Structure of SHOC2-KRAS-PP1C complex reveals RAS isoform-specific determinants and insights into targeting complex assembly by RAS inhibitors

Domain: biology
Published: 2026-01-10
Editorial outcome: minor_revision

Abstract
RAF activation is essential for MAPK signaling and is mediated by RAS binding and the dephosphorylation of a conserved phosphoserine by the SHOC2–RAS–PP1C complex. MRAS forms a high-affinity SHOC2–MRAS–PP1C (SMP) complex, while canonical RAS isoforms (KRAS, HRAS, NRAS) form analogous but lower-affinity assemblies. Yet, cancers driven by oncogenic KRAS, HRAS, or NRAS remain strongly SHOC2-dependent, suggesting that these weaker complexes contribute to tumorigenesis. To elucidate how canonical RAS proteins form lower-affinity ternary complexes, the cryo-EM structure of the SHOC2–KRAS–PP1C (SKP) complex stabilized by Noonan syndrome mutations is described. The SKP architecture is similar to the SMP complex but forms fewer contacts and buries less surface area due to the absence of MRAS-specific structural features in KRAS that enhance complex stability. RAS inhibitors MRTX1133 and RMC-6236 alter Switch-I/II conformations, thereby blocking SKP assembly more effectively than they disrupt pre-formed complexes. These RAS inhibitors do not affect SMP formation because they do not bind MRAS. Since MRAS is upregulated in resistance to KRAS inhibition, we characterize a MRAS mutant capable of binding MRTX1133. This MRAS mutant can form an SMP complex, but MRTX1133 blocks its assembly, demonstrating the feasibility of dual SKP and SMP targeting. Overall, our findings define isoform-specific differences in SHOC2–RAS–PP1C complex formation and support a strategy to prevent both SKP and SMP assemblies to overcome resistance in RAS-driven cancers.

Introduction
The RAS–RAF–MEK–ERK signaling cascade transmits mitogenic signals from the cell surface to the nucleus, orchestrating key cellular programs such as proliferation, differentiation, and survival1. Dysregulation of this pathway, often through mutations in RAS or RAF, contributes to approximately 20–30% of human cancers2. Activation begins when receptor tyrosine kinases stimulate GDP-to-GTP exchange on RAS proteins, enabling them to recruit RAF kinases to the plasma membrane. RAF then undergoes conformational changes and dimerization1,3, activating downstream MEK and ERK kinases through phosphorylation1,4.

RAF activation is tightly regulated by phosphorylation at conserved serine residues in conserved regions 2 (CR2-pS) and 3 (CR3-pS), which flank the kinase domain5. In the inactive state, these sites are bridged by a 14-3-3 dimer, stabilizing RAF in an autoinhibited conformation6–8. Upon GTP loading, membrane-bound RAS engages the RAF RAS-binding domain (RBD) and cysteine-rich domain (CRD), facilitating RAF membrane localization and exposing CR2-pS for site-specific dephosphorylation by the SHOC2–RAS–PP1C ternary complex9–11. This dephosphorylation relieves autoinhibition and permits dimerization of RAF protomers, allowing a single 14-3-3 dimer to bridge CR3-pS sites across the dimer interface.

The SHOC2–RAS–PP1C ternary complex is composed of the leucine-rich repeat scaffold protein SHOC2, the catalytic phosphatase PP1C, and an active RAS isoform12. Our biophysical and structural studies, along with those of others, have shown that MRAS, which shares ~50% sequence identity with canonical RAS isoforms (KRAS, HRAS, and NRAS), forms a high-affinity SHOC2–MRAS–PP1C (SMP) complex and revealed the architecture of the ternary complex13–16. Canonical RAS isoforms form analogous but lower-affinity complexes.

SHOC2 acts as a scaffolding protein to assemble the ternary complex and contains 20 leucine-rich repeats (LRRs) along with an N-terminal intrinsically disordered region harboring an RVxF PP1C-binding motif. PP1C, a serine/threonine phosphatase with three isoforms (PP1CA, PP1CB, PP1CC), specifically dephosphorylates RAF CR2-pS when activated by SHOC2 and GTP-bound RAS12,13,16.

Despite their reduced binding affinity, canonical RAS containing SHOC2–H/K/NRAS–PP1C complexes remain functionally important in cancer. Notably, (i) the lower-affinity SHOC2–H/K/NRAS–PP1C complexes are still capable of dephosphorylating RAF CR2-pS substrates17; (ii) MRAS knockout mice exhibit no apparent phenotype, suggesting functional compensation by canonical RAS isoforms18,19; and (iii) oncogenic mutants of H/K/NRAS, particularly Q61 mutants, show strong dependence on SHOC2 for cancer cell growth and survival, indicating that SHOC2–PP1C complexes with oncogenic RAS can overcome weaker binding and effectively promote RAF dephosphorylation and activation15,16,20,21. However, the structural basis for ternary complex formation by canonical RAS isoforms remains unclear due to their biochemical instability. Recent advances in targeting KRAS include allele-specific inhibitors, such as sotorasib and adagrasib, which target the KRAS-G12C mutation22,23. However, resistance frequently arises through multiple mechanisms, including MRAS mutations and upregulation, emphasizing the need for strategies that simultaneously target KRAS and MRAS24–27.

In this work, we use Noonan syndrome-(SHOC2-M173I and PP1CA-P50R) and cancer-associated mutations (KRAS-Q61R) to assemble a stabilized SHOC2–KRAS–PP1C (stabilized SKP) complex. The stabilized SKP complex shares a similar overall architecture with SMP but reveals isoform-specific contacts that account for the reduced binding affinity of KRAS relative to MRAS. We evaluate MRTX1133, a KRAS-G12D-selective inhibitor effective against both nucleotide states, and RMC-6236, a pan-RAS inhibitor that binds active RAS via cyclophilin A28–30, as inhibitors of the formation of wild-type SHOC2–KRAS–PP1C (SKP) and SMP complexes. Both inhibitors bind KRAS with high affinity and prevent SKP assembly more potently than they disrupt preformed SKP complexes. Neither inhibitor binds MRAS or disrupts the SMP complex. An MRTX1133-sensitive MRAS variant that retains high-affinity binding to SHOC2 and PP1C is used to evaluate dual inhibition of SMP and SKP complexes, establishing proof-of-concept for dual targeting of MRAS and KRAS. These findings reveal RAS isoform-specific features of SHOC2 and PP1C complexes and suggest that disrupting both SKP and SMP may help limit MAPK pathway reactivation in RAS-driven cancers.

Results
SHOC2 dependency in RAS-mutant cancers and stabilized SKP complex

We and others have previously reported (summarized in Supplementary Table 1) distinct differences in binding affinity between SHOC2–PP1C complexes formed with MRAS versus those formed with the canonical RAS isoforms HRAS, KRAS, and NRAS by different biophysical techniques13–16. Among these, MRAS forms the highest-affinity complex with SHOC2 and PP1C (KD ≈ 120 nM by SPR), while H/K/NRAS form analogous ternary complexes with significantly lower affinity (KD ≈ 0.7–4 μM by SPR)13. Despite this weaker binding, genome-wide CRISPR/Cas9 fitness screens from the Cancer Dependency Map (DepMap; https://depmap.org/portal/) revealed that cancer cell lines harboring oncogenic mutations (G12X, G13X, Q61X) in H/K/NRAS display a marked dependency on SHOC2 for proliferation and survival15,16,21,31. Paradoxically, no comparable dependency was observed for MRAS despite its higher affinity binding to SHOC2 and PP1C. Chronos score correlations further support these findings, showing strong co-dependency between SHOC2 and mutant RAS isoforms, particularly those with Q61 and G13 mutations, with a weaker correlation for G12 mutations. (Fig. 1a, b). These observations suggest that while the SHOC2–PP1C interaction with canonical RAS isoforms is biochemically less stable, it is functionally more critical in the context of RAS-driven cancers. This disconnect between affinity and cellular dependency may reflect a requirement for dynamic or transient SHOC2–H/K/NRAS–PP1C engagement to support efficient RAF dephosphorylation and sustained MAPK pathway activity in oncogenic settings.

To define the structural basis of canonical RAS interaction with SHOC2 and PP1C, we aimed to determine the structure of the SHOC2–KRAS–PP1C complex. Previous efforts using X-ray crystallography and cryo-electron microscopy (cryo-EM) were unsuccessful due to the dissociation of the low-affinity complex during sample preparation16,32. To overcome this challenge, we applied a rational mutagenesis approach to stabilize complex formation, building on prior insights into SHOC2–RAS–PP1C interactions (Fig. 1c). The SHOC2-M173I and PP1CA-P50R mutations, both associated with Noonan syndrome, had previously been shown to enhance SMP complex affinity by ~2–3 fold13,33. Additionally, the Noonan syndrome-associated MRAS-Q71R mutation, which introduces an arginine at the interface, strengthens SMP complex formation by forming additional polar contacts15. SMP and SKP affinities measured by ITC, conducted in 500 mM NaCl and 5% glycerol to maintain PP1C solubility, are higher (900 nM for SMP, 7 µM for SKP) than those measured earlier by SPR at 150 mM NaCl (120 nM for SMP, 0.7 µM for SKP), reflecting the effect of ionic strength rather than intrinsic affinity differences (see Methods for details). The corresponding cancer-associated mutation in KRAS (Q61R), when combined with Noonan syndrome mutations SHOC2-M173I and PP1CA-P50R, led to a substantial ~40-fold increase in SKP complex affinity, reducing the KD from 7 µM to 154 nM (Fig. 1d), which is tighter than the SMP complex affinity as measured by ITC under high salt conditions.

This stabilized SKP complex, in combination with optimized graphene oxide grids, enabled structure determination by cryo-EM at 3.0 Å resolution (Fig. 1e; Supplementary Fig. 1a–g and Supplementary Table 2). The resulting Coulomb potential map enabled detailed visualization of the molecular interfaces that stabilize the ternary complex and allowed direct comparison with the SMP complex, thereby providing insight into the conserved and isoform-specific features of SHOC2–RAS–PP1C assembly.

Cryo-EM structure of the stabilized SKP complex

The overall architecture of the stabilized SKP complex reveals that SHOC2 adopts a curved, horseshoe-shaped structure, serving as a scaffold that brings KRAS and PP1CA into proximity, resembling previously determined SMP complex structures13–16. All three proteins—SHOC2, KRAS, and PP1CA—are well resolved in the cryo-EM Coulomb potential map (Fig. 1e). SHOC2 shows progressively lower local resolution toward the C-terminal end of its LRR domain, likely due to increased flexibility (Fig. 2a). The active site channels of manganese-bound PP1CA remain fully exposed within the stabilized SKP complex (Fig. 2b). SHOC2 engages PP1CA through an RVxF motif located in its N-terminal intrinsically disordered region, which binds to a conserved hydrophobic pocket on PP1CA used by other RVxF-containing regulatory proteins (Fig. 2b)34.

The LRR domain of SHOC2 forms a curved solenoid, with its concave surface partially wrapped around PP1CA. The Coulomb potential map for 19 of the 20 LRRs is well resolved. KRAS, like other RAS isoforms, adopts a G-domain fold comprising a six-stranded β-sheet and five α-helices. Within the complex, the Switch-I (residues 30-38) and Switch-II (residues 60-76) regions of KRAS interact with the upper concave face of the LRR domain of SHOC2, while additional contacts are made between KRAS and PP1CA through residues in the pre-Switch-I and interswitch regions (Fig. 2b). These interactions contribute to complex stability, burying a total of ~5200 Å² of surface area and forming 10 salt bridges and 17 hydrogen bonds.

Although all three pathogenic mutations enhanced complex affinity biochemically, only one forms a direct contact in the stabilized SKP structure. PP1CA-P50R engages in van der Waals interactions with SHOC2 residues N202 and E224 (Fig. 2c). The SHOC2-M173I mutation, previously shown to increase SMP complex affinity, does not form direct contacts in the stabilized SKP complex (Fig. 2d). The substitution of methionine by isoleucine likely increases local hydrophobicity within the LRR domain, indirectly contributing to complex stabilization. Similarly, the KRAS-Q61R mutation does not make direct contacts at the KRAS–SHOC2 interface (Fig. 2e), unlike the MRAS-Q71R mutant SMP complex structure, which engages through both direct and water-mediated interactions with SHOC2 residues N265 and R28815. These water-mediated interactions may also be present in the stabilized SKP complex but are unresolved at the current cryo-EM resolution.

Mechanistic insights into KRAS versus MRAS engagement with SHOC2 and PP1C

The stabilized SKP, and SMP complexes superimpose with a low root mean square deviation (RMSD) of 1.2 Å over Cα atoms, indicating a conserved overall architecture (Fig. 3a). Despite this similarity, the stabilized SKP complex buries approximately 1000 Å² less surface area and forms 6 fewer salt bridges and 12 fewer hydrogen bonds compared to the SMP complex as calculated by PISA35 and PDBSum36, consistent with the lower binding affinity of wild-type KRAS relative to MRAS for SHOC2 and PP1C (Figs. 1d and 3b, c and Supplementary Figs. 2a, b and 3). The most substantial loss occurs at the SHOC2–KRAS interface, which buries 450 Å² less surface area due to reduced contacts with the Switch-I and -II regions of KRAS (Fig. 3d). Only three hydrogen bonds are observed between SHOC2 and KRAS, involving KRAS residues E37 from Switch-I and E63 and Q70 from Switch-II, which interact with SHOC2 residues R177, N265, and D106, respectively. In contrast, the SHOC2–MRAS interface contains seven hydrogen bonds (Fig. 3d and Supplementary Fig. 4a).

