LZTR1: How it recognizes, binds, and degrades RAS proteins

RAS proteins act as molecular switches that regulate cellular growth and development¹. They cycle between an inactive “off” state bound to guanosine diphosphate (GDP) and an active “on” state bound to guanosine triphosphate (GTP). Activation is facilitated by guanine nucleotide exchange factors, which catalyze the exchange of bound GDP to GTP, while inactivation is accelerated by GTPase-activating proteins, which stimulate RAS to hydrolyze GTP back to GDP^2,3. In the GTP-bound state, two flexible regions, called switch I and switch II, undergo conformational changes that allow RAS to interact with downstream effector proteins and initiate signaling pathways⁴. Maintaining the proper balance between active and inactive RAS is essential, as mutations in HRAS, KRAS, or NRAS that favor the GTP-bound state can drive uncontrolled cellular growth and cancer.

Recently, a novel regulatory mechanism was discovered in which certain RAS proteins are targeted for degradation through ubiquitination^5-7. The adaptor protein Leucine Zipper-like Transcription Regulator 1 (LZTR1) uses its N-terminal Kelch domain to bind RIT1 and MRAS, and to a lesser extent HRAS, KRAS, and NRAS. LZTR1 then links these RAS proteins to the Cullin3–RING E3 ubiquitin ligase complex via its C-terminal BTB-BACK domains, leading to their degradation by the proteasome. Notably, LZTR1 binds only the inactive GDP-bound state, unlike other RAS effectors that recognize the active GTP-bound form. Our recent publication in Science addressed how LZTR1 selects for specific RAS proteins in the GDP-bound inactive state, outlined the mechanisms by which RIT1 and LZTR1 mutations contribute to human disease, and explored the feasibility of modeling these pathogenic mutations in animal models⁸. Collectively, these findings point to a promising new strategy for therapeutically targeting RAS-driven cancers by harnessing the cell’s own degradation machinery.

To investigate how LZTR1 distinguishes among RAS proteins, we first measured binding affinities with purified proteins. RIT1 and MRAS bound LZTR1 tightly (dissociation constant, KD, of 4–5 µM), while HRAS, KRAS, and NRAS exhibited weaker binding (KD around 20 µM), and none interacted in the GTP-bound state. To understand the structural basis for this selectivity, we used X-ray crystallography to determine the structures of RIT1– and MRAS–LZTR1 complexes. Initial crystallization attempts failed, but AlphaFold modeling indicated that the N-terminal 50 residues of LZTR1 are disordered, and a 50-residue loop within the Kelch domain is flexible. Removing these regions did not affect the GDP-specific binding preference and allowed successful crystallization. The crystal structures, solved at ~3 Å resolution, revealed that LZTR1’s Kelch domain forms a six-bladed β-propeller, with each blade made up of four antiparallel β-strands (Kelch motif) stabilized by hydrophobic interactions between neighboring motifs (Figure 1). This β-propeller has a truncated cone shape with a central channel, and both RIT1 and MRAS bind identically at the narrow end, using their switch I and switch II regions to contact all six Kelch motifs. In the GTP-bound state, these switch regions position themselves to sterically clash with LZTR1, explaining its exclusive recognition of the inactive GDP-bound form.

As the canonical RAS proteins bind LZTR1 more weakly than RIT1 and MRAS, we tested whether mutations could increase KRAS affinity to stabilize the interaction for structural studies. Mutating T35A or changing E62 to the equivalent residue in RIT1 increased KRAS binding to LZTR1 to 8 µM and 2 µM, respectively, and the double mutant T35A/E62A bound at ~2 µM, allowing determination of the KRAS–LZTR1 structure. The structure closely resembles RIT1- and MRAS-bound LZTR1. Higher affinity also promoted degradation: KRAS-E62A levels decreased in cells with LZTR1 but not when LZTR1 was silenced, suggesting that small molecules could trap oncogenic KRAS in its inactive form, leading to its breakdown. This strategy would potentially lower the population of oncogenic KRAS in the cell and dampen signaling.

