Clustering and a conformational switch drive activation of the mammalian receptor tyrosine kinase ROS1

Domain: chemistry
Published: 2026-02-17
Editorial outcome: minor_revision

Abstract
Receptor tyrosine kinases (RTKs) are key regulators of cellular signaling and are often co-opted in cancer. ROS1 is an orphan RTK aberrantly expressed in multiple tumors, yet no approved biologic therapies target it, and its activation mechanism remains unknown. Here, we present Cryo-EM structures of mammalian ROS1 in ligand-free and NELL2-bound states, revealing how trimeric NELL2 induces both receptor clustering and a conformational switch that relieves receptor autoinhibition – both mechanisms are required for ROS1 activation. These structures, along with biochemical characterization, reflect a striking evolutionary divergence in regulatory logic compared to the invertebrate ortholog Sevenless (dROS1S), highlighting how conserved RTKs can adopt fundamentally different activation strategies. Guided by these structural insights, we develop monoclonal antibodies that either block ligand binding or trap ROS1 in an inactive conformation. These agents potently suppress ROS1 signaling, representing distinct mechanistic classes of biologics that directly target ROS1 activity. Our findings elucidate a distinct mode of RTK regulation and establish a therapeutic framework for cancers driven by ROS1.

Introduction
Receptor tyrosine kinases (RTKs) are cell-surface receptors that survey the extracellular environment for cues to properly coordinate cellular signaling and orchestrate development1. Dysregulated RTK signaling is associated with various cancers and represents an important therapeutic target in oncology1,2. ROS1 (Rochester Sarcoma) was first discovered as part of a viral avian oncogene isolated from a spontaneous tumor in a Connecticut white rock chicken3,4. The Drosophila homolog of ROS1, Sevenless, is regulated through trans-cellular interactions with the G protein-coupled receptor (GPCR) BOSS5,6. Recent structural studies of Sevenless’ extracellular region revealed an N-terminal “bent-over” conformation and showed that receptor activation is promoted under acidic conditions7,8. However, mammals lack a BOSS homologue and its mechanism of regulation remains uncharacterized. Structurally, ROS1 distinguishes itself among human RTKs as having the largest extracellular region (ECR) – composed of nine Fibronectin type III (FNIII) domains and three β -propeller (YWTD) domains 9,10 (Fig. 1a). This domain configuration is unique among RTKs, suggesting a distinct mechanism of action and regulation1.

However, the absence of detailed structural information has left a significant gap in our understanding of ROS1’s activation and regulatory process9.

Under normal physiological conditions, ROS1 is essential for development of the epididymis11,12. In ROS1 knock-out mice, the epididymis is poorly differentiated, resulting in infertility due to impaired sperm maturation12. In cancer, ROS1 is often associated with gene fusion-driven (ligand independent) activation13, as seen in approximately 1% of glioblastomas14,15 and < 2% of non-small-cell lung cancers16-18. However, full-length (non-fusion) ROS1 has been reported to be expressed in approximately 30% of primary brain tumors19,20, 30% of non-small cell lung cancer (NSCLC)21, and 80-100% of oral squamous cell carcinomas22. Additionally, full-length ROS1 is upregulated in E-cadherin-deficient breast cancers, where inhibition of ROS1 with tyrosine kinase inhibitors leads to synthetic lethality23. Consequently, targeting the extracellular region of ROS1 presents a promising strategy for controlling multiple types of cancer. Despite this potential, the absence of experimental evidence regarding the structure and regulation of ROS1’s extracellular region (ECR) has hindered the development of therapeutic biologics.

Recently, NELL2 (Neuronal EIGF-Like-Like 2) has been identified as a possible agonist for mammalian ROS124. NELL2 is a multimeric, multidomain secreted glycoprotein composed of an N-terminal laminin G domain, a coiled-coil domain, five von Willebrand factor type C (VWC) domains, and six epidermal growth factor (EGF) repeats25,26 (Fig. 1a). Previous studies have shown that NELL2 plays a role in axon guidance by interacting with ROBO327,28. However, the mechanism by which NELL2 regulates ROS1 activation remains unclear. In this study, we demonstrate that NELL2 binds directly to ROS1 with high affinity and enhances the receptor’s intracellular kinase activity. Using cryo-electron microscopy (Cryo-EM), we determine the structures of ROS1's extracellular region (ECR) both in the absence and presence of NELL2, revealing significant ligand-induced conformational changes essential for ROS1 activation. The activation of ROS1 integrates two distinct regulatory mechanisms where NELL2’s oligomerization and its induction of a ROS1 conformational change are required for activation. Furthermore, we identify two inhibitory antibodies that target ROS1’s critical regulatory mechanisms. Our findings provide a molecular basis for the regulation of ROS1 activity, highlight the pivotal role of NELL2 in inducing these structural transitions, and open avenues for therapeutic interventions targeting ROS1-expressing cancers.

Results
NELL2 is a direct agonist for ROS1

Previous studies have established that the lumicrine factor NELL2 plays a role in modulating ROS1 related cell differentiation within the mouse epididymis24. Our research aimed to further delineate this pathway by investigating whether NELL2 could directly interact with and influence ROS1 activation. We engineered expression constructs for the entire extracellular region (ECR) of murine ROS1 and full-length human NELL2 (Fig. 1a, Supplementary Fig. 1a-1d). Through biolayer interferometry (BLI) analysis, we demonstrate that NELL2 binds directly to the N-terminal ECR of ROS1 with high-affinity, yielding an equilibrium dissociation constant (K ) of 116 nM (Fig. 1b). This affinity is similar to other soluble growth factors binding to their receptors (e.g. the K of EGF binding to EGFR is 175 nM) 29.

Having established direct binding, we sought to determine if NELL2 alone could stimulate ROS1 phosphorylation in cells. A significant challenge for studying ROS1 activity in cells is that no cell lines are known to endogenously express full length ROS1. Moreover, repeated attempts to generate stable ROS1-expressing heterologous cell lines by transfection have failed to yield detectable protein.9. To overcome this challenge, we devised a chimeric construct combining the ECR of ROS1 with the kinase domain of ALK, resulting in a NIH/3T3 stable cell line that expresses ROS1’s ECR with ALK’s kinase domain (designated ROS1 ). This design allowed the use of well-established phospho-ALK antibodies for detection and is particularly relevant since ROS1 and ALK’s kinase domains are phylogenetically closest to each other9,13,30 (Supplementary Fig. 2a-2c). Utilizing this ROS1 stable cell line, we demonstrate that NELL2 triggers robust receptor and ERK phosphorylation (Fig. 1c) in a dose- and time-dependent manner (Supplementary Fig. 2d), a response not observed in the control ALK expressing cell line (Supplementary Fig. 2c).

RTK signaling is typically stimulated by clustering receptors along with their intracellular tyrosine kinase domains. Consequently, oligomeric ligands commonly mediate activation of different RTKs1,31. Therefore, we investigated the role of NELL2’s oligomerization on ROS1 activity by generating a deletion mutant that removes NELL2’s coiled-coil domain (NELL2 ). NELL2 expressed well and purified as a monomer (Supplementary Fig. 3a).NELL2 was able to bind with the same affinity as wild type NELL2 to ROS1 as determined by BLI (Fig. 1c). However, NELL2 was unable to stimulate ROS1 and ERK phosphorylation in ROS1 stable cells (Fig. 1d). Taken together, these findings establish NELL2 as a direct agonist for ROS1 and show that ligand oligomerization is necessary for inducing ROS1 activation.

To further define the stoichiometry of the NELL2-ROS1 receptor interaction, we performed size-exclusion chromatography coupled with multi-angle light scattering (SEC–MALS) (Supplementary Fig. 3b). NELL2 alone eluted at it s expected trimeric mass (276.9 kDa). Titrating increasing amounts of ROS1 into a fixed concentration of trimeric NELL2 revealed robust formation of 1:1 (trimeric NELL2:ROSE1) and 1:2 complexes. Further increasing ROS1 to a 1:3 ratio produced a species of ~711.8 kDa, smaller than the predicted mass of a fully saturated NELL2-ROS1 complex, consistent with incomplete occupancy of NELL2’s third binding site. Together, these data indicate that a single NELL2 trimer can engage multiple ROS1 receptors, thereby promoting ROS1 clustering.