A distinguishing feature of MRAS is the β5–α4 helical loop within its allosteric lobe, which forms van der Waals interactions and a hydrogen bond between MRAS-H132 and SHOC2-E428 (Fig. 3e). This loop is one residues shorter and more aliphatic in KRAS, resulting in the absence of equivalent contacts with SHOC2 (Fig. 3e and Supplementary Fig. 4b). The interswitch region also diverges significantly between KRAS and MRAS (Fig. 3f, g and Supplementary Fig. 4c). In KRAS, Q43 forms a single main-chain hydrogen bond with PP1CA-D179, while R41 makes minimal contact due to an internal hydrogen bond with E3 that reorients its side chain away from the PP1CA surface (Fig. 3f and Supplementary Fig. 4c). In contrast, MRAS-H53 (equivalent to KRAS-Q43) forms two hydrogen bonds through both backbone and side chain with PP1CA-D179, and MRAS-L51 (equivalent to KRAS-R41) engages in additional van der Waals interactions with PP1CA (Fig. 3g).

Smaller but notable losses (~300 Å² each) are also observed at the KRAS–PP1C and SHOC2–PP1C interfaces in the stabilized SKP complex. KRAS lacks the N-terminal extension present in MRAS, which in the SMP complex contributes van der Waals contacts and a hydrogen bond between MRAS-S4 and PP1CA-E218 (Fig. 3h). This extension is further stabilized by internal van der Waals interactions between MRAS residues P7 and W60 (Fig. 3h). Additionally, the SHOC2 RVxF motif and twelve flanking residues form a β-hairpin that engages PP1CA in the SMP complex. In the stabilized SKP structure, only the RVxF motif and five adjacent residues are resolved, and this region adopts a shorter β-strand conformation (Fig. 3a). The comparative analysis suggests that the cumulative loss of buried surface area, salt bridges, hydrogen bonds, and van der Waals contacts in the stabilized SKP complex is not due to the stabilization mutations but sequence differences between MRAS and KRAS. This underlies the reduced stability and lower affinity of the wild-type SKP complex relative to the SMP complex.

Distinct switch-II conformations define effector-specific RAS interactions

Effector binding to RAS is essential for propagating downstream signaling and is typically mediated through its GTP-bound conformation, which exposes key interaction surfaces in the Switch-I and, in some cases, Switch-II regions. Switch-I adopts a conserved conformation across diverse effectors, including CRAF, PI3Kα, Rgl2, Sin1, SHOC2–PP1C, the allosteric site of RasGEF SOS1, and RasGAP NF1, optimized for high-affinity engagement (Fig. 4)11,13,37–40.

In contrast, Switch-II is markedly more flexible in both GDP- and GTP-bound states and is often unresolved in unbound structures unless stabilized by crystal packing. Effector or regulator binding typically stabilizes this region, but its conformation varies depending on the binding partner. For example, in the KRAS–CRAF complex, Switch-II does not directly engage the RBD–CRD but is resolved as a flexible loop (residues 60–64) followed by a short α2 helix (residues 65–75) (Fig. 4a). In contrast, effectors such as Sin1, Rgl2, and RasGAP NF1 make direct contacts with Switch-II, leading to a shortened loop (residue 60) and an elongated, bent α2 helix (residues 61–75) (Fig. 4b–d). A similar conformation is observed in HRAS bound to the allosteric site of RasGEF SOS1, with a slight variation in loop positioning compared to KRAS–CRAF (Fig. 4e)41.

PI3Kα and SHOC2–PP1C, in both SKP and SMP complexes, engage Switch-II extensively, inducing a distinct conformation with a shortened α2 helix (residues 68–75 in KRAS; 78–85 in MRAS) and an extended loop (residues 60–67 in KRAS; 70–77 in MRAS) (Fig. 4f–h). These comparisons highlight the structural plasticity of Switch-II, which, rather than adopting a single conserved conformation, serves as a dynamic interface that assumes distinct structural states depending on the mode of interaction with subsets of effectors or regulators.

Structural basis for MRTX1133-mediated inhibition of SKP complex assembly

KRAS-G12C inhibitors, such as sotorasib and adagrasib, function by covalently modifying the cysteine at position 12 in the GDP-bound state, thereby preventing nucleotide exchange and subsequent activation. However, resistance to these inhibitors frequently arises through the emergence of other active KRAS mutations27,42. More recently, adaptive resistance has been attributed to the mislocalization of Scribble (SCRIB) from the membrane-associated SHOC2–SCRIB–PP1C complex to the cytoplasm24,25. Cytoplasmic SCRIB inactivates the Hippo pathway, enabling YAP nuclear translocation and transcription of target genes, including MRAS. MRAS upregulation, along with nucleotide exchange via SOS, contributes to the feedback reactivation of MAPK signaling24,26 and MRAS-regulated resistance pathways43,44. It is currently unknown whether MRTX1133 induces similar resistance mechanisms, though studies suggest it can trigger EGFR-mediated feedback and promote upregulation of RAS pathway genes such as Kras, Yap1, Myc, and Cdk6/Abcb1a/b45–48. These observations raise the possibility that co-targeting oncogenic KRAS and MRAS could prevent SHOC2–RAS–PP1C (SRP) complex–driven escape mechanisms.

To investigate whether pharmacological targeting of RAS proteins could disrupt the assembly of SHOC2–RAS–PP1C complexes, we evaluated the effects of RAS inhibitors on SKP and SMP formation. Given the importance of these complexes in MAPK signaling, including potential roles in adaptive resistance to KRAS inhibitors, we sought a proof-of-concept approach to identify small molecules capable of blocking both KRAS- and MRAS-mediated complex assembly.

MRTX1133 is a selective, non-covalent KRAS-G12D inhibitor that binds with high affinity to both GDP- and GTP-bound forms, engaging a pocket formed by Switch-II and the α3 helix29. MRTX1133 can also bind wild-type and other oncogenic mutants of KRAS, but with relatively lower affinity49. Using ITC (with 500 mM NaCl, see Methods for details), we confirmed that MRTX1133 binds both wild-type KRAS and KRAS-G12D with low nM affinity in both nucleotide states, albeit weaker than measured by SPR under lower salt conditions29,49, with tighter binding to the GDP- than GMPPNP-bound state in both cases (Fig. 5a and Supplementary Fig. 5a).

To better understand the structural basis of MRTX1133 binding to wild-type KRAS and how this might impact complex formation, we determined the crystal structures of wild-type KRAS(GDP) and KRAS(GMPPNP) bound to MRTX1133 at 1.4 Å and 1.9 Å resolution, respectively (Fig. 5b and Supplementary Fig. 5b–d). These structures allow direct comparison with existing KRAS-G12D–MRTX1133 structures and provide a model system for analyzing inhibitor effects in the context of non-mutant SKP complex assembly. They superimpose with a low RMSD (0.3 Å) and show minor differences in Switch-I and -II conformations (Supplementary Fig. 5e). MRTX1133 occupies the same Switch-II pocket as in KRAS-G12D. In wild-type KRAS, the absence of an aspartate at position 12 is compensated by bridging water molecules and crystallization ions (e.g., sulfate or chloride) that help coordinate the P-loop and nucleotide with MRTX1133’s bicyclic piperazine group (Supplementary Fig. 5f).

Despite its weaker binding to wild-type KRAS, MRTX1133 effectively blocked SKP complex assembly in ITC experiments and weakly dissociated pre-formed complexes (Fig. 5c and Supplementary Fig. 5g). Structural modeling revealed that MRTX1133 binding displaces the α2 helix and alters Switch-I, resulting in steric clashes with SHOC2-Y129 and impaired SHOC2 interaction (Fig. 5d). Thus, MRTX1133 binding to KRAS not only inhibits RAF binding but also prevents SKP complex formation.

Proof-of-concept for dual inhibition of KRAS and MRAS to disrupt ternary complex assembly

To explore broader inhibition of RAS isoforms, we tested the pan-RAS inhibitor RMC-6236, which forms a tight binary complex with cyclophilin A (CypA) and then binds H/K/NRAS proteins, blocking Switch-I/II engagement with effectors28. ITC measurements showed that RMC-6236 binds CypA with a KD of 108 nM and forms a ternary complex with KRAS at 127 nM (Fig. 6a and Supplementary Fig. 6a). Like MRTX1133, RMC-6236–CypA disrupted and prevented SKP complex formation, through blocking of Switch-I engagement with SHOC2 and CypA occupying the SHOC2-binding site (Fig. 6b, c and Supplementary Fig. 6b). However, it did not bind MRAS or interfere with SMP formation, suggesting that resistance to RMC-6236 could similarly arise through MRAS upregulation (Fig. 6a, b and Supplementary Fig. 6b).

KRAS and MRAS share 53% sequence identity, though only six residues within the MRTX1133 Switch-II binding pocket differ between the two proteins: D21A, F74Y (the only residue that also forms van der Waal interactions in the stabilized SKP, and SMP structures), E79D, R105H, F106Y, and L109Q (MRAS numbering) (Fig. 7a and Supplementary Fig. 7a). As such, MRTX1133 does not bind MRAS in either GDP- or GMPPNP-bound states (Fig. 7b and Supplementary Fig. 7b), nor does it affect SMP complex assembly (Supplementary Fig. 7c). While ligand redesign could be explored to accommodate MRAS, we pursued a simpler approach: introducing mutations into MRAS Switch-II pocket to enable MRTX1133 binding. The MRAS-R105H single mutant failed to bind MRTX1133, and the MRAS-F74Y/R105H double mutant showed only weak binding in the GTP-bound state. However, the triple mutant F74Y/R105H/L109Q exhibited high-nanomolar binding in both nucleotide states, and the quadruple mutant F74Y/R105H/F106Y/L109Q (MRASmut) bound MRTX1133 with low-nanomolar affinity in both GDP- and GMPPNP-bound forms (Fig. 7b and Supplementary Fig. 7b). Crystal structures of MRASmut bound to MRTX1133 in both nucleotide states were determined at 1.5 Å and 1.9 Å resolution and closely resembled the wild-type KRAS–MRTX1133 structures (Supplementary Table 3; Fig. 7c and Supplementary Fig. 8a–d).

Despite these mutations, MRASmut retained the ability to assemble the SMP complex with SHOC2 and PP1C, with affinity similar to wild-type MRAS (Fig. 7d). However, MRTX1133 successfully prevented SHOC2-MRASmut-PP1C complex formation, although it did not disassemble pre-formed complexes (Fig. 7d and Supplementary Fig. 8e). Structural modeling showed that MRTX1133 binding in MRASmut displaces the α2 helix and disrupts Switch-I engagement by SHOC2, mimicking the mechanism observed for SKP disruption (Fig. 7e). Although MRTX1133 prevented the assembly of SHOC2-MRASmut-PP1C and SHOC2-KRAS-PP1C complexes in biophysical assays, its effect on catalytic activity remained unclear. To address this, we used an in vitro dephosphorylation assay, which monitors the release of phosphate from a CRAF CR2-pS peptide substrate. PP1C alone exhibited weak dephosphorylation activity (Fig. 7f), whereas SHOC2 and MRAS together synergistically activated PP1C activity to a significantly higher level. SHOC2 alone weakly stimulated PP1C, while MRAS alone did not stimulate PP1C (Fig. 7f). MRTX1133 had no effect on the catalytic activity of the SHOC2-MRAS-PP1C complex, as MRTX1133 cannot bind MRAS (Fig. 7g). The catalytic activity of the SHOC2-MRASmut-PP1C and SHOC2-KRAS-PP1C complexes was partially and completely inhibited by MRTX1133, respectively, consistent with its higher binding affinity for KRAS than for MRASmut (Fig. 7h, i). Furthermore, we observe that a RAS-less cell line devoid of HRAS, KRAS, NRAS, and MRAS transfected with MRASmut-Q71L treated with MRTX1133 could prevent dephosphorylation of CRAF, relative to MRAS-Q71L (Supplementary Fig. 9).

Recently, the Novartis team reported several compounds that bind to a pocket on SHOC2, thereby preventing RAS engagement with SHOC2 and PP1C50. These compounds block complex assembly by all RAS proteins, including MRAS. They reported a crystal structure of SHOC2 bound to Compound (R)-5 (Cmpd (R)-5), which has an affinity of ~1 µM for SHOC250. In our dephosphorylation assay, we observe that Cpmd (R)-5 partially inhibits the activity of SHOC2-MRAS-PP1C and SHOC2-MRASmut-PP1C complexes and strongly inhibits the activity of SHOC2-KRAS-PP1C complex due to the weaker affinity of KRAS than MRAS for SHOC2 and PP1C (Supplementary Fig. 10).

