Ubiquitination is a highly regulated post-translational modification that controls almost every process in the cell. The linear ubiquitin chain assembly complex (LUBAC) is the only enzyme forming Met1-linked (linear) ubiquitin chains and is a key regulator of immunity, inflammation, and cell death downstream of the TNF receptor. LUBAC consists of three proteins, HOIP, HOIL-1L and SHARPIN. The RBR (RING-between-RING) E3 ubiquitin ligase HOIP is the catalytic center of the LUBAC. RBR E3 ligases catalyze the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to the substrate in a 2-step mechanism. We previously solved the crystal structure of the HOIP-RBR/E2-ubiquitin/ubiquitin transfer complex [1], which revealed key aspects of the catalytic mechanism of RBR E3 ligases. We also identified that HOIP is allosterically regulated by ubiquitin.
We now present new biochemical and biophysical data showing that the allosteric ubiquitin specifically activates the 1st step of the RBR reaction, the ubiquitin transfer from the E2 to the HOIP active site, but not the 2nd step. Surprisingly, the allosteric activation of HOIP by ubiquitin is controlled by the cofactor HOIL-1L. HOIL-1L, via its NZF domain, increases the binding affinity of HOIP for allosteric ubiquitin and induces specificity for allosteric linear ubiquitin linkages over other ubiquitin chains. These observations suggest that LUBAC is regulated by a positive feedback loop. We further identified a role of HOIL-1L as an activator of the 2nd step of the HOIP RBR reaction, the ubiquitin transfer from HOIP to the substrate. Finally, we present data suggesting that HOIP and HOIL-1L form a stable 2:1 complex in which a HOIP dimer binds a single HOIL-1L molecule. Our data further define the intricate regulatory mechanism of HOIP by its cofactor HOIL-1L and allosteric ubiquitin chains, and pave the way for future studies of LUBAC regulation in a cellular context.