Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208–215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the “new Pi site” attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation.

A novel yeast-based high-throughput method for the identification of protein palmitoylation inhibitors

Protein S-acylation or palmitoylation is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of proteins through a thioester bond. Palmitoylation and palmitoyltransferases (PATs) have been linked to several types of cancers, diseases of the central nervous system and many infectious diseases where pathogens use the host cell machinery to palmitoylate their effectors. Despite the central importance of palmitoylation in cell physiology and disease, progress in the field has been hampered by the lack of potent-specific inhibitors of palmitoylation in general, and of individual PATs in particular. Herein, we present a yeast-based method for the high-throughput identification of small molecules that inhibit protein palmitoylation. The system is based on a reporter gene that responds to the acylation status of a palmitoylation substrate fused to a transcription factor. The method can be applied to heterologous PATs such as human DHHC20, mouse DHHC21 and also a PAT from the parasite Giardia lamblia . As a proof-of-principle, we screened for molecules that inhibit the palmitoylation of Yck2, a substrate of the yeast PAT Akr1. We tested 3200 compounds and were able to identify a candidate molecule, supporting the validity of our method.

Examining the Underappreciated Role of S-Acylated Proteins as Critical Regulators of Phagocytosis and Phagosome Maturation in Macrophages

Phagocytosis is a receptor-mediated process used by cells to engulf a wide variety of particulates, including microorganisms and apoptotic cells. Many of the proteins involved in this highly orchestrated process are post-translationally modified with lipids as a means of regulating signal transduction, membrane remodeling, phagosome maturation and other immunomodulatory functions of phagocytes. S-acylation, generally referred to as S-palmitoylation, is the post-translational attachment of fatty acids to a cysteine residue exposed topologically to the cytosol. This modification is reversible due to the intrinsically labile thioester bond between the lipid and sulfur atom of cysteine, and thus lends itself to a variety of regulatory scenarios. Here we present an overview of a growing number of S-acylated proteins known to regulate phagocytosis and phagosome biology in macrophages.

Mode of inhibitory binding of epigallocatechin gallate to the ubiquitin-activating enzyme Uba1 via accelerated molecular dynamics

The hinge-like movement of the SCCH domain upon ligand binding closes the ubiquitin binding site and disrupts the interfaces crucial for thioester bond formation.

Offloading Role of a Discrete Thioesterase in Type II Polyketide Biosynthesis

ABSTRACT Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KSα), chain length factor (KSβ), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KSα or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

Protein folding modulates the adhesion strategy of Gram positive pathogens

Gram positive bacteria colonize mucosal tissues against large mechanical perturbations, such as coughing, which generate large shear forces that exceed the ability of non-covalent bonds to remain attached. To overcome these challenges, the pathogen Streptococcus pyogenes utilizes the protein Cpa, a pilus tip-end adhesin equipped with a Cys-Gln thioester bond. The reactivity of this bond towards host surface ligands enables covalent anchoring of the bacterium, allowing it to resist large mechanical shocks; however, colonization also requires cell migration and spreading over surfaces. The molecular mechanisms underlying these seemingly incompatible requirements remain unknown. Here, we demonstrate a magnetic tweezers force spectroscopy assay that resolves the dynamics of Cpa thioester bond under force. While folded at forces < 6 pN, Cpa thioester bond reacts reversibly with amine ligands, of common occurrence in inflammation sites; however, mechanical unfolding and exposure to forces higher than 35 pN blocks thioester reactivity entirely. We propose that this folding-coupled thioester reactivity switch allows the adhesin to hop and sample host surface ligands at low force (nomadic mobility phase), and yet gets covalently anchored in place while under mechanical stress (locked phase). We dub such bonds “smart covalent bonds”, adding a novel class to the known repertoire of non-covalent adhesion strategies that include slip bonds, and catch bonds.

Structural basis for adenylation and thioester bond formation in the ubiquitin E1

The ubiquitin (Ub) and Ub-like (Ubl) protein-conjugation cascade is initiated by E1 enzymes that catalyze Ub/Ubl activation through C-terminal adenylation, thioester bond formation with an E1 catalytic cysteine, and thioester bond transfer to Ub/Ubl E2 conjugating enzymes. Each of these reactions is accompanied by conformational changes of the E1 domain that contains the catalytic cysteine (Cys domain). Open conformations of the Cys domain are associated with adenylation and thioester transfer to E2s, while a closed conformation is associated with pyrophosphate release and thioester bond formation. Several structures are available for Ub E1s, but none has been reported in the open state before pyrophosphate release or in the closed state. Here, we describe the structures ofSchizosaccharomyces pombeUb E1 in these two states, captured using semisynthetic Ub probes. In the first, with a Ub-adenylate mimetic (Ub-AMSN) bound, the E1 is in an open conformation before release of pyrophosphate. In the second, with a Ub-vinylsulfonamide (Ub-AVSN) bound covalently to the catalytic cysteine, the E1 is in a closed conformation required for thioester bond formation. These structures provide further insight into Ub E1 adenylation and thioester bond formation. Conformational changes that accompany Cys-domain rotation are conserved for SUMO and Ub E1s, but changes in Ub E1 involve additional surfaces as mutational and biochemical analysis of residues within these surfaces alter Ub E1 activities.