Cyclization of ubiquitin chains reinforces their recognition by ZNF216.

Lys48-linked ubiquitin chains, regulating proteasomal protein degradation, are known to include cyclized forms. This cyclization hinders recognition by many downstream proteins by occluding the Ile44-centered patch. In contrast, the A20-like Znf domain of ZNF216 (a ubiquitin-binding protein, A20 Znf) is expected to bind to cyclic ubiquitin chains via constitutively solvent-exposed surfaces. However, the underlying interaction mechanism remains unclear. Here, our ITC and NMR experiments collectively showed that cyclization did not interfere with and even slightly enhance the molecular recognition of diubiquitin by A20 Znf. This effect is explained by the cyclization-induced repression of conformational dynamics in diubiquitin and an enlarged molecular interface in the complex. Thus, these results suggest that cyclic ubiquitin chains can be involved in regulation of ZNF216-dependent proteasomal protein degradation.

An integrated approach of NMR experiments and MD simulations visualizes structural dynamics of a cyclic multi-domain protein.

Cyclization can stabilize the structure of proteins, as previously demonstrated in single-domain proteins. Although Lys48-linked polyubiquitin, a multi-domain protein, is also known to be cyclized in human cells, the structural effects of cyclization remain unclear. Here, we examined the impact of cyclization on the structural stability and dynamics of cyclic Lys48-linked diubiquitin (Ub2 ). As expected, cyclization increased the thermal stability of Ub2 and its resistance to proteolytic digestion, indicating that cyclization stabilized the structure of Ub2 . Furthermore, cyclization repressed the interdomain motion in Ub2 , but cyclic Ub2 still exhibited microsecond conformational exchange in NMR relaxation dispersion experiments. A series of long coarse-grained (CG) MD simulations visualized how cyclization slowed down the intrinsic nanosecond open-closed domain motion of Ub2 to microseconds. Thus, CG-MD analysis helped to explain the unexpected NMR relaxation results, thereby facilitating characterization of the structural stabilization of cyclic Ub2 .

Expression, solubility monitoring, and purification of the co-folded LUBAC LTM domain by structure-guided tandem folding in autoinducing cultures.

The linear ubiquitin chain assembly complex tethering motif (LUBAC-LTM) domain is composed of two different accessory LUBAC components (HOIL-1L and SHARPIN) but folds as a single globular domain. Targeted disruption of the intricate LTM-LTM interaction destabilizes LUBAC in lymphoma cells, thereby attenuating LUBAC stability, which highlights that targeting the interaction between the two LTM motifs is a promising strategy for the development of new agents against cancers that depend on LUBAC activity for their survival. To further screen for small-molecule inhibitors that can selectively disrupt the LTM-LTM interaction, it is necessary to obtain high-purity samples of the LTM domain. Ideally, such a sample would not contain any components other than the LTM itself, so that false positives (molecules binding to other parts of LUBAC) could be eliminated from the screening process. Here we report a simple strategy that enabled successful bacterial production of the isolated LUBAC LTM domain in high yield and at high purity. The strategy combines (1) structural analysis highlighting the possibility of tandem expression in the SHARPINL™ to HOIL-1LL™ direction; (2) bacterial expression downstream of EGFP to efficiently monitor expression and solubility; (3) gentle low-temperature folding using autoinduction. Formation of stably folded LTM was verified by size-exclusion chromatography and heteronuclear NMR spectroscopy. From 200-ml cultures sufficient quantities (~7 mg) of high-purity protein for structural studies could be obtained. The presented strategy will be beneficial for LUBAC LTM-based drug-screening efforts and likely serve as a useful primer for similar cases, i.e., whenever a smaller folded fragment is to be isolated from a larger protein complex for site-specific downstream applications.

Molecular recognition and deubiquitination of cyclic K48-linked ubiquitin chains by OTUB1.

Conjugation of K48-linked ubiquitin chains to intracellular proteins mainly functions as a signal for proteasomal degradation. The conjugating enzyme E2-25K synthesizes not only canonical (noncyclic) but also cyclic K48-linked ubiquitin chains. Although the cyclic conformation is expected to repress molecular recognition by ubiquitin binding proteins due to restricting the flexibility of the ubiquitin subunits in a chain, multiple proteins are reported to associate with cyclic ubiquitin chains similar to noncyclic chains. However, the molecular mechanism of how cyclic ubiquitin chains are recognized remains unclear. Here we investigated the effect of cyclization on ubiquitin-chain cleavage and molecular recognition by a K48-linkage specific deubiquitinating enzyme OTUB1 for cyclic diubiquitin by NMR spectroscopic analyses. Compared to noncyclic diubiquitin, we observed slow but unambiguously detectable cleavage of cyclic diubiquitin to monoubiquitin by OTUB1. Intriguingly, upon ubiquitin chain cleavage, cyclic diubiquitin appeared to alter its "autoinhibited" conformation to an incompletely but partially accessible conformation, induced by interaction with OTUB1 via the ubiquitin-subunit specific recognition patches and adjacent surfaces. These data imply that cyclic ubiquitin chains may exist stably in cells in spite of the presence of deubiquitinating enzymes and that these chains can be recognized by intracellular proteins in a manner distinct from that of noncyclic ubiquitin chains.