Leucine-rich repeat kinase 2 allosteric modulators

The present invention relates to binding agents of human Leucine-rich Repeat Kinase 2 (LRRK2). More particular, allosteric modulators of LRRK2 activity have been identified, for targeting LRRK2 in human cells, while leaving LRRK2 subcellular localisation unaffected. Even more specifically, protein binding agents for allosteric modulation of LRRK2 kinase activity are disclosed, comprising immunoglobulin single variable domains (ISVDs) binding to human LRRK2 with nanomolar affinity. The invention thus reveals means and methods for a novel LRRK2 targeting approach through allosteric modulation of its activity for use in treatment of LRRK2-related pathologies, such as Parkinson's disease, as well as for use in detection of LRRK2 in vitro and in vivo, and for use as a diagnostic

Structural and functional insights into tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue

Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A designed ankyrin-repeat protein that targets Parkinsons disease-associated LRRK2.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinsons disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase

Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase

Aromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydrolysis reaction. The effect of the aromatic stacking on catalysis is peculiar in that uracil, the aromatic moiety influenced by the aromatic interaction is relatively distant from the site of hydrolysis at the alpha-phosphate group. Using crystallographic, kinetics, optical spectroscopy and thermodynamics calculation approaches we delineate a possible mechanism by which rate acceleration is achieved through the remote π–π interaction. The abundance of similarly positioned aromatic interactions in various nucleotide hydrolyzing enzymes (e.g. most families of ATPases) raises the possibility of the reported phenomenon being a general component of the enzymatic catalysis of phosphate ester hydrolysis

MnmE, a GTPase That Drives a Complex tRNA Modification Reaction

MnmE is a multi-domain GTPase that is conserved from bacteria to man. Together with its partner protein MnmG it is involved in the synthesis of a tRNA wobble uridine modification. The orthologues of these proteins in eukaryotes are targeted to mitochondria and mutations in the encoding genes are associated with severe mitochondrial diseases. While classical small GTP-binding proteins are regulated via auxiliary GEFs and GAPs, the GTPase activity of MnmE is activated via potassium-dependent homodimerization of its G domains. In this review we focus on the catalytic mechanism of GTP hydrolysis by MnmE and the large scale conformational changes that are triggered throughout the GTPase cycle. We also discuss how these conformational changes might be used to drive and tune the complex tRNA modification reaction. (C) 2016 Wiley Periodicals, Inc

The Universally Conserved Prokaryotic GTPases

Members of the large superclass of P-loop GTPases share a core domain with a conserved three-dimensional structure. In eukaryotes, these proteins are implicated in various crucial cellular processes, including translation, membrane trafficking, cell cycle progression, and membrane signaling. As targets of mutation and toxins, GTPases are involved in the pathogenesis of cancer and infectious diseases. In prokaryotes also, it is hard to overestimate the importance of GTPases in cell physiology. Numerous papers have shed new light on the role of bacterial GTPases in cell cycle regulation, ribosome assembly, the stress response, and other cellular processes. Moreover, bacterial GTPases have been identified as high-potential drug targets. A key paper published over 2 decades ago stated that, "It may never again be possible to capture [GTPases] in a family portrait" (H. R. Bourne, D. A. Sanders, and F. McCormick, Nature 348:125-132, 1990) and indeed, the last 20 years have seen a tremendous increase in publications on the subject. Sequence analysis identified 13 bacterial GTPases that are conserved in at least 75% of all bacterial species. We here provide an overview of these 13 protein subfamilies, covering their cellular functions as well as cellular localization and expression levels, three-dimensional structures, biochemical properties, and gene organization. Conserved roles in eukaryotic homologs will be discussed as well. A comprehensive overview summarizing current knowledge on prokaryotic GTPases will aid in further elucidating the function of these important proteins.status: publishe