Together, these findings establish a structural and functional basis for dual targeting of oncogenic KRAS and MRAS. As a proof-of-concept strategy, they demonstrate that inhibitors capable of blocking both SKP and SMP complex formation, either by engaging Switch-II directly, by stabilizing conformations incompatible with SHOC2 binding, or blocking SHOC2 binding, can effectively interfere with SHOC2-mediated signaling. Both MRTX1133 and RMC-6236 robustly inhibited SKP formation but only weakly dissociated existing ternary complexes. These results provide key mechanistic insights for therapeutic implications: preventing nascent complex assembly is comparatively more tractable than attempting to dislodge stable ternary complexes. The ability to prevent both SKP and SMP formation expands the scope of SHOC2-targeted RAS pathway inhibition, providing a framework for the rational development of dual-target inhibitors to overcome adaptive resistance in RAS-driven cancers.

Methods
Cloning of expression constructs
The Entry clones for the single expression constructs PP1CA (2–300), PP1CA (7–300), MRAS (1–178), MRAS mutants (1–178), KRAS (1–169), KRAS mutant (1–169), and CypA (1–165) were synthesized (ATUM, Inc.) as codon-optimized fragments for Escherichia coli expression, downstream of a TEV protease site (ENLYFQ/G). Entry clones were transferred to Expression clones containing an amino-terminal His6-tag (pDest-527, Addgene no. 1518) or an amino-terminal His6-Maltose Binding Protein (MBP) tag (pDest-566, Addgene no. 11517) by Gateway LR recombination.

The Entry clones for the two polycistronic plasmids, SHOC2-M173I (2–582)-KRAS-Q61R (1-169)-PP1CA-P50R (2-330)+SUGT1 and SHOC2 (2-582)+SUGT1, were synthesized and codon optimized for insect expression (with the exception of SHOC2(2-582) which was PCR amplified from an MGC cDNA template plasmid (Horizon, MHS6278-20275921) with flanking attB1 and attB2 sites and an amino-terminal TEV protease cleavage site). The Entry clones were combined with pDest-623 (Addgene no. 161878) or pDest-624 (Addgene no. 161879) in a Gateway multisite LR recombination reaction to generate Expression plasmids60. The Bac-to-Bac system (Thermo Fisher Scientific) was used to generate bacmid DNA in strain DE9561.

Protein expression and purification
SHOC2 and stabilized SKP complex were expressed with the SUGT1 chaperone in insect cells as described before13,17. Briefly, 1.5×106 cells/ml of serum-free adapted Sf9 cells grown in SF900 III medium (100 mL) were transfected with DNA–cellfectin II lipid complex (70 μL of bacmid DNA: 250 μL cellfectin II: 500 μL SF900 III medium). The culture was incubated for 120 h at 27°C before the cell culture supernatant was isolated, and the baculovirus titer was determined by qPCR (TaqMan Gene Expression assay for the baculovirus GP64 protein). 1–2 L of Tni-FNL cells in SF900 III medium (7×105 cells/ml) were grown for 24 h at 21°C to allow doubling before baculovirus infection with a MOI of 362. Cell cultures were grown for 72 h at 21°C before collection and lysis (100 ml/L of cells) in buffer A (20 mM HEPES, pH 7.4, 300 mM sodium chloride, and 1 mM TCEP) using a Microfluidizer. Clarified lysates (100,000×g, 30 min at 4°C) were filtered (0.45-μm high flow PES filter) and captured using a 5-ml Ni Sepharose High-Performance column (GE Healthcare) equilibrated in 20 mM HEPES, pH 7.4, 300 mM sodium chloride, 1 mM TCEP, and 35 mM imidazole on an NGC chromatography system (Bio-Rad). The column was washed for 5 column volumes (CVs) with 7% buffer B (20 mM HEPES, pH 7.4, 300 mM sodium chloride, 1 mM TCEP, 500 mM imidazole) before a gradient elution over 20 CVs to 100% buffer B. The elution peak was dialyzed into buffer A and digested with His-tagged TEV (ratio of 1:20 v/v protease:pooled protein) overnight at 4°C. His-tagged TEV and cleaved His-MBP were captured by a second Ni Sepharose column while cleaved proteins eluted in the flow-through and by running a shallow gradient of 0–10% buffer B over 10 CVs. Cleaved proteins were concentrated and purified further by size-exclusion chromatography using a 16/600 Superdex S200 column (Cytiva) equilibrated with 20 mM HEPES, pH 7.4, 150 mM sodium chloride, and 1 mM TCEP.

CypA, RAS proteins, and mutants were expressed in E. coli and purified as before63. Briefly, 1–2 L cells in Dynamite media were grown at 37°C until an OD 600nm of 6–8 was reached before induction with 0.5 mM IPTG. Cells were grown for a further 18–20 h at 16°C. Cells were collected, lysed, and purified as described above for the SHOC2 and stabilized SKP complex, except for the RAS proteins. For the RAS proteins, the S200 equilibration buffer contained 5 mM magnesium chloride.

PP1CA was expressed and purified in E. coli as described above for RAS proteins, except the cells also harbored the GroEL-expression plasmid, pG-tf2 (Takara Bio USA), and expression was conducted at 10°C. The lysis buffer was 20 mM Tris-HCl, pH 8.0, 700 mM sodium chloride, 10% w/v glycerol, 1 mM manganese sulfate, 1 mM TCEP, and 0.5% w/v Triton X-100. Immobilized metal affinity chromatography was performed in this buffer without Triton X-100. PP1CA was purified in an identical way to SHOC2 or stabilized SKP, except a 5 mL MBPTrap HP column (Cytiva) was used to capture undigested fusion protein after TEV digestion, which failed to immobilize on the Ni Sepharose column. Furthermore, the S200 equilibration buffer was 20 mM Tris-HCl, pH 8.0, 500 mM sodium chloride, 1.0 mM manganese sulfate, and 1 mM TCEP.

Nucleotide exchange of RAS GTPases
The bound GDP nucleotide in all recombinant RAS proteins was exchanged with a non-hydrolysable GTP analog, GMPPNP. 200 μL of ~15 mg/ml RAS protein was diluted with 720 μL of 40 mM Tris, pH 8.0, 200 mM ammonium sulfate, and 0.1 mM zinc chloride. 20 μL of 200 mM GMPPNP (tetralithium salt, Jena Bioscience) and 60 μL of alkaline phosphatase-agarose beads (Sigma) were added, and the reaction was mixed for 3 hours at room temperature. The beads were removed by centrifugation (1500×g for 2 min) before the addition of 12 μL of 200 mM GMPPNP and 20 μL of 1 M magnesium chloride. The reaction was mixed overnight at 4°C. Excess nucleotide was removed through desalting (PD-10 desalting column, Cytiva) equilibrated with 20 mM HEPES, 150 mM sodium chloride, 5 mM magnesium chloride, 1 mM TCEP, pH 7.4. Nucleotide analysis was performed to confirm the exchange of GDP to GMPPNP.

Cryo-EM sample preparation and data acquisition
The purified SHOC2-KRAS-PP1CA complex was initially screened via negative-stain TEM on a FEI Tecnai T20 TEM, utilizing a uranyl formate stain to estimate concentration for blotting on graphene oxide before proceeding to Cryo-EM. For cryo-EM, 2.7 μl of 0.08 mg/mL of the SHOC2-KRAS-PP1CA complex was plunge frozen in liquid ethane cooled by liquid nitrogen onto Quantifoil R 2/2 200 mesh gold grids (Quantifoil Micro Tools GmbH) covered with a monolayer of graphene oxide (Graphene Supermarket, NY); these grids were produced in-house by following a published protocol64. Grids were briefly exposed to UV/Ozone for 10 min using a Helios 500 UV Ozone Cleaner (UVFAB, CA) immediately before blotting. Vitrification was performed using a Thermo Fisher Scientific Vitrobot Mark IV plunger with a chamber temperature of 4°C and a chamber humidity of 95%. Grids were then transferred to the FEI Titan Krios (Thermo Fisher Scientific) TEM microscope at the National Cryo-EM Facility (NCEF) at the Frederick National Laboratory for Cancer Research operating at 300 kV with a Gatan K3 direct electron detector (Gatan Inc., Pleasanton, CA). Dose-fractionated movie stacks of 40 frames were collected in counting mode using the Latitude software (Gatan Inc., Pleasanton, CA) at a nominal magnification of 105,000, a defocus range of −0.75 to −2.25 μm, and a pixel size of 0.873 Å. Image shift was utilized as an imaging strategy using 4 images per hole with one focus position, with an exposure time of 2.29 s and a total dose of 52.3 e−/Å2. One dataset was collected, resulting in a total of 6860 micrographs.

Cryo-EM data processing, model building, and analysis
Data processing was carried out in CryoSPARC65. A total of 6860 exposures were preprocessed with the Patch Motion Correction and Patch CTF Estimation jobs. A combination of manual picking and iterative rounds of Topaz training was performed using the wrapper included with CryoSPARC, and after particle inspection 518,550 particles were extracted using a box size of 256 66. These particles were subjected to iterative rounds of 2D classification to remove junk particles, and 199,681 particles were used to generate an initial model via the Ab-Initio Reconstruction Job. After 1 round of homogeneous refinement followed by non-uniform refinement, we were able to obtain a 3 Å reconstruction. Resolution was estimated using the gold-standard Fourier shell correlation (GSFSC) of 0.143.

The volume was sharpened with CryoSPARC B-factor-based sharpening as well as DeepEMhancer v0.13 using its wideTarget training model67. SHOC2 and PP1CA from PDB 7TVF and KRAS from PDB 5UFE were combined, and rigid-body fit into the sharpened map using Chimera 1.668. The combined model was then subjected to flexible fitting with IMODFIT 1.0369. Sections that did not have sufficient detail were removed, and the model was iteratively refined with Coot, Phenix, and ISOLDE 1.470–72. The local resolution map was calculated using CryoSPARC’s Local Resolution Estimation job. Data collection and refinement statistics for the structure are shown in Supplementary Table 2. For the analysis of the anisotropy of the cryo-EM map, the 3DFSC validation server (https://3dfsc.salk.edu/)73 was used to generate a histogram and directional FSC plot, and CryoSPARC was used to assess the orientation diagnostics.

Crystallization of RAS proteins with inhibitors
Crystallization screening was conducted at 20°C using the sitting-drop vapor diffusion method. RAS proteins were mixed with an equal volume of reservoir solution (200 nL:200 nL) using an ARI Crystal Phoenix Robot (Hudson Lab Automation).

KRAS (1-169) GDP + MRTX1133: 620 μL of 192 μM of KRAS was mixed with 20 μL of 7.42 mM MRTX1133 (final concentration of DMSO 3.1%) and incubated overnight at 4°C before concentration to a final volume of 160 μL (14.4 mg/ml). The complex was then screened. Crystals grew in 25.5% w/v PEG 4000, 170 mM ammonium sulfate, 15% v/v glycerol (condition C6 of the Wizard III/IV screen, Molecular Dimensions). Crystals were cryoprotected with 20% v/v glycerol. A 1.56 Å dataset was collected on beamline 24-ID-C at the Advanced Photon Source (Argonne).

KRAS (1-169) GMPPNP + MRTX1133: 500 μL of 515 μM of exchanged KRAS was mixed with 35 μL of 7.42 mM MRTX1133 (final concentration of DMSO 6.5%, 9.5 mg/ml) and incubated overnight at 4°C. The complex was then screened. Crystals grew in 2 M lithium sulfate, 50 mM sodium cacodylate, pH 6.0, 15 mM magnesium chloride, 5 mM spermidine (condition H6 of the Nucleix screen, Hampton Research). Crystals were cryoprotected with 2 M lithium sulfate. A 1.9 Å dataset was collected on beamline 24-ID-C at the Advanced Photon Source (Argonne).

MRASmut (1-178) GDP + MRTX1133: 1800 μL of 200 μM of MRASmut was mixed with 54 μL of 7.42 mM MRTX1133 (final concentration of DMSO 2.9%) and incubated overnight at 4°C before concentration to a final volume of 280 μL (26.3 mg/ml). The complex was then screened. Crystals grew in 20% w/v PEG 3350, 200 mM ammonium fluoride (condition A3 of the PEG/Ion screen, Hampton Research). Crystals were cryoprotected with 20% v/v glycerol. A 1.6 Å dataset was collected on beamline iO3 at Diamond.

MRASmut (1-178) GMPPNP + MRTX1133: 650 μL of 192 μM of exchanged MRASmut was mixed with 20 μL of 7.42 mM MRTX1133 (final concentration of DMSO 3.0%) and incubated overnight at 4°C before concentration to a final volume of 200 μL (12.0 mg/ml). The complex was then screened. Crystals grew in 1.6 M magnesium sulfate, 100 mM MES, pH 6.5 (condition E6 of the Top96 screen, Molecular Dimensions). Crystals were cryoprotected with 20% v/v glycerol. A 1.8 Å dataset was collected on beamline iO3 at Diamond.