Mutations in RIT1 are linked to Noonan syndrome, a RASopathy, and various cancers. RIT1 mutations (F82L, T83P and M90I) in the GDP-bound state abolish LZTR1 binding by disrupting protein-protein contacts and local structure, preventing RIT1 degradation. Other RIT1 mutations, such as A57G, Y89H, and Q79L, still bind LZTR1 but favor the active GTP-bound form, also escaping degradation in cells.

Pathogenic LZTR1 mutations are found in Noonan syndrome and LZTR1-related Schwannomatosis. Testing 25 Kelch-domain mutations showed that most weaken or abolish RIT1 binding, while two cause mis-splicing and mRNA decay. Structural analysis reveals three mechanisms (Figure 1):

• Type I: Directly disrupts contacts or creates steric clashes (e.g., Y119C, G248R, R412C).
• Type II: Indirectly by destabilization of loops essential for RIT1 binding (e.g., R248C).
• Type III: Introduction of charged residues that destabilize the overall Kelch domain.

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We further investigated the most common Noonan syndrome mutation in LZTR1 (G248R, a Type I mutation) in an experimental model to understand the effects of disrupting the LZTR1-RIT1 interaction in vivo. We observe that homozygous cells carrying the G248R mutation exhibit increased RIT1 abundance and MAPK signaling. Furthermore, homozygous Lztr1^G248R is embryonically lethal in mice, and both RIT1 and MRAS are more abundant. This observation phenocopies mice where Lztr1 has been deleted^9,10.

Our work shows that LZTR1 preferentially binds RIT1 and MRAS over HRAS, KRAS, and NRAS in the GDP-bound inactive state. Crystal structures reveal that this selectivity is driven by the nucleotide-dependent conformation of switch I and II and explain how mutations in RIT1 or LZTR1 cause Noonan syndrome, Schwannomatosis, and certain cancers. Several strategies currently exist for preventing oncogenic RAS engaging with effector proteins and eliciting signaling, such as direct inhibitors targeting the switch-II pocket, compounds that lock RAS in a non-functional complex with cyclophilin A, and PROTACs (Proteolysis-Targeting Chimeras) designed to actively degrade mutant RAS proteins^11,12. We have shown that stronger KRAS binding to LZTR1 promotes its degradation in cells. As RAS proteins are bona fide substrates for degradation by LZTR1, our proposed strategy of enhancing LZTR1 affinity with molecular glues for oncogenic RAS proteins could promote their degradation through the cell’s own machinery. This approach does not rely on PROTACs or rendering RAS nonfunctional, but uses a more natural cellular pathway, potentially providing better specificity and a complementary tool to existing targeted protein degradation technologies.

References

1. Simanshu, D.K., Nissley, D.V. & McCormick, F. RAS Proteins and Their Regulators in Human Disease. Cell 170, 17–33 (2017).
2. Vigil, D., Cherfils, J., Rossman, K.L. & Der, C.J. Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nat Rev Cancer 10, 842–57 (2010).
3. Hennig, A., Markwart, R., Esparza-Franco, M.A., Ladds, G. & Rubio, I. Ras activation revisited: role of GEF and GAP systems. Biol Chem 396, 831–48 (2015).
4. Mozzarelli, A.M., Simanshu, D.K. & Castel, P. Functional and structural insights into RAS effector proteins. Mol Cell 84, 3163–3164 (2024).
5. Castel, P. et al. RIT1 oncoproteins escape LZTR1-mediated proteolysis. Science 363, 1226–1230 (2019).
6. Bigenzahn, J.W. et al. LZTR1 is a regulator of RAS ubiquitination and signaling. Science 362, 1171–1177 (2018).
7. Steklov, M. et al. Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination. Science362, 1177–1182 (2018).
8. Dharmaiah, S. et al. Structural basis for LZTR1 recognition of RAS GTPases for degradation. Science 389, 1112–1117 (2025).
9. Cuevas-Navarro, A. et al. Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs. Elife 11, e76495 (2022).
10. Chen, S. et al. Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation. Cancer Discov 12, 2434–2453 (2022).
11. Jiang, J. et al. Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers. Cancer Discov 14, 994–1017 (2024).
12. Popow, J. et al. Targeting cancer with small-molecule pan-KRAS degraders. Science 385, 1338–1347 (2024).