action in IMPase1 inhibition is not fully understood, preventing rational design of IMPase1 inhibitors. To fill this gap, we performed crystallographic and biochemical studies to investigate how ebselen inhibits IMPase1. We obtained a structure of IMPase1 in space group P21 after treatment with ebselen that revealed three key active-site loops (residues 33–44, 70–79, and 161–165) that are either disordered or in multiple conformations, supporting a hypothesis whereby dynamic conformational changes may be important for catalysis and ebselen inhibition. Using the thermal shift assay, we confirmed that ebselen significantly destabilizes the enzyme. Molecular docking suggests that ebselen could bind in the vicinity of His217. Investigation of the role of IMPase1 residues His217 and Cys218 suggests that inhibition of IMPase1 by ebselen may not be mediated via covalent modification of the active-site cysteine (Cys218) and is not affected by the covalent modification of other cysteine residues in the structure. Our results suggest that effects previously ascribed to ebselen-dependent inhibition likely result from disruption of essential active-site architecture, preventing activation of the IMPase1–Mg2+ complex.

Open Access

Show Abstract ▼

Abstract: CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.

Show Abstract ▼

Abstract: Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. Here, we report that this inhibition is dependent on incubation time, the solution conditions and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which our simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis, and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.