Structure determination
All crystallographic data were indexed and integrated with XDS, except for MRASmut (1-179) GDP + MRTX1133, which was indexed and integrated with DIALS74,75. All data were scaled, truncated, and converted to structural factors using Aimless74,76. Molecular replacement was performed with MOLREP using KRAS (1-169) GDP-G12D bound to MRTX1133 (PDB 7RPZ) for KRAS datasets and MRAS (1-178) GDP (PDB 1X1R) for MRASmut datasets76. Molecular replacement was performed with MOLREP using KRAS (1-169) GDP-G12D bound to MRTX1133 (PDB 7RPZ) for KRAS datasets and MRAS (1–178) GDP (PDB 1X1R) for MRASmut datasets76. The models were rebuilt with Coot72. Refinement was initially carried out by Refmac5, followed by Phenix.Refine71,76,77. Interactions, buried surface areas, and contacts were analyzed using Protein interfaces, surfaces, and assemblies (PISA) and PDBSum server at the European Bioinformatics Institute35,36. Figures were generated with PyMOL. All crystallographic and structural analysis software was supported by the SBGrid Consortium78. Data collection and refinement statistics for the structures are shown in Supplementary Table 3.

Binding affinity measurements using isothermal titration calorimetry
Technical duplicate isothermal titration calorimetry measurements were performed on a MicroCal PEAQ-ITC instrument (Malvern Panalytical). MRTX1133 and RMC6236 (Chemgood) were dissolved in DMSO to a final concentration of 7.4 mM and 18 mM, respectively. Most experiments were conducted in 30 mM HEPES, 500 mM sodium chloride, 1 mM magnesium chloride, 0.5 mM TCEP, 0.1 mM manganese chloride, 5% v/v glycerol, pH 7.5 by dialysis of the proteins. The high salt and glycerol were required to keep PP1CA soluble in isolation and allow a direct comparison across datasets. Previous SPR measurements were made in 150 mM sodium chloride13.

RAS proteins binding to MRTX1133: MRTX1133 was placed in the cell (50 μM), and RAS protein was in the syringe (500 μM). DMSO was added to a final concentration of 5% v/v.

SMP and SKP formation: PP1CA and RAS proteins were placed in the cell (20–50 μM) with SHOC2 at a 10-fold higher concentration in the syringe (200–500 μM).

Inhibition of SMP and SKP formation by MRTX1133 or RMC6236-CypA: PP1CA, RAS protein, and either MRTX1133 or RMC6236-CypA were placed in the cell (40 μM) with SHOC2 at a 10-fold higher concentration in the syringe (400 μM). DMSO was added to a final concentration of 5% v/v.

The remaining experiments were conducted in 20 mM HEPES, 150 mM sodium chloride, 2 mM magnesium chloride, 1 mM TCEP, pH 7.5.

RMC6236 binding to CypA: RMC6236 was placed in the cell (45 μM), and CypA protein was in the syringe (450 μM). DMSO was added to a final concentration of 5% v/v.

RMC6236-CypA binding to RAS proteins: A 1:1 complex of RMC6236-CypA was placed in the cell (50 μM) and RAS protein in the syringe (500 μM). DMSO was added to a final concentration of 5% v/v.

RMC6236-CypA and MRTX1133 disassembly of SKP and SMP complexes: Prepared 1:1:1 of the SKP and SMP complexes from the individual proteins. These were dialyzed into the above buffer. SKP or SMP were placed in the cell (40–50 μM), and either RMC6236-CypA or MRXT1133 were placed in the syringe (400–500 μM). DMSO was added to a final concentration of 5% v/v.

All experiments were measured at 25°C, with 19 injections (1×0.4 μL and 18×2.2 μL) with 175 s spacings and a stirring speed of 750 rpm. Data were analyzed using the MicroCal PEAQ-ITC analysis software (v1.41, Malvern Panalytical) and a “one set of sites” model. Data were plotted with Prism 10. A representative run of each experiment is shown in the figures with the average KD reported for technical duplicate experiments. All ITC parameters (KD, ΔH, and -TΔS) have been tabulated as averages with their ranges. Furthermore, previous important measurements from the literature have been recorded (Supplementary Table 1).

Phosphatase assays
For peptide dephosphorylation assays, an 800 µl stock of 1 nM PP1CA, 1 µM SHOC2, and 1 µM RAS was prepared in 20 mM Tris, 150 mM sodium chloride, 1 mM magnesium chloride, 0.5 mM TCEP, 0.1 mM manganese chloride, pH 7.4, using stocks of 160 nM, 40 µM, and 40 µM, respectively, of each protein. The 40 µM RAS stock solution (MRAS, MRASmut or KRAS in the GMPPNP-bound state) contained 40 µM RAS protein with either 5% DMSO or 50 µM of either MRTX1133 or Compound (R)-5 in 5% DMSO. Compound (R)-5 was synthesized by Enamine. The SHOC2-RAS-PP1C complexes were incubated at room temperature for at least 15 min. In solution™ Microcystin-LR Microcystis aeruginosa (Sigma, 475821), an inhibitor of PP1CA, was diluted to 10 µM in water, and 1 µl was aliquoted to each well of a 96 Black Well Assay Plate (Corning 3603) to inhibit the reaction at the various time points. 10 mg of CRAF-CR2pS peptide (Ac-SQRQRSTpSTPNVHMV, Biomatik) was dissolved in 100 mM Tris, pH 8.0 to a final concentration of 8 mM. The SHOC2-RAS-PP1C complex solution was added to 10 µl of peptide, and 80 µl of this solution was taken and added to a Microcystin-LR coated well at various time points (0, 3, 6, 9, 12, 15, 30, 45, and 60 min). Assays were performed in triplicate at room temperature. Duplicate controls of buffer, water, 100 µM CRAF-CR2pS peptide, and Microcystin-LR were included. Free phosphate was measured using the Malachite Green assay kit from Sigma (MAK307) by the addition and mixing of 20 µl of working reagent to each well, incubation for 30 min followed by absorbance measurements at 620 nm on a FLUOstar Omega plate reader (BMG Labtech). Absorbances were converted to free phosphate using a standard curve generated from the phosphate standards. Dephosphorylation assays were also performed with the binary complex of SHOC2-PP1CA and MRAS-PP1CA, and PP1CA alone at the same concentrations as above. Graphs and fits were generated with Prism 10.

Generation of quadruple knockout HRAS/NRAS/KRAS/MRAS 293 cells
“RASless” (HRAS/NRAS/KRAS triple knockout) 293 cells were transfected with sgRNAs to knockout (KO) MRAS (sgMRAS-1: GGAGCAATACATGCGCACGG, sgMRAS-2: GTCATTCCCGATGATCCTCG), following the same procedure79. Single-cell clones were validated via Western Blotting and sequencing of the sgRNA target region. DNA from cell pellets was extracted using the Qiagen Blood and Cell DNA extraction kit according to manufacturer’s instructions. DNA was amplified using Pfu Ultra II polymerase and corresponding primers (Table 1).

