Chemical proteomics: a powerful tool for exploring protein lipidation

The study of post-translational modifications such as protein lipidation is a non-trivial challenge of the post-genomic era. In recent years the field of chemical proteomics has greatly advanced our ability to identify and quantify protein lipidation. In the present review, we give a brief overview of the tools available to study protein acylation, prenylation and cholesterylation, and their application in the identification and quantification of protein lipidation in health and disease.</jats:p

Peptidomimetic inhibitors of N-myristoyltransferase from human malaria and leishmaniasis parasites

N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex

Quantitative Lipoproteomics in Clostridium difficile Reveals a Role for Lipoproteins in Sporulation

This is the final version of the article. Available from Elsevier via the DOI in this record.Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.T.C. was funded by an EPSRC grant to the Institute of Chemical Biology, Imperial College London. A.K.-S. was funded by a European Union Seventh Framework Program (EU MCN Grant agreement 237942)

Modulation of Amide Bond Rotamers in 5-Acyl-6,7-dihydrothieno[3,2-c]pyridines

2-Substituted <i>N</i>-acyl-piperidine is a widespread and important structural motif, found in approximately 500 currently available structures, and present in nearly 30 pharmaceutically active compounds. Restricted rotation of the acyl substituent in such molecules can give rise to two distinct chemical environments. Here we demonstrate, using NMR studies and density functional theory modeling of the lowest energy structures of 5-acyl-6,7-dihydrothieno­[3,2-<i>c</i>]­pyridine derivatives, that the amide <i>E</i>:<i>Z</i> equilibrium is affected by non-covalent interactions between the amide oxygen and adjacent aromatic protons. Structural predictions were used to design molecules that promote either the <i>E</i>- or <i>Z</i>-amide conformation, enabling preparation of compounds with a tailored conformational ratio, as proven by NMR studies. Analysis of the available X-ray data of a variety of published <i>N</i>-acyl-piperidine-containing compounds further indicates that these molecules are also clustered in the two observed conformations. This finding emphasizes that directed conformational isomerism has significant implications for the design of both small molecules and larger amide-containing molecular architectures

Peptidomimetic inhibitors of N-myristoyltransferase from human malaria and leishmaniasis parasites

N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex

Conformational transition of FGFR kinase activation revealed by site-­specific unnatural amino acid reporter and single molecule FRET

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo