A C2HC zinc finger is essential for the RING-E2 interaction of the ubiquitin ligase RNF125

The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2(120-128)) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2(120-128) region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2

A population shift between sparsely populated folding intermediates determines amyloidogenicity

The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of non-uniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and non-amyloidogenic variants of ?2-microglobulin (?2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the ITrans state and population of a less stable, more dynamic species ablates amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding

An oxygen-sensitive toxin-antitoxin system

The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin-antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SOxH- containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle

Triplet NOAH supersequences optimised for small molecule structure characterisation

A series of NMR supersequences are presented for the time‐efficient structure characterisation of small molecules in the solution state. These triplet sequences provide HMBC, HSQC, and one homonuclear correlation experiment of choice according to the NMR by Ordered Acquisition using 1H detection principle. The experiments are demonstrated to be compatible with non‐uniform sampling schemes and may be acquired and processed under full automation

Triplet NOAH supersequences optimised for small molecule structure characterisation

A series of NMR supersequences are presented for the time‐efficient structure characterisation of small molecules in the solution state. These triplet sequences provide HMBC, HSQC, and one homonuclear correlation experiment of choice according to the NMR by Ordered Acquisition using 1H detection principle. The experiments are demonstrated to be compatible with non‐uniform sampling schemes and may be acquired and processed under full automation