UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

Crystal structure of a novel domain of the motor subunit of the Type I restriction enzyme EcoR124 involved in complex assembly and DNA binding.

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein-protein and protein-DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.

pHluorin-assisted expression, purification, crystallization and X-ray diffraction data analysis of the C-terminal domain of the HsdR subunit of the Escherichia coli type I restriction-modification system EcoR124I.

The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = ß = γ = 90.00° and two molecules in the asymmetric unit (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å.

Crystallization behaviour of glyceraldehyde dehydrogenase from Thermoplasma acidophilum.

The glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH) is a microbial enzyme that catalyzes the oxidation of D-glyceraldehyde to D-glycerate in the artificial enzyme cascade designed for the conversion of glucose to the organic solvents isobutanol and ethanol. Various mutants of TaAlDH were constructed by a random approach followed by site-directed and saturation mutagenesis in order to improve the properties of the enzyme that are essential for its functioning within the cascade. Two enzyme variants, wild-type TaAlDH (TaAlDHwt) and an F34M+S405N variant (TaAlDH F34M+S405N), were successfully crystallized. Crystals of TaAlDHwt belonged to the monoclinic space group P1211 with eight molecules per asymmetric unit and diffracted to a resolution of 1.95 Å. TaAlDH F34M+S405N crystallized in two different space groups: triclinic P1 with 16 molecules per asymmetric unit and monoclinic C121 with four molecules per asymmetric unit. These crystals diffracted to resolutions of 2.14 and 2.10 Šfor the P1 and C121 crystals, respectively.