Secondary interactions in ubiquitin-binding domains achieve linkage or substrate specificity

Small ubiquitin-binding domains (UBDs) recognize small surface patches on ubiquitin with weak affinity, and it remains a conundrum how specific cellular responses may be achieved. Npl4-type zinc-finger (NZF) domains are ∼30 amino acid, compact UBDs that can provide two ubiquitin-binding interfaces, imposing linkage specificity to explain signaling outcomes. We here comprehensively characterize the linkage preference of human NZF domains. TAB2 prefers Lys6 and Lys63 linkages phosphorylated on Ser65, explaining why TAB2 recognizes depolarized mitochondria. Surprisingly, most NZF domains do not display chain linkage preference, despite conserved, secondary interaction surfaces. This suggests that some NZF domains may specifically bind ubiquitinated substrates by simultaneously recognizing substrate and an attached ubiquitin. We show biochemically and structurally that the NZF1 domain of the E3 ligase HOIPbinds preferentially to site-specifically ubiquitinated forms of NEMO and optineurin. Thus, despite their small size, UBDs may impose signaling specificity via multivalent interactions with ubiquitinated substrates.