Transfection and co-immunoprecipitation
Cells were transiently transfected with pDest302-EF1-3XFLAG-MRAS Q71L and pDest-Myc-SHOC2 plasmids using JetOPTIMUS transfection reagent according to the manufacturer’s protocol. 48 h later, cells were treated with 3 µM MRTX1133 or DMSO control for 4 h. Cells were washed with PBS and lysed in cold Lysis Buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 5 mM magnesium chloride, 1% Triton-X-100, 1 mM DTT, protease (Roche), and phosphatase inhibitors (Millipore-Sigma). After centrifuging at 27,000×g for 10 min at 4°C, the cleared supernatant was split between tubes containing either anti-FLAG agarose or protein A/G agarose and anti-CRAF antibody. An aliquot of lysate was saved with 4X NuPAGE LDS. After 1 h rotating at 4°C, tubes were centrifuged briefly to pellet beads. Supernatant was aspirated and beads were washed with cold wash buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 5 mM magnesium chloride, 1% Triton-X-100) 3 times. 1.5X NuPAGE LDS was added to drained beads, and samples were heated at 70°C for 10 min prior to SDS-PAGE. Gels were transferred to a nitrocellulose membrane, which was then blocked with 5% milk/TBS-T for 1 h. Primary antibodies (Table 2) were incubated with membranes overnight in 3% BSA/TBS-T. Membranes were washed 3×5 min with TBS-T and were incubated with anti-rabbit or anti-mouse secondary antibodies at room temperature for 1 h. Following three washes with TBS-T, membranes were scanned on a Li-COR Odyssey scanner.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Structure of SHOC2-KRAS-PP1C complex Reveals RAS isoform-Specific Determinants and Insights into Targeting complex Assembly by RAS Inhibitors\nThe SHOC2-MRAS-PP1C (SMP) complex is integral to RAF activation in the MAPK signaling pathway. It facilitates RAF dephosphorylation, promoting dimerization and RAF activation. While KRAS can form a ternary complex with SHOC2 and PP1C, its affinity is significantly lower than that of MRAS, making it difficult to obtain a complex structure. To address this, researchers introduced single-point mutations into each complex component to enhance the affinity of the SHOC2-KRAS-PP1C variant (SKP') complex, enabling successful cryo-EM reconstruction.\nTo explore therapeutic strategies targeting this complex, two RAS inhibitors were employed: MRTX1133, a KRAS G12D-specific inhibitor, and RMC-6236, a pan-RAS inhibitor. These inhibitors disrupted SKP' assembly without affecting SMP formation. Additionally, mutations in MRAS (MRASmut) were engineered to enhance its affinity for a KRAS G12D inhibitor. The resulting MRASmut complex assembly was impeded by these inhibitors, suggesting a potential approach to downregulate aberrant MAPK signaling upstream of RAF.\nResults from these studies indicate that targeting GTPase interactions with the SHOC2-PP1 complex could serve as a novel mechanism to inhibit MAPK pathway activation. However, the novelty of this approach is tempered by a recent Nature publication focusing on targeting NRAS Q61R-SHOC2 interactions using novel SHOC2 inhibitors that prevent native complex formation. Putting this study in context with the current findings could provide a more comprehensive understanding of targeting RASopathies and RAS-driven cancers. Further validation of these results will be needed to confirm the therapeutic potential of disrupting SHOC2-GTPase interactions in clinical settings.\nComments:\n1. The investigators should cite the published SHOC2/NRAS Q61R paper (Hauseman et al., Nature 2025), and put the published findings in context with the current study. Moreover, testing the recently identified SHOC2 inhibitor to examine inhibition of the native SKP, SKP’ and SMP complex would elevate the impact of the work.\n2. Crystallographic statistics are generally adequate, though the I/sigma I is borderline low in the high-resolution shells for KRAS(GMPPCP)MRTX1133 and MRASmut(GDP)MRTX1133.\n3. A key objective of this work is to understand how structural differences between SKP and SMP complexes give rise to differences in binding affinity. While the structural details associated with SKP’ is informative, a fairly rare KRAS Q61R mutant and SHOC2/PP1 with Noonen mutations is used. This non-native complex has a comparable binding affinity with SMP (150 nM vs 120 nM), so it is unclear how this structural comparison explains native differences between SKP and SMP.\n4. The earlier reported binding affinity of SMP is mentioned by the investigators to be 120 nM, while the calculated Kd by ITC in this paper (Fig. 1D left panel) is 900 nM. Some explanation is needed here.\n5. All ITC measurements are stated to be averages of 2 runs, yet error analyses and type of replicates (biological vs technical) are not provided for any of these measurements.\na. The reported ITC data lack error values, which are necessary for interpreting the presented Kd values.\nb. Given the large amount of ITC data, it would be helpful to tabulate these results and compare with earlier reported Kd values and associated references (relevant ternary complexes, WT KRAS, KRAS-G12D with MRTX1133, etc.)\nc. It is ambiguous in Figures 1D, 5C, 6B and 7D which component is being titrated and which components were premixed (cell). Clarification in the results section or Figure legends would be helpful.\nd. For ITC runs that are not productive, both “No assembly” and “No binding” are used to describe the results. Consistent terminology (binding for RAS inhibitor complexes vs assembly for the ternary complexes) would be helpful.\ne. Figure 1d – for the thermogram in the middle (SHOC2+KRAS+PP1CA). Has the heat of dilution has been subtracted in the Wiseman plot (plateau doesn’t seem to be a zero)?\nf. Figure 5a – The lack of data points in the sigmoidal region makes Kd determination questionable.\n6. In SI Fig 4a, the Kd results appear to contradict current literature. Why does MRTX1133 bind WT tighter than G12D and why does MRTX1133 prefer active-state G12D over inactive? In the main text (middle of p9 – it is stated that ITC determined sub-nM affinity for MRTX1133 and KRAS G12D) – please reference current literature for comparison and provide justification.\na. ITC for KRAS(GMPPCP)G12D in Figure 4a –The lack of data points in the sigmoidal region makes Kd determination questionable.\nb. The SKP' complex was assembled using the KRAS Q61R mutant. Was the binding affinity of this mutant for MRTX1133 assessed?\n7. Mutations in MRAS were introduced by making KRAS substitutions to enable MRTX1133 binding. While innovative, why was this approach not applied to RMC-6236?\n8. The author mentioned that 6 residues of the SWII binding pocket are different in MRAS and KRAS, with mutations made rendering MRTX1133 sensitive to MRAS binding. It would be helpful to describe whether the residues mutated are involved in additional interactions in the SKP/SMP complex and how they differ between SKP’ and SMP.\n9. The authors claim proof of concept for dual drug targeting. If key residues within MRAS are mutated to make it look like KRAS, then it's expected that the MRASmut would bind the KRAS inhibitor. A stronger proof of concept would be doing SAR on the KRAS inhibitor to make it a MRAS binder. While this is likely beyond the scope of this manuscript, a discussion would heighten impact of the work.\n10. Clarification is needed- bottom of p11. The investigators state that “Active KRASmut promotes formation of SHOC2-KRASmut-PP1C complexes…” all active KRAS mutants? A certain subset?\n11. Page 18, “Refinement was initially…by Phenix. Refine” this sentence is repeated.\n12. It is difficult to follow Figure 1e, as both surface and cartoon models are used. It would be helpful to either change the transparency or present the surface and cartoon model side by side.\n13. It is difficult to follow Figure 3e, as similar color schemes are used for both SMP and SKP’ complexes. The investigators should either change the color scheme or present a superposition of the individual proteins in the SMP and SKP’ complex.\n14. Figure 6a, 7b – the nucleotide state of KRAS and MRAS should be described.\n15. Throughout the text, the authors use SKP to refer to the SKP' variant. It would be helpful to clarify the nomenclature to clearly distinguish between native SKP and SKP'.\n16. In Figure 8d, the investigators provide an illustration to convey the concept that- ‘A pan-RAS inhibitor targeting both KRAS and MRAS would block RAF recruitment’. However, the figure does not clearly demonstrate that inhibiting MRAS and KRAS leads to the inhibition of RAF activation.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The manuscript, “Structure of SHOC2–KRAS–PP1C Complex Reveals RAS Isoform-Specific Determinants and Insights into Targeting Complex Assembly by RAS Inhibitors,” presents a 3.0 Å cryo-EM structure of the SHOC2–KRAS–PP1C (SKP) ternary complex, stabilized by pathogenic mutations associated with Noonan syndrome and cancer. The structure offers detailed modeling of SHOC2’s LRRs, KRAS, and PP1C, and reveals isoform-specific interactions that account for the lower stability of the SKP complex relative to the higher-affinity SHOC2–MRAS–PP1C (SMP) complex.\nThe authors also explore the therapeutic potential of targeting complex assembly with RAS inhibitors. They demonstrate that MRTX1133 and RMC-6236 disrupt SKP formation while sparing the SMP complex. To test whether SMP formation could also be inhibited, they engineered a mutant MRAS (MRASmut) capable of binding MRTX1133 and showed that formation of the SHOC2–MRASmut–PP1C (SMutP) complex was blocked. This suggests a potential strategy for dual inhibition—targeting both SKP and SMP complexes—if MRAS-selective RAS inhibitor analogs can be developed, offering an avenue for a possible oncogenic treatment.\nThe findings described in this manuscript are clearly presented and the structural analyses is well done. The fact that the SKP complex looks quite similar to the corresponding complex with MRAS (SMP) might raise the question of just how impactful is the advance here, although there are some notable differences that seem to be significant. Additional strengths of the work include the authors’ structure of the SKP complex using pathogenic mutations associated with Noonan syndrome and cancer offers a nice starting point to understand the conserved SHOC2-RAS interface that could be targeted for drug development. The differential impact of MRTX1133 and RMC-6236 on canonical versus MRAS-dependent complexes also offers potentially important implications toward combatting therapeutic resistance. Moreover, engineering an MRTX1133-sensitive MRAS (MRASmut) variant offers a proof-of-principle that possible analogs of RAS of pan-RAS inhibitors with selectivity for MRAS could target SMP complex formation and potentially lead an oncogenic treatment.\nHowever, a limitation of this study lies in the lack of physiological validation of the engineered MRASmut variant. While structural fidelity of the mutant-SMP complex is inferred from cryo-EM analysis between the SKP and SMP complexes, the functional competence of the mutant complex—particularly its ability to catalyze RAF CR2-pS dephosphorylation—was not explored. Given the centrality of this enzymatic function to SHOC2-RAS-PP1C signaling, biochemical assays and downstream signaling integrity, as demonstrated by proper de-phosphorylation activity within the mutant SMP complex, would represent important functional data to go along with the ability of MRTX1133 to bind the engineered MRASmut complex. Thus, the authors should address this issue by performing in vitro assays comparing the dephosphorylation of CR2-pS-containing RAF by wild-type versus mutant MRASmut complexes and Western blots from cell-based functional assays, showing phosphorylation readouts of RAF in MRAS-knockout cells reconstituted with either wild-type or MRASmut. These experiments would validate the mutant complex's functional equivalence to the wild-type SMP complex and support the biological significance of its observed inhibition by MRTX1133.\nMinor comments:\n1. A through proof-reading of the manuscript is needed. There are various errors, for example in the Title: “Structure of SHOC2–KRAS–PP1C complex Reveals RAS Isoform-Specific Determinants and Insights into Targeting Complex Assembly by RAS Inhibitors”;“complex” should be capitalized.\n2. In the “Results”, the sentence MRTX1133 successfully prevented SMmutP complex formation…” “SMmutP” was not mentioned in the text, is this a typo?\n3. In the “Discussion”, the sentence “Active KRASmut promotes formation of SHOC2-KRASmut-PP1C complexes, driving MAPK activation through dephosphorylatio” needs a citation.", "reviewer_5": "Bonsor et al. present the structure determination and analysis of the SHOC2-KRAS-PP1CA (SKP) complex, complementing earlier results using SHOC2-MRAS-PP1CA (SMP) complexes. To stabilise the SKP assembly, known disease mutations were used. It is assumed that the resulting structures are still informative for these complexes in general, rather than specifically only for complexes carrying disease mutations, which appears reasonable but is not directly demonstrated. Due to the involvement of these signalling complexes in cancer, these new findings are important and may enable discovery of next-generation therapeutics.\nThe proof-or-principle experiment on SKP/SMP complex disruption using inhibitors presented in the manuscript is interesting, though new compounds would have to be developed to target un-mutated MRAS. It is, however, understandable that this exceeds the scope of the current work.\nThe paper is well written and reports interesting new findings that advance our understanding of the assembly of RAS-signalling complexes and pave the way for the discovery of future cancer therapeutics. As such, the paper is a possible candidate for publication in Nature Communications. However, the comments below will need to be addressed in order for the paper to adhere to the technical standards established in the field. Some minor comments and questions listed below should also be addressed.\nCryo-EM validation: The authors should add the following items to the manuscript to support the accuracy of cryo-EM structure determination:\n- FSC curves between the model and the cryo-EM map (model vs. map FSC), evluated at FSC = 0.5\n- Analysis of the anisotropy of the cryo-EM map, e.g. using the 3D FSC validation server\n- Figures showing the cryo-EM map and the refined model for important sections of the structure or where atomic interactions have been deduced (e.g. Fig. 2c-e; Fig. 3d-i) to support the accuracy of the modelling.\nX-ray structures: The ligand densities are convincing, indicating that high-quality electron density maps have been obtained (as expected at the stated resolutions). However, Table 2 shows that the Rfree values are virtually identical across the X-ray structures, even though one might have expected the higher-resolution structures to exhibit lower Rfree values because the structures can be built more accurately. At the same time, the validation reports indicate somewhat high Ramachandran outlier statistics for some structures. Could the authors double check their full models to ensure that they are built as well as possible at the reported resolutions?\nMinor comments and questions:\nPage 4, bottom: Are the cellular concentrations of these proteins high enough to allow complex formation, given the affinities cited, or is it possible that H/K/NRAS only gain access to ternary complex formation in the presence of affinity-increasing mutations?\nPage 7, top: Is the number of salt bridges and hydrogen bonds taken directly from PISA output or has this been re-evaluated visually using the molecular model? If the latter, what criteria were applied?\nPage 18, near the bottom: Three sentences are present twice (referencing MOLREP, REFMAC refinement, and PISA).