Molecular basis for recognition of Gly/N-degrons by CRL2ZYG11B and CRL2ZER1

N-degron pathways are a set of proteolytic systems that target the N-terminal destabilizing residues of substrates for proteasomal degradation. Recently, the Gly/N-degron pathway has been identified as a new branch of the N-degron pathway. The N-terminal glycine degron (Gly/N-degron) is recognized by ZYG11B and ZER1, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we present the crystal structures of ZYG11B and ZER1 bound to various Gly/N-degrons. The structures reveal that ZYG11B and ZER1 utilize their armadillo (ARM) repeats forming a deep and narrow cavity to engage mainly the first four residues of Gly/N-degrons. The α-amino group of the Gly/N-degron is accommodated in an acidic pocket by five conserved hydrogen bonds. These structures, together with biochemical studies, decipher the molecular basis for the specific recognition of the Gly/N-degron by ZYG11B and ZER1, providing key information for future structure-based chemical probe design. [Display omitted] •Crystal structures of ZYG11B and ZER1 bound to various Gly/N-degrons are solved•ZYG11B and ZER1 use a deep and narrow cavity to engage Gly/N-degron•The first four residues of substrates are the key recognition elements•ZYG11B and ZER1 prefer bulky aromatic residues following the first glycine CRL2ZYG11B and CRL2ZER1 are responsible for degradation of substrates bearing an N-terminal glycine through the proteasome-dependent Gly/N-degron pathway. Yan et al. provide structural insights into recognition of the Gly/N-degron by ZYG11B and ZER1.