Revealing the cullin-RING ligase code

Cullin-RING ligases (CRLs), representing one-half of all ubiquitin ligases in humans, are notable for their roles in biology and in the induced-proximity drug discovery platform targeted protein degradation. CRLs collaborate with ubiquitin-carrying enzymes to promote protein ubiquitylation, the forging of poly-ubiquitin chains onto substrates, causing their degradation by the proteasome. Substrates are selected by CRL subunits that recognize amino acid sequence motifs as well as post-translational modifications that distinguish bona fide substrates from the thousands of proteins contained in cells. Recently, we uncovered the unexpected finding that ubiquitin-carrying enzymes, the catalytic component for CRL-dependent substrate ubiquitylation, also appear to play important roles during substrate selection. Here it was found that the carrying enzyme physically contacts the CRL substrate receptor subunit, helping to place the substrate Lysine residue that is modified by ubiquitin into the active site. We hypothesize the existence of a CRL-ubiquitin carrying enzyme code, where subsets of CRLs recognize one or a small subset of the CRL-dedicated ubiquitin-carrying enzymes. In this grant cycle, we seek to further uncover the CRL code using kinetics, structural biology, proteomics, single- molecule microscopy and cell biology. CRL dynamics, in particular the RBX1/2 subunit that recognizes ubiquitin-carrying enzymes, are critical to the function of the code and will be explored using single-molecule fluorescence resonance energy transfer between dyes appended to CRL subunits or ubiquitin-carrying enzymes. Structural biology, particularly cryo-electron microscopy, will be employed to study how CRL- ubiquitin carrying enzyme complex formation is stabilized and how the CRL code manifests through direct interaction between ubiquitin-carrying enzymes and CRL substrate receptors. High resolution pre-steady state kinetics will be performed on in vitro reconstituted ubiquitylation reactions to estimate the rates of ubiquitin transfer from ubiquitin-carrying enzymes to CRL-bound substrates, testing the hypothesis that the CRL code imparts efficient substrate ubiquitylation only for physiologically optimized CRL-carrying-enzyme partners. Substrate discovery proteomic screens will lead to new investigations into biologically interesting yet uncharacterized CRLs, including for natural biological substrates as well as the neo-substrates of targeted protein degradation. In combination, these studies will expand the CRL-ubiquitin carrying enzyme code, further explain how CRL dynamics enable physical bridging between the CRL and the carrying enzyme and define the structural underpinnings of the code. Project Number: 1R35GM163591-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Gary Kleiger | Institution: UNIVERSITY OF NEVADA LAS VEGAS, LAS VEGAS, NV | Award Amount: $411,739 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award B Study Section[MRAB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11329288