No-nonsense:insights into the functional interplay of nonsense-mediated mRNA decay factors

Nonsense-mediated messenger RNA decay (NMD) represents one of the main surveillance pathways used by eukaryotic cells to control the quality and abundance of mRNAs and to degrade viral RNA. NMD recognises mRNAs with a premature termination codon (PTC) and targets them to decay. Markers for a mRNA with a PTC, and thus NMD, are a long a 3′-untranslated region and the presence of an exon-junction complex (EJC) downstream of the stop codon. Here, we review our structural understanding of mammalian NMD factors and their functional interplay leading to a branched network of different interconnected but specialised mRNA decay pathways. We discuss recent insights into the potential impact of EJC composition on NMD pathway choice. We highlight the coexistence and function of different isoforms of up-frameshift protein 1 (UPF1) with an emphasis of their role at the endoplasmic reticulum and during stress, and the role of the paralogs UPF3B and UPF3A, underscoring that gene regulation by mammalian NMD is tightly controlled and context-dependent being conditional on developmental stage, tissue and cell types

Covalent inhibitors of LgtC: a blueprint for the discovery of non-substrate-like inhibitors for bacterial glycosyltransferases

Non-substrate-like inhibitors of glycosyltransferases are sought after as chemical tools and potential lead compounds for medicinal chemistry, chemical biology and drug discovery. Here, we describe the discovery of a novel small molecular inhibitor chemotype for LgtC, a retaining α-1,4-galactosyltransferase involved in bacterial lipooligosaccharide biosynthesis. The new inhibitors, which are structurally unrelated to both the donor and acceptor of LgtC, have low micromolar inhibitory activity, comparable to the best substrate-based inhibitors. We provide experimental evidence that these inhibitors react covalently with LgtC. Results from detailed enzymological experiments with wild-type and mutant LgtC suggest the non-catalytic active site residue Cys246 as a likely target residue for these inhibitors. Analysis of available sequence and structural data reveals that non-catalytic cysteines are a common motif in the active site of many bacterial glycosyltransferases. Our results can therefore serve as a blueprint for the rational design of non-substrate-like, covalent inhibitors against a broad range of other bacterial glycosyltransferases

Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications

Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR

Specificity in protein-DNA recognition arises from the synergy of several factors that stem from the structural and chemical signatures encoded within the targeted DNA molecule. Here, we deciphered the nature of the interactions driving DNA recognition and binding by the bacterial transcription factor PdxR, a member of the MocR family responsible for the regulation of pyridoxal 5 & PRIME;-phosphate (PLP) biosynthesis. Single particle cryo-EM performed on the PLP-PdxR bound to its target DNA enabled the isolation of three conformers of the complex, which may be considered as snapshots of the binding process. Moreover, the resolution of an apo-PdxR crystallographic structure provided a detailed description of the transition of the effector domain to the holo-PdxR form triggered by the binding of the PLP effector molecule. Binding analyses of mutated DNA sequences using both wild type and PdxR variants revealed a central role of electrostatic interactions and of the intrinsic asymmetric bending of the DNA in allosterically guiding the holo-PdxR-DNA recognition process, from the first encounter through the fully bound state. Our results detail the structure and dynamics of the PdxR-DNA complex, clarifying the mechanism governing the DNA-binding mode of the holo-PdxR and the regulation features of the MocR family of transcription factors

Serine hydroxymethyltransferase: A model enzyme for mechanistic, structural, and evolutionary studies.

Serine hydroxymethyltransferase is a ubiquitous representative of the family of fold type I, pyridoxal 5'-phosphate-dependent enzymes. The reaction catalyzed by this enzyme, the reversible transfer of the Cβ of serine to tetrahydropteroylglutamate, represents a link between amino acid and folates metabolism and operates as a major source of one-carbon units for several essential biosynthetic processes. Serine hydroxymethyltransferase has been intensively investigated because of the interest aroused by the complex mechanism of the hydroxymethyltransferase reaction and its broad substrate and reaction specificity. Although the increasing availability of crystallographic data and the characterization of several site-specific mutants helped in understanding previous functional and structural studies, they also represent the starting point of novel investigations. This review will focus on recently highlighted catalytic, structural, and evolutionary aspects of serine hydroxymethyltransferase. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology