Komander, D. | Search bioRxiv

bioRxiv2017

Active site alanine substitutions can convert deubiquitinating enzymes into avid ubiquitin-binding domains

A common strategy for studying the biological role of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

biochemistry↗

bioRxiv2017

An "invisible" ubiquitin conformation is required for efficient phosphorylation by PINK1

The Ser/Thr protein kinase PINK1 phosphorylates the well-folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We had previously shown that Ser65-phosphorylation results in a conformational change, in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation in which the last {beta}-strand is retracted to extend the Ser65 loop and shorten the C-terminal tail. We here show using Chemical Exchange Saturation Transfer (CEST) NMR experiments, that a similar, C-terminally retracted (Ub-CR) conformation exists in wild-type Ub. Ub point mutations in the moving {beta}5-strand and in neighbouring strands shift the Ub/Ub-CR equilibrium. This enabled functional studies of the two states, and we show that the Ub-CR conformation binds to the PINK1 kinase domain through its extended Ser65 loop and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub-CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded proteins or domains.

molecular biology↗