\nFig. 2a: Local resolution maps are typically coloured such that high resolution regions are blue and low-resolution regions are red (mirroring low/high b-factors from crystallography). The authors should also consider changing the colour palette used because the current spectrum (the default in Chimera) can cause difficulty for those with impaired colour vision (e.g. Crameri et al., Nat. Commun. 2020; https://www.nature.com/articles/s41467-020-19160-7).\nFig. 4: Switch I should be labelled in the figure panels (like Switch II)."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most concerns, as well as those raised by other reviewers. Substantial revisions have been made to the manuscript, including modifications to figures and text, with new data in response to reviewer requests. Major revisions include:\n1. Inclusion of Compound 5 in the study, demonstrates its effect on SHOC2–RAS–PP1C complex activity toward a CRAF substrate via dephosphorylation assays.\n2. Addition of the standard error of the mean values for all ITC data in the Table, along with the Kd values reported by others.\n3. Addition of “titrant → cell” labels in all ITC figures.\n4. Repetition of ITC experiments to obtain additional data points in the sigmoidal region.\n5. Improved visualization through updated surface and cartoon models.\n6. Execution of in vitro dephosphorylation assays (with or without MRTX1133) using SHOC2 and PP1CA with MRAS, MRASmut, or KRAS against a phosphopeptide of CRAF-CR2pS, with a new figure included.\n7. Addition of a new figure showing Western blot analysis in RAS-less cells transfected with GTP-locked MRAS(Q71L) or MRASmut(Q71L).\n8. Inclusion of FSC evaluation at 0.5 in the relevant figure.\n9. Rebuilding of MRASmut(GDP)+MRTX1133 structural regions to address Ramachandran outliers, with updated Ramachandran statistics included in the Table for all rebuilt structures.\nIn addition to these major updates, the authors have implemented several minor textual and figure improvements throughout the manuscript. However, one point mentioned in the rebuttal that remains unaddressed in the main text is that the authors have not clarified whether the ITC replicates are biological or technical. This information should be added to the figure legends. Moreover, the justification for the large discrepancy in Kd values (previously 120 nM vs. 900 nM by ITC) remains unclear. The explanation provided does not fully resolve this issue. Additionally, the authors’ statement that “all other instances refer to wild-type SKP” appears inconsistent, since the SKP complex structures reportedly include one mutation each in SHOC2, KRAS, and PP1C—thus, these do not represent wild-type SKP.\nAside from these minor concerns, the revised manuscript is significantly improved and otherwise ready for publication.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I have carefully reviewed the revised manuscript and the rebuttal provided by the authors. I feel that the authors have made a good response to my original critiques with my main issue being that they perform in vitro de-phosphorylation assays, which the authors have done.", "reviewer_5": "Bonsor, Finci, et al. have revised their manuscript according to the comments of several anonymous reviewers. While the manuscript has been improved, additional changes will be needed before it can be considered for publication.\nMain comments:\n1) The authors have generally addressed the reviewers' comments in a suitable manner in their response to reviewers, but in some cases, the changes to the manuscript itself are rather minimal. This applies particularly to some valid issues raised by reviewer #1, e.g. points 3, 4.\n2) Along the same lines: From the rebuttal: \"All ITC measurements reported are technical replicates, and the standard error of the mean has been calculated for each measurement. This information has now been explicitly stated in the Materials and Methods.\" - I could not find the terms \"technical replicate\" or \"standard error of the mean\" anywhere in the manuscript text (the latter appears in a supplementary table). Could the authors specify where this is stated in the methods (maybe line numbers would help)?\n3) The validation of the structure determined by cryo-EM requires further adjustments:\ni) The local resolution estimation in Fig. 2a is uninformative - almost everything is blue (3 Å), nothing is red (7 Å). The range of resolutions displayed within the color ramp should probably be 3 Å to 5 Å, not 3 Å to 7 Å.\nii) Supplementary Table 2: The model resolution determined by model vs. map FSC at FSC = 0.143 is meaningless. This value should be removed. The value at FSC = 0.5 should be retained, as this is the correct threshold.\niii) The use of DeepEMhancer and other advanced post-processing programs is not a problem as such, but use of DeepEMhancer maps for structure refinement and validation (e.g. phenix.real_space_refine and model vs. map FSC) is not generally an accepted practice in the field. Can the authors confirm that a conventionally post-processed map was used for determination of the model vs. map FSC? If not, this should be changed and the updated curve/cutoff value reported.\niv) In the interest of transparency, the authors may want to consider disclosing the use of DeepEMhancer in the figure legends where the maps treated with this algorithm are shown, rather than just in Supplementary Table 2 and the methods section.\nOther minor comments:\nThe paper on 3D FSC validation (Tan et al., Nature Methods, 2017) should be cited.\nSupplementary Fig. 10: Showing the compound in question (compound 5) would be helpful to the non-specialist reader."}, "author_response": "Point-by-point response to reviewer’s comments:\nWe sincerely appreciate the reviewers’ thorough and constructive feedback. In this revised\nmanuscript, we have addressed all concerns raised, resulting in an improved and more robust\nversion. Below, we provide point-by-point responses to the reviewers’ comments (highlighted\nin blue).\nReviewer #1:\nStructure of SHOC2-KRAS-PP1C complex Reveals RAS isoform-Specific Determinants and\nInsights into Targeting complex Assembly by RAS Inhibitors\nThe SHOC2-MRAS-PP1C (SMP) complex is integral to RAF activation in the MAPK signaling\npathway. It facilitates RAF dephosphorylation, promoting dimerization and RAF activation.\nWhile KRAS can form a ternary complex with SHOC2 and PP1C, its affinity is significantly\nlower than that of MRAS, making it difficult to obtain a complex structure. To address this,\nresearchers introduced single-point mutations into each complex component to enhance the\naffinity of the SHOC2-KRAS-PP1C variant (SKP') complex, enabling successful cryo-EM\nreconstruction.\nTo explore therapeutic strategies targeting this complex, two RAS inhibitors were\nemployed: MRTX1133, a KRAS G12D-specific inhibitor, and RMC-6236, a pan-RAS inhibitor.\nThese inhibitors disrupted SKP' assembly without affecting SMP formation. Additionally,\nmutations in MRAS (MRASmut) were engineered to enhance its affinity for a KRAS G12D\ninhibitor. The resulting MRASmut complex assembly was impeded by these inhibitors,\nsuggesting a potential approach to downregulate aberrant MAPK signaling upstream of RAF.\nResults from these studies indicate that targeting GTPase interactions with the SHOC2-\nPP1 complex could serve as a novel mechanism to inhibit MAPK pathway activation. However,\nthe novelty of this approach is tempered by a recent Nature publication focusing on targeting\nNRAS Q61R-SHOC2 interactions using novel SHOC2 inhibitors that prevent native\ncomplex formation. Putting this study in context with the current findings could provide a more\ncomprehensive understanding of targeting RASopathies and RAS-driven cancers. Further\nvalidation of these results will be needed to confirm the therapeutic potential of disrupting\nSHOC2-GTPase interactions in clinical settings.\nComments:\n1. The investigators should cite the published SHOC2/NRAS Q61R paper (Hauseman et al.,\nNature 2025) and put the published findings in context with the current study. Moreover, testing\nthe recently identified SHOC2 inhibitor to examine inhibition of the native SKP, SKP’ and SMP\ncomplex would elevate the impact of the work.\nThe publication by Hauseman et al. (Nature, 2025) on SHOC2/NRAS Q61R appeared in the\npublic domain during the review process. The structures of SHOC2 bound to a cyclic peptide\nand Compound 5 were released by the PDB approximately one month before their publication.\naffinity SHOC2 binder described in the manuscript is Compound 6. While Compound 6 can be\nsynthesized commercially, its lead time is more than 12 weeks, and thus, we proceeded with\ntesting Compound 5 to avoid significant experimental delays.\nIn the revised manuscript, we have now cited this work. Due to its limited solubility (as\nnoted in Hauseman et al.), we were unable to assess its effect on SHOC2-RAS-PP1C\nassembly using ITC. However, we demonstrate that Compound 5 affects the activity of SHOC2-\nRAS-PP1C complexes on a CRAF substrate via dephosphorylation assays. These data have\nnow been included (Supplementary Fig. 10), alongside the effects of MRTX1133 on the activity\nof SHOC2-RAS-PP1C complexes, as requested by reviewer #2.\n2. Crystallographic statistics are generally adequate, though the I/sigma I is borderline low in\nthe high-resolution shells for KRAS(GMPPCP)MRTX1133 and MRASmut(GDP)MRTX1133.\nWe thank the reviewer for this comment. The final resolution was selected based on both CC1/2\nand I/σI from the Aimless summary, rather than relying solely on I/σI.\n3. A key objective of this work is to understand how structural differences between SKP and\nSMP complexes give rise to differences in binding affinity. While the structural details\nassociated with SKP’ is informative, a fairly rare KRAS Q61R mutant and SHOC2/PP1 with\nNoonen mutations is used. This non-native complex has a comparable binding affinity with\nSMP (150 nM vs 120 nM), so it is unclear how this structural comparison explains native\ndifferences between SKP and SMP.\nThe stabilization of the SKP through three pathogenic mutations does result in an affinity similar\nto WT SMP. However, our structural analysis reveals several differences in the SKP’ interface\ncompared to SMP, including loss of hydrogen bonds and van der Waal interactions. As\ndescribed in the main text, only one of the mutations, PP1C-P50R, forms direct de novo\ncontacts at an interface, while the others do so indirectly but still locally e.g. SHOC2-M173I is\nfound at the interface, no direct contacts form with KRAS and the mutation does not affect distal\nresidues in SHOC2, demonstrating that the mutations stabilize the interaction locally, while the\nremainder of the interface remains largely unchanged from native SKP. Although the KRAS\nQ61R mutant is relatively rare, the structure of the SMP complex by Hauseman et al (Nature,\n2022) was determined using this stabilization mutation (as well as SHOC2-M173I) to increase\ncomplex affinity, which motivated its use in our study. Comparisons of the SMP complexes\nacross all four research groups show practically identical interfaces, except for local\nconformational changes at the mutation site, further highlighting that stabilization does not alter\nthe native interface.\n4. The earlier reported binding affinity of SMP is mentioned by the investigators to be 120 nM,\n2\nwhile the calculated Kd by ITC in this paper (Fig. 1D left panel) is 900 nM. Some explanation\nis needed here.\nThe reported Kd values were obtained using two different techniques—SPR in the earlier study\n(120 nM) and ITC in this work (900 nM). Differences in immobilization, mass transport, and\ndetection principles between SPR and ITC often lead to variations in absolute affinities.\nFurthermore, ITC required higher concentrations of salt to keep PP1C soluble at the\nconcentrations required; however, the relative rank order of affinities across complexes is\npreserved in both cases.\n5. All ITC measurements are stated to be averages of 2 runs, yet error analyses and type of\nreplicates (biological vs technical) are not provided for any of these measurements.\nAll ITC measurements reported are technical replicates, and the standard error of the mean\nhas been calculated for each measurement. This information has now been explicitly stated in\nthe Materials and Methods.\na. The reported ITC data lack error values, which are necessary for interpreting the presented\nKd values.\nThe standard error of the mean has been calculated, and the mean values are reported in the\ntext. A summary of all the ITC data, along with their associated errors, has now been included\nin Supplementary Table 1.\nb. Given the large amount of ITC data, it would be helpful to tabulate these results and compare\nwith earlier reported Kd values and associated references (relevant ternary complexes, WT\nKRAS, KRAS-G12D with MRTX1133, etc.)\nWe agree with the reviewer and have now tabulated all our ITC data (Supplementary Table 1),\nalongside previously published measurements of binary and ternary protein interactions, as\nwell as MRTX1133 and RMC6236 binding of KRAS and CypA proteins, respectively.\nc. It is ambiguous in Figures 1D, 5C, 6B and 7D which component is being titrated, and which\ncomponents were premixed (cell). Clarification in the results section or Figure legends would\nbe helpful.\nWe thank the reviewer for this helpful suggestion. All ITC traces now show the titrant being\ninjected into the cell as “titrant→cell” in their titles.\nd. For ITC runs that are not productive, both “No assembly” and “No binding” are used to\ndescribe the results. Consistent terminology (binding for RAS inhibitor complexes vs assembly\nfor the ternary complexes) would be helpful.\nWe have revised the figures to show “No binding” for RAS inhibitor complexes and “No\nassembly” for ternary complexes.\n3\ne. Figure 1d – for the thermogram in the middle (SHOC2+KRAS+PP1CA). Has the heat of\ndilution has been subtracted in the Wiseman plot (plateau doesn’t seem to be a zero)?\nThe reviewer is correct that the heat of dilution had not been subtracted; this has now been\ncorrected.\nf. Figure 5a – The lack of data points in the sigmoidal region makes Kd determination\nquestionable.\nWe agree with the reviewer. This data has been recollected with more injections of a smaller\nvolume to accurately measure the thermodynamics of binding.\n6. In SI Fig 4a, the Kd results appear to contradict current literature. Why does MRTX1133 bind\nWT tighter than G12D and why does MRTX1133 prefer active-state G12D over inactive? In the\nmain text (middle of p9 – it is stated that ITC determined sub-nM affinity for MRTX1133 and\nKRAS G12D) – please reference current literature for comparison and provide justification.\nWe appreciate the reviewer’s comment. As suggested, the ITC data have been recollected with\nmore injections of a smaller volume to accurately measure the thermodynamics of binding. As\nstated in the methods, the ITC data were collected in 500 mM sodium chloride and 5% glycerol\nto facilitate comparisons when MRTX1133 is used in conjunction with SKP and SMP\ncomplexes, as PP1C precipitates at the concentrations required in the ITC at low salt\nconcentrations. KRAS G12D binds MRTX1133 slightly weaker than WT under these\nconditions, most likely due to the higher concentration of salt interfering with the aspartic group\nforming hydrogen bonds to MRTX1133. With repeated and more accurate measurements, the\nGMPPNP-bound G12D mutant now binds slightly weaker than the GDP-bound G12D mutant.\nMinor discrepancies in K values with previously published values may also reflect the different\nD\nsensitivity ranges of biophysical methods, such as SPR and ITC, among others. We have now\ncited relevant literature for comparison and provided justification for the observed differences\nin the main text and Supplementary Table 1.