Structure of mammalian ROS1

To determine the structure of ROS1’s ECR, we expressed and purified the full extracellular domain of murine ROS1 (ROS1 , residues 28-1846) and used Cryo-EM for structure determination (Supplementary Fig. 4a-4e). The density map allowed us to model ROS1 from the N-terminal CATCH domain to the eighth FNIII domain (FNIII-8). Only the final juxtamembrane FNIII-9 domain could not be modeled confidently. The Cryo-EM structure yields a largely rigid and elongated ROS1 with dimensions of 294 Å × 80 Å (Fig. 1e-1f).

The N-terminal half of ROS1 adopts a bent-over configuration, in which the CATCH domain and first two FNIII domains fold over onto the second YWTD (YWTD-B) and the fourth FNIII domain (Fig. 1f). This bent-over state resembles our prior structure of Sevenless8. A series of interactions stabilizes this bent over state and involves two main contact points (denoted as interfaces 1 and 2) (Supplementary Fig. 5a). Interface 1 involves the CATCH domain and first FNIII domain making contacts with the second β-propeller (Supplementary Fig. 5b-5c). At interface 2, a long extended loop from the third FNIII domain (residues 592-601) inserts into a hydrophobic groove formed by the YWTD-A and second FNIII domains (Supplementary Fig. 5d).

The C-terminal half of ROS1’s ECR, distal to FNIII-4, transitions into a continuous, extended structure that includes a series of additional FNIII domains and the third YWTD domain (YWTD-C) (Fig. 1f). This rigid organization stands in contrast to prior structures of ROS1’s Drosophila homolog, Sevenless, in which only the N-terminal bent-over portion was resolved – while the remainder of the ectodomain appeared disordered and could not be modeled7,8. Unlike the invertebrate structure, the mammalian ROS1 ectodomain reveals a rigidly projected C-terminal "leg" extending from the N-terminal region.

Structure of ligand bound ROS1

To understand the activation mechanism of ROS1 by NELL2, we collected Cryo-EM micrographs of full length trimeric NELL2 with the entire ECR of ROS1 and determined their complex structure. The Cryo-EM analysis revealed a highly heterogeneous particle population (Supplementary Fig. 6a-6g). The final refined map, capturing nearly the entire ROS1 ECR is shown in Figure 1g. The overall dimentions of the complex (330 Å × 100 Å) are larger than the unliganded state.

The model reveals an interaction between ROS1 and NELL2 with a 1:1 stoichiometry (Fig. 1g). Ligand binds at the N-terminus of ROS1 with the first two FNIII domains and the first β -propeller (YWTD-A) (Fig. 1h). Notably, this site is distinct from the invertebrate ligand binding site which is isolated to the third FNIII domain of dROS1. In the complex, the first 2 VWC domains, and first 2 EGF domains of NELL2 are resolved. These four domains form a loop structure with the first VWC domain (VWC-1) interacting with the first EGF domain (EGF-1) to stabilize the quaternary loop structure (Fig. 1h, Supplementary Fig. 3c). The second VWC domain (VWC-2) largely mediates the turn of the loop. Furthermore, VWC-2 sits atop the first beta-propeller (YWTD-A) of ROS1 (Fig. 1h). ROS1’s first and second FNIII domains additionally support the interaction with NELL2’s EGF domains (Fig. 1h). Despite using full length NELL2 constructs, only the domains involved in complex formation are resolved in the reconstruction (Fig. 1a, 1g). This likely indicates that the oligomer (coiled-coil) of NELL2 is not rigidly connected to the ROS1 binding domains, yielding only a single NELL2 protomer interacting with a single ROS1 after averaging.

Notