Protein ubiquitination regulates various cellular events, such as protein degradation, immune signalling and DNA repair. This functional versatility arises from the ability of ubiquitin (Ub) to form distinct polymers linked through one of its eight primary amines (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63 and the N terminus of Met1). Discrimination of these differently linked polyUb chains by linkage-specific ubiquitin-binding domains (UBDs) is crucial to ensure an appropriate cellular response to a particular stimulus. While Lys48- and Lys63-linked chains have been studied extensively and their functions are well-described, the remaining, so-called atypical Ub chains have largely resisted characterisation. The broad aim of this thesis was to better our understanding of atypical Ub modifications with a focus on Ub recognition by linkage-specific binders. In the first part, the UBD family of Npl4-like zinc fingers (NZFs) will be discussed, as this family has previously been shown to be capable of linkage-specific recognition. A candidate-based approach was used to screen UBDs for linkage-preference for atypical Ub chains. This revealed that TRABID NZF1 is specific for Lys29/Lys33 chains and is the first UBD to specifically recognise these linkages. Moreover, [a protein] was found to be specific for Lys6/Lys63 chains and is the first UBD with specificity for Lys6 chains. Biochemical and structural characterisation of these UBDs in complex with their preferred chain types uncovered the molecular mechanisms that confer linkage specificity. Using insights gained from studying NZF UBDs, further investigations into the versatility of the NZF scaffold were performed. Unexpectedly, this revealed that [a protein] is able to recognise substrate ubiquitination of [a protein] in a substrate- and site-specific manner and this interaction was structurally and biochemically characterised.

In the second part, the UBDs of all selective autophagy receptors were screened for interesting interactions with Ub. None of the UBDs tested exhibited linkage-specific interaction with Ub, except for OPTN, which prefers Met1-linked chains. Moreover, because autophagy receptors can also act in mitophagy, these UBDs were tested for preferential interaction with Ub phosphorylated on Ser65. However, none of the UBDs tested prefer phosphorylated Ub. Two structures of these UBDs in complex with Ub are presented, which together with previously published structures, allowed us to rationalise the observed binding behaviours.

In the final part, the generation of linkage-specific tools for Lys6- and Lys33-/Lys11-linked chain is described. In collaboration with Avacta, linkage-specific affimer binders against Lys6- and Lys33/Lys11-linked polyUb chains have been developed. Crystal structures of the affimers in complex with their cognate diUb linkages revealed the basis of linkage specificity and further allowed structure-guided improvements. These improved affimers prefer their cognate diUb >106-fold over other Ub linkages. Furthermore, affimers were validated in a number of applications: using the Lys6-affimer as a pull-down reagent allowed enrichment of Lys6 chains over 100-fold compared to other linkages. This led us to identify the HECT E3 ligase HUWE1 as a major source of cellular Lys6 chains. Furthermore, the Lys6-affimer was used in confocal imaging where it localised to depolarized mitochondria in a Parkin-dependent manner, consistent with Parkin assembling Ub chains, including Lys6 linkages, upon mitochondrial damage. Together, these affimers are the first linkage-specific tools for these atypical Ub chains and enable targeted investigations into the biological functions of specific atypical Ub linkages.

Taken together, the research presented in this thesis identified novel, linkage-specific components of the Ub system. Moreover, the developed tools will lay the basis for further, mechanistic investigations into the functions of atypical Ub linkages in important signalling pathways which will hopefully shed light on the biological roles of these understudied chain types.