\na. ITC for KRAS(GMPPCP)G12D in Figure 4a –The lack of data points in the sigmoidal region\nmakes Kd determination questionable.\nWe agree with the reviewer. This data has been recollected with more injections of a smaller\nvolume to accurately measure the thermodynamics of binding.\nb. The SKP' complex was assembled using the KRAS Q61R mutant. Was the binding affinity\nof this mutant for MRTX1133 assessed?\nWe have not measured MRTX1133 binding to KRAS Q61R by ITC, as the KRAS Q61R mutant\nwas not used in any experiments related to MRTX1133. Recently, in a separate study (PMID:\n4\n40473215), KRAS Q61R was assessed by SPR and found to bind MRTX1133 with reduced\naffinity compared to KRAS WT.\n7. Mutations in MRAS were introduced by making KRAS substitutions to enable MRTX1133\nbinding. While innovative, why was this approach not applied to RMC-6236?\nWe selected one of the two inhibitors for this proof-of-concept study. Considering that\nMRTX1133 is well characterized and a direct binder of RAS GTPase, we chose it for this study.\nFurthermore, MRTX1133 was easier to work with as we only needed to mutate the Switch-II\npocket. To conduct RMC6236 measurements we would need to mutate both Switch-I, to allow\nthe CypA-MRAS interface to form, and Switch-II to allow RMC6236 interaction to occur.\n8. The author mentioned that 6 residues of the SWII binding pocket are different in MRAS and\nKRAS, with mutations made rendering MRTX1133 sensitive to MRAS binding. It would be\nhelpful to describe whether the residues mutated are involved in additional interactions in the\nSKP/SMP complex and how they differ between SKP’ and SMP.\nOnly one residue, Y64 (KRAS) or F74 (MRAS), in the SWII pocket interacts with both\nMRTX1133 and SHOC2 in the SMP or SKP complexes. These interactions occur through van\nder Waals interactions and have been noted in the main text.\n9. The authors claim proof of concept for dual drug targeting. If key residues within MRAS are\nmutated to make it look like KRAS, then it's expected that the MRASmut would bind the KRAS\ninhibitor. A stronger proof of concept would be doing SAR on the KRAS inhibitor to make it a\nMRAS binder. While this is likely beyond the scope of this manuscript, a discussion would\nheighten impact of the work.\nWe would like to clarify that this point is addressed in the Results section, where we explain\nwhy MRAS was mutated:- “While ligand redesign could be explored to accommodate MRAS,\nwe pursued a simpler approach: introducing mutations into MRAS Switch-II pocket to restore\nMRTX1133 binding.” We also highlight in the Discussion that “rational design of RAS or pan-\nRAS inhibitors, such as MRTX1133 or RMC-6236 analogs, could yield compounds with\nselective or dual affinity for both KRAS and MRAS,” acknowledging the potential for future SAR\nstudies to extend this proof-of-concept.\n10. Clarification is needed- bottom of p11. The investigators state that “Active KRASmut\npromotes formation of SHOC2-KRASmut-PP1C complexes…” all active KRAS mutants? A\ncertain subset?\nWe thank the reviewer for this correction. We have rewritten this sentence and the next to state\ninto a single sentence: “Active KRASmut, such as Q61 or G13 mutations that exhibit a strong\ndependency on SHOC2 as seen in CRISPR dependency data, promotes formation of SHOC2-\nKRASmut-PP1C complexes, driving MAPK activation through dephosphorylation.”\n5\n11. Page 18, “Refinement was initially…by Phenix. Refine” this sentence is repeated.\nWe thank the reviewer for their careful reading of the material and methods. The duplicated\nsentence has been removed.\n12. It is difficult to follow Figure 1e, as both surface and cartoon models are used. It would be\nhelpful to either change the transparency or present the surface and cartoon model side by\nside.\nWe thank the reviewer for their suggestion and have increased the transparency of the map to\nallow better visualization of the cartoon model.\n13. It is difficult to follow Figure 3e, as similar color schemes are used for both SMP and SKP’\ncomplexes. The investigators should either change the color scheme or present a superposition\nof the individual proteins in the SMP and SKP’ complex.\nWe combined Figures 3d and 3e into a single, larger figure that clearly shows the differences\nin Switch engagement of SHOC2 in SMP and SKP complexes.\n14. Figure 6a, 7b – the nucleotide state of KRAS and MRAS should be described.\nThe nucleotide state of KRAS and MRAS is now described in the titles.\n15. Throughout the text, the authors use SKP to refer to the SKP’ variant. It would be helpful\nto clarify the nomenclature to clearly distinguish between native SKP and SKP'.\nWe would like to keep the nomenclature as is because SKP’ is only used in a single ITC\nexperiment to measure affinity and in our structural determination; all other instances refer to\nwild-type SKP. We note that previously reported SMP complex structures by four different\ngroups contained mutations in MRAS and truncations in SHOC2, yet were referred to as SMP\ncomplexes throughout. For consistency, we would prefer to retain the name SKP complex, and\nwe have clearly stated at the outset that SKP complex structures include one mutation each in\nSHOC2, KRAS, and PP1C.\n16. In Figure 8d, the investigators provide an illustration to convey the concept that- ‘A pan-\nRAS inhibitor targeting both KRAS and MRAS would block RAF recruitment’. However, the\nfigure does not clearly demonstrate that inhibiting MRAS and KRAS leads to the inhibition of\nRAF activation.\nWe thank the reviewer for their careful review of our figures. We agree that the figure did not\nexplicitly convey RAF recruitment, and we have now updated it to clearly indicate that inhibition\nof both KRAS and MRAS blocks RAF recruitment and activation.\nThe manuscript, “Structure of SHOC2–KRAS–PP1C Complex Reveals RAS Isoform-Specific\nDeterminants and Insights into Targeting Complex Assembly by RAS Inhibitors,” presents a\n3.0 Å cryo-EM structure of the SHOC2–KRAS–PP1C (SKP) ternary complex, stabilized by\npathogenic mutations associated with Noonan syndrome and cancer. The structure offers\ndetailed modeling of SHOC2’s LRRs, KRAS, and PP1C, and reveals isoform-specific\ninteractions that account for the lower stability of the SKP complex relative to the higher-affinity\nSHOC2–MRAS–PP1C (SMP) complex.\nThe authors also explore the therapeutic potential of targeting complex assembly with\nRAS inhibitors. They demonstrate that MRTX1133 and RMC-6236 disrupt SKP formation while\nsparing the SMP complex. To test whether SMP formation could also be inhibited, they\nengineered a mutant MRAS (MRASmut) capable of binding MRTX1133 and showed that\nformation of the SHOC2–MRASmut–PP1C (SMutP) complex was blocked. This suggests a\npotential strategy for dual inhibition—targeting both SKP and SMP complexes—if MRAS-\nselective RAS inhibitor analogs can be developed, offering an avenue for a possible oncogenic\ntreatment.\nThe findings described in this manuscript are clearly presented and the structural\nanalyses is well done. The fact that the SKP complex looks quite similar to the corresponding\ncomplex with MRAS (SMP) might raise the question of just how impactful is the advance here,\nalthough there are some notable differences that seem to be significant. Additional strengths\nof the work include the authors’ structure of the SKP complex using pathogenic mutations\nassociated with Noonan syndrome and cancer offers a nice starting point to understand the\nconserved SHOC2-RAS interface that could be targeted for drug development. The differential\nimpact of MRTX1133 and RMC-6236 on canonical versus MRAS-dependent complexes also\noffers potentially important implications toward combatting therapeutic resistance. Moreover,\nengineering an MRTX1133-sensitive MRAS (MRASmut) variant offers a proof-of-principle that\npossible analogs of RAS of pan-RAS inhibitors with selectivity for MRAS could target SMP\ncomplex formation and potentially lead an oncogenic treatment.\nHowever, a limitation of this study lies in the lack of physiological validation of the engineered\nMRASmut variant. While structural fidelity of the mutant-SMP complex is inferred from cryo-\nEM analysis between the SKP and SMP complexes, the functional competence of the mutant\ncomplex—particularly its ability to catalyze RAF CR2-pS dephosphorylation—was not\nexplored. Given the centrality of this enzymatic function to SHOC2-RAS-PP1C signaling,\nbiochemical assays and downstream signaling integrity, as demonstrated by proper de-\nphosphorylation activity within the mutant SMP complex, would represent important functional\ndata to go along with the ability of MRTX1133 to bind the engineered MRASmut complex. Thus,\nthe authors should address this issue by performing in vitro assays comparing the\ndephosphorylation of CR2-pS-containing RAF by wild-type versus mutant MRASmut\ncomplexes and Western blots from cell-based functional assays, showing phosphorylation\n7\nreadouts of RAF in MRAS-knockout cells reconstituted with either wild-type or MRASmut.\nThese experiments would validate the mutant complex's functional equivalence to the wild-type\nSMP complex and support the biological significance of its observed inhibition by MRTX1133.\nWe thank the reviewer for this suggestion. To address this, we performed in vitro\ndephosphorylation assays using SHOC2 and PP1CA with either MRAS, MRASmut or KRAS\nagainst a phosphopeptide of CRAF-CR2pS. All three RAS proteins (MRAS, MRASmut, and\nKRAS) synergistically stimulated PP1CA activity in the presence of SHOC2, whereas SHOC2\nalone only weakly stimulated activity, and RAS alone had no effect (Figure 7f).\nWe also performed these assays in the presence of MRTX1133. As expected,\nMRTX1133 had no effect on SMP activity because it does not bind MRAS (Figure 7g).\nConsistent with previous observations, MRTX1133 inhibits SKP activity and partially inhibits\nSHOC2-MRASmut-PP1C complex activity, likely reflecting the higher affinity of the SHOC2-\nMRASmut-PP1C complex compared to SKP (Figure 7h and 7i).\nWe also performed Western blots in RAS-less cells (lacking HRAS, KRAS, NRAS, and\nMRAS), transfected with MRAS or MRASmut, both carrying the Q71L mutation to maintain the\nGTP-bound state. MRTX1133 had no effect on RAF dephosphorylation in MRAS-expressing\ncells, whereas a partial reduction was observed in MRASmut cells (Supplementary Figure 9).\nMinor comments:\n1. A through proof-reading of the manuscript is needed. There are various errors, for example\nin the Title: “Structure of SHOC2–KRAS–PP1C complex Reveals RAS Isoform-Specific\nDeterminants and Insights into Targeting Complex Assembly by RAS Inhibitors”;“complex”\nshould be capitalized.\nWe thank the reviewer for their careful reading of our manuscript. We have capitalized\n“complex” in the title and have thoroughly reviewed the manuscript to correct any other errors.\n2. In the “Results”, the sentence MRTX1133 successfully prevented SMmutP complex\nformation…” “SMmutP” was not mentioned in the text, is this a typo?\nWe thank the reviewer for pointing this out. This was a typo, and “SMmutP” to “SHOC2-\nMRASmut-PP1C complex”.\n3. In the “Discussion”, the sentence “Active KRASmut promotes formation of SHOC2-\nKRASmut-PP1C complexes, driving MAPK activation through dephosphorylatio” needs a\ncitation.\nWe have added the Hauseman et al (Nature, 2025) as a citation to support this statement.\nBonsor et al. present the structure determination and analysis of the SHOC2-KRAS-PP1CA\n(SKP) complex, complementing earlier results using SHOC2-MRAS-PP1CA (SMP)\ncomplexes. To stabilise the SKP assembly, known disease mutations were used. It is assumed\nthat the resulting structures are still informative for these complexes in general, rather than\nspecifically only for complexes carrying disease mutations, which appears reasonable but is\nnot directly demonstrated. Due to the involvement of these signalling complexes in cancer,\nthese new findings are important and may enable discovery of next-generation therapeutics.\nThe proof-or-principle experiment on SKP/SMP complex disruption using inhibitors\npresented in the manuscript is interesting, though new compounds would have to be developed\nto target un-mutated MRAS. It is, however, understandable that this exceeds the scope of the\ncurrent work.\nThe paper is well written and reports interesting new findings that advance our\nunderstanding of the assembly of RAS-signalling complexes and pave the way for the\ndiscovery of future cancer therapeutics. As such, the paper is a possible candidate for\npublication in Nature Communications. However, the comments below will need to be\naddressed in order for the paper to adhere to the technical standards established in the field.\nSome minor comments and questions listed below should also be addressed.\nCryo-EM validation: The authors should add the following items to the manuscript to support\nthe accuracy of cryo-EM structure determination:\n- FSC curves between the model and the cryo-EM map (model vs. map FSC), evluated at FSC\n= 0.5.\nWe thank the reviewer for the suggestion. The FSC evaluated at 0.5 is now shown in\nSupplementary Figure 1.\n- Analysis of the anisotropy of the cryo-EM map, e.g. using the 3D FSC validation server.\nWe thank the reviewer for the suggestion. For the analysis of the anisotropy in the cryo-EM\nmap, we utilized the 3D FSC validation server to generate a histogram and directional FSC\nplot. We also utilized orientation diagnostics in cryoSPARC to examine the relative signal,\nvisualized in a 2D azimuth-elevation chart and a 3D scatter plot, as well as the relative signal\nwithin the twelve regions of the viewing sphere. Both plots are shown in Supplementary Figure\n1.\n- Figures showing the cryo-EM map and the refined model for important sections of the\nstructure or where atomic interactions have been deduced (e.g. Fig. 2c-e; Fig. 3d-i) to support\nthe accuracy of the modelling.\naround the mutation instead of the van der Waals. For Figure 3, we now show the map around\nthe key interfaces and residues as a separate new Supplementary Figure (Supplementary\nFigure 4).\nX-ray structures: The ligand densities are convincing, indicating that high-quality electron\ndensity maps have been obtained (as expected at the stated resolutions). However, Table 2\nshows that the Rfree values are virtually identical across the X-ray structures, even though one\nmight have expected the higher-resolution structures to exhibit lower Rfree values because the\nstructures can be built more accurately.\nWe carefully re-examined all four structures in response to the reviewer’s suggestion.\nRefinement strategies for KRASwt(GDP)+MRTX1133 and MRASmut(GDP)+MRTX1133 were\nchecked by PDB-REDO. For KRASwt(GDP)+MRTX1133 (1.40Å), we were using isotropic B-\nfactor refinement with TLS, however, Hamilton tests (a way to test the significance in the\nimprovement of R/Rfree from the inclusion of more parameters, in this case anisotropic vs\nisotropic B-factor refinement, PMID: 22505269) conclusively selected anisotropic B-factor\nrefinement. Re-refinement of the KRASwt(GDP)+MRTX1133 structure with anisotropic\nrefinement for protein and ligands and isotropic refinement for water has lowered R/Rfree to\n16.0/19.8. The Ramachandran outliers have been fixed, and this structure has been replaced\nin the PDB, and Supplementary Table 3 has been updated. For MRASmut(GDP)+MRTX1133\n(1.50Å), Hamilton tests conclusively selected isotropic B-factor refinement, but no improvement\nof R/Rfree was observed. The maps show no significant difference peaks, and all residues are\nmodeled with no additional waters required. However, the higher R/Rfree may be due to poor\nelectron density surrounding Switch-II of the second copy of MRASmut(GDP) in the\nasymmetric unit (chain B). We rebuilt regions of this structure (see our response to the next\ncomment) and also replaced it in the PDB.\nWe agree with the reviewer’s suggestion. If the data quality were nearly identical across\nthe three datasets, the R and Rfree values would be expected to vary according to resolution.\nHowever, this is not the case here. Despite using similar refinement protocols, the similar Rfree\nvalues across structures of differing resolution likely reflect a combination of factors, including\ndifferences in data quality (e.g., completeness, redundancy, and I/σ(I)) and other parameters,\nwhich can reduce variation in Rfree despite differences in nominal resolution.\nAt the same time, the validation reports indicate somewhat high Ramachandran outlier\nstatistics for some structures. Could the authors double check their full models to ensure that\nthey are built as well as possible at the reported resolutions?\nWe thank the reviewer for this suggestion. We have rebuilt regions of the\nMRASmut(GDP)+MRTX1133 structure due to the few Ramachandran outliers. This has\nimproved the Molprobity scores in the PDB report. The re-refined structure has been deposited\n10\nto the PDB, and Supplementary Table 3 has been updated. The outliers for the\nKRASwt(GDP)+MRTX1133 were fixed during the anisotropic B-factor refinement (described\nabove) and have also been redeposited. We have also added Ramachandran details to\nSupplementary Table 3 for all structures.\nPage 4, bottom: Are the cellular concentrations of these proteins high enough to allow complex\nformation, given the affinities cited, or is it possible that H/K/NRAS only gain access to ternary\ncomplex formation in the presence of affinity-increasing mutations?\nPhysiological total RAS levels are low-micromolar to sub-micromolar, and PP1C/SHOC2 are\npresent at moderate abundance (PMID: 11923843; BioNumbers ID 100889). Given the\nsubstantially higher affinity of MRAS for SHOC2 and PP1C, as well as the concentrating effect\nof membrane localization, MRAS can nucleate ternary assembly at endogenous expression\nlevels. It is possible that due to their lower affinity, wild-type H/K/NRAS are unable to form a\nstable ternary complex unless their local concentration, GTP-bound concentration, or affinity\nfor SHOC2 and PP1C is increased (PMID: 35831509; PMID: 36175670). Oncogenic mutations\nin KRAS/HRAS/NRAS can substantially increase the fraction of GTP-bound RAS and, together\nwith membrane localization, crowding, and clustering, can overcome the relatively weak\naffinities observed in vitro, making ternary assembly at the membrane more favorable than\npredicted from solution K s alone (PMID: 15860728; PMID: 31674905).\nD\nPage 7, top: Is the number of salt bridges and hydrogen bonds taken directly from PISA output\nor has this been re-evaluated visually using the molecular model? If the latter, what criteria\nwere applied?\nThe number of salt bridges and hydrogen bonds was taken directly from PDBSum\n(https://www.ebi.ac.uk/thornton-srv/software/PDBsum1/). We have modified the sentence to\n“…SKP complex buries approximately 1000 Å² less surface area and forms 6 fewer salt bridges\nand 12 fewer hydrogen bonds compared to the SMP complex (as calculated by the structural\nanalysis programs PISA and PDBSum),…” for clarification.\nPage 18, near the bottom: Three sentences are present twice (referencing MOLREP, REFMAC\nrefinement, and PISA).\nWe thank the reviewer for pointing out these duplicated sentences. They have now been\nremoved.\nFig. 2a: Local resolution maps are typically coloured such that high resolution regions are blue\nand low-resolution regions are red (mirroring low/high b-factors from crystallography). The\nauthors should also consider changing the colour palette used because the current spectrum\n(the default in Chimera) can cause difficulty for those with impaired colour vision (e.g.\nCrameri et al., Nat. Commun. 2020; https://www.nature.com/articles/s41467-020-19160-7).\nupdated with a color palette that is friendly for individuals with impaired color vision.\nFig. 4: Switch I should be labelled in the figure panels (like Switch II).\nWe have labelled Switch I in Figure 4 as the reviewer suggested.\nWe sincerely appreciate the reviewers’ thorough and constructive feedback. In this revised\nmanuscript, we have addressed all concerns raised, resulting in an improved and more robust version.\nBelow, we provide point-by-point responses to the reviewers’ comments (highlighted in blue).\nReviewer #1 (Remarks to the Author):\nThe authors have addressed most concerns, as well as those raised by other reviewers. Substantial\nrevisions have been made to the manuscript, including modifications to figures and text, with new data\nin response to reviewer requests. Major revisions include:\n1. Inclusion of Compound 5 in the study, demonstrates its effect on SHOC2–RAS–PP1C complex\nactivity toward a CRAF substrate via dephosphorylation assays.\n2. Addition of the standard error of the mean values for all ITC data in the Table, along with the Kd\nvalues reported by others.\n3. Addition of “titrant → cell” labels in all ITC figures.\n4. Repetition of ITC experiments to obtain additional data points in the sigmoidal region.\n5. Improved visualization through updated surface and cartoon models.\n6. Execution of in vitro dephosphorylation assays (with or without MRTX1133) using SHOC2 and\nPP1CA with MRAS, MRASmut, or KRAS against a phosphopeptide of CRAF-CR2pS, with a new figure\nincluded.\n7. Addition of a new figure showing Western blot analysis in RAS-less cells transfected with GTP-\nlocked MRAS(Q71L) or MRASmut(Q71L).\n8. Inclusion of FSC evaluation at 0.5 in the relevant figure.\n9. Rebuilding of MRASmut(GDP)+MRTX1133 structural regions to address Ramachandran outliers,\nwith updated Ramachandran statistics included in the Table for all rebuilt structures.\nIn addition to these major updates, the authors have implemented several minor textual and figure\nimprovements throughout the manuscript. However, one point mentioned in the rebuttal that remains\nunaddressed in the main text is that the authors have not clarified whether the ITC replicates are\nbiological or technical. This information should be added to the figure legends. Moreover, the\njustification for the large discrepancy in Kd values (previously 120 nM vs. 900 nM by ITC) remains\nunclear. The explanation provided does not fully resolve this issue. Additionally, the authors’ statement\nthat “all other instances refer to wild-type SKP” appears inconsistent, since the SKP complex structures\nreportedly include one mutation each in SHOC2, KRAS, and PP1C—thus, these do not represent\nwild-type SKP.\nWe apologize to both Reviewer #1 and Reviewer #5 for the oversight. We have now revised the\nMethods section to clearly state: “Technical duplicate isothermal titration calorimetry measurements\nwere performed on a MicroCal PEAQ-ITC instrument.” Additionally, “A representative run of each\nexperiment is shown in the figures with the average K reported for technical duplicate experiments.\nD\nAll ITC parameters (K , ΔH and -TΔS) have been tabulated as averages with their ranges.” All figure\nD\nlegends have been updated to indicate: “The K is calculated from two technical replicates.”\nD\nMoreover, the justification for the large discrepancy in Kd values (previously 120 nM vs. 900 nM by\nITC) remains unclear. The explanation provided does not fully resolve this issue.\nWe measured the K of SMP formation as 120 nM by SPR, using 150 mM NaCl and up to 1 µM PP1C\nD\nin the titration, a concentration at which PP1C remains stable in solution. For ITC, much higher PP1C\nconcentrations (~35–50 µM) are required; at these concentrations, PP1C aggregates and precipitates\nin 150 mM NaCl. To maintain solubility, we used 500 mM NaCl and 5% glycerol, conditions commonly\nused for PP1C in ITC (PMID: 37002212, 22284538, 27572260). Thus, the difference in K values\nD\narises from the higher ionic strength used in ITC measurements. Similarly, the SKP complex shows\nweakening from 700 nM by SPR (150 mM NaCl) to 7 µM by ITC (500 mM NaCl). We have added a\nsentence in the Methods to clarify: “Previous SPR measurements were made in 150 mM sodium\n1\nchloride.” Furthermore, we have included “SMP and SKP affinities measured by ITC, conducted in\n500 mM NaCl and 5% glycerol to maintain PP1C solubility, are higher (900 nM for SMP, 7 µM for SKP)\nthan those measured earlier by SPR at 150 mM NaCl (120 nM for SMP, 0.7 µM for SKP), reflecting\nthe effect of ionic strength rather than intrinsic affinity differences (see Methods for details).” in the\nmain text as well.\nAdditionally, the authors’ statement that “all other instances refer to wild-type SKP” appears\ninconsistent, since the SKP complex structures reportedly include one mutation each in SHOC2,\nKRAS, and PP1C—thus, these do not represent wild-type SKP.\nWe have revised the manuscript so that the SHOC2(M173I)-KRAS(Q61R)-PP1CA(P50R) complex is\nnow referred to as the “stabilized SKP,” while the wild-type SHOC2-KRAS-PP1C complex is referred\nto as SKP throughout the manuscript.\nWe have altered the final two sentences of the ‘Mechanistic insights into KRAS versus MRAS\nengagement with SHOC2 and PP1C’ section to “The comparative analysis suggests that the\ncumulative loss of buried surface area, salt bridges, hydrogen bonds, and van der Waals contacts in\nthe stabilized SKP complex is not due to the stabilization mutations but sequence differences between\nMRAS and KRAS. This underlies the reduced stability and lower affinity of the wild-type SKP complex\nrelative to the SMP complex.” to summarize these observations.\nAside from these minor concerns, the revised manuscript is significantly improved and otherwise ready\nfor publication.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nI have carefully reviewed the revised manuscript and the rebuttal provided by the authors. I feel that\nthe authors have made a good response to my original critiques with my main issue being that they\nperform in vitro de-phosphorylation assays, which the authors have done.\nReviewer #5 (Remarks to the Author):\nBonsor, Finci, et al. have revised their manuscript according to the comments of several anonymous\nreviewers. While the manuscript has been improved, additional changes will be needed before it can\nbe considered for publication.\n1) The authors have generally addressed the reviewers' comments in a suitable manner in their\nresponse to reviewers, but in some cases, the changes to the manuscript itself are rather minimal.\nThis applies particularly to some valid issues raised by reviewer #1, e.g. points 3, 4.\nFor Point #3, we have revised the manuscript so that the SHOC2(M173I)-KRAS(Q61R)-PP1CA(P50R)\ncomplex is now referred to as the “stabilized SKP,” while the wild-type SHOC2-KRAS-PP1C complex\nis referred to as SKP throughout the manuscript. We have altered the final two sentences of the\n‘Mechanistic insights into KRAS versus MRAS engagement with SHOC2 and PP1C’ section to “The\ncomparative analysis suggests that the cumulative loss of buried surface area, salt bridges, hydrogen\nbonds, and van der Waals contacts in the stabilized SKP complex is not due to the stabilization\nmutations but sequence differences between MRAS and KRAS. This underlies the reduced stability\nand lower affinity of the wild-type SKP complex relative to the SMP complex.” to summarize these\nobservations.\nFor Point #4, we measured the K of SMP formation as 120 nM by SPR, using 150 mM NaCl and up\nD\nto 1 µM PP1C in the titration, a concentration at which PP1C remains stable in solution. For ITC, much\nhigher PP1C concentrations (~35–50 µM) are required; at these concentrations, PP1C aggregates\nand precipitates in 150 mM NaCl. To maintain solubility, we used 500 mM NaCl and 5% glycerol,\nconditions commonly used for PP1C in ITC (PMID: 37002212, 22284538, 27572260). Thus, the\ndifference in K values arises from the higher ionic strength used in ITC measurements. Similarly, the\nD\nSKP complex shows weakening from 700 nM by SPR (150 mM NaCl) to 7 µM by ITC (500 mM NaCl).\nWe have added a sentence in the Methods to clarify: “Previous SPR measurements were made in\n150 mM sodium chloride.” Furthermore, we have included “SMP and SKP affinities measured by ITC,\nconducted in 500 mM NaCl and 5% glycerol to maintain PP1C solubility, are higher (900 nM for SMP,\n7 µM for SKP) than those measured earlier by SPR at 150 mM NaCl (120 nM for SMP, 0.7 µM for\nSKP), reflecting the effect of ionic strength rather than intrinsic affinity differences (see Methods for\ndetails).” in the main text as well.\n2) Along the same lines: From the rebuttal: \"All ITC measurements reported are technical replicates,\nand the standard error of the mean has been calculated for each measurement. This information has\nnow been explicitly stated in the Materials and Methods.\" - I could not find the terms \"technical\nreplicate\" or \"standard error of the mean\" anywhere in the manuscript text (the latter appears in a\nsupplementary table). Could the authors specify where this is stated in the methods (maybe line\nnumbers would help)?\nWe apologize to both Reviewer #1 and Reviewer #5 for the oversight. We have now revised the\nMethods section to clearly state: “Technical duplicate isothermal titration calorimetry measurements\nwere performed on a MicroCal PEAQ-ITC instrument.” Additionally, “A representative run of each\nexperiment is shown in the figures with the average K reported for technical duplicate experiments.\nD\nAll ITC parameters (K , ΔH and -TΔS) have been tabulated as averages with their ranges.” All figure\nD\nlegends have been updated to indicate: “The K is calculated from two technical replicates.”\nD\n3) The validation of the structure determined by cryo-EM requires further adjustments:\ni) The local resolution estimation in Fig. 2a is uninformative - almost everything is blue (3 Å), nothing\n3\nis red (7 Å). The range of resolutions displayed within the color ramp should probably be 3 Å to 5 Å,\nnot 3 Å to 7 Å.\nWe thank the reviewer for their comment. The revised local resolution color ramp in Fig. 2a now runs\nfrom 3 Å to 5 Å, resulting in a more informative figure.\nii) Supplementary Table 2: The model resolution determined by model vs. map FSC at FSC = 0.143\nis meaningless. This value should be removed. The value at FSC = 0.5 should be retained, as this is\nthe correct threshold.\nWe have removed the FSC=0.143 entry from Supplementary Table 2.\niii) The use of DeepEMhancer and other advanced post-processing programs is not a problem as\nsuch, but use of DeepEMhancer maps for structure refinement and validation (e.g.\nphenix.real_space_refine and model vs. map FSC) is not generally an accepted practice in the field.\nCan the authors confirm that a conventionally post-processed map was used for determination of the\nmodel vs. map FSC? If not, this should be changed and the updated curve/cutoff value reported.\nWe thank the reviewer for this suggestion. The volume was sharpened separately with both\nCryoSPARC B-factor-based sharpening and DeepEMhancer v0.13 using its wideTarget training\nmodel. We have now updated the model vs. map FSC with a B-Factor-sharpened map and have\nupdated the table to reflect this.\niv) In the interest of transparency, the authors may want to consider disclosing the use of\nDeepEMhancer in the figure legends where the maps treated with this algorithm are shown, rather\nthan just in Supplementary Table 2 and the methods section.\nWe thank the reviewer for this suggestion. In the interest of transparency, we have now indicated the\nuse of DeepEMhancer maps in the figure legends.\nOther minor comments:\nThe paper on 3D FSC validation (Tan et al., Nature Methods, 2017) should be cited.\nThe reference for the 3D FSC validation has now been cited.\nSupplementary Fig. 10: Showing the compound in question (compound 5) would be helpful to the non-\nspecialist reader.\nWe have added the skeletal structures of Compound5, MRTX1133, and RMC6236 to the relevant\nSupplementary figures.\n4"}]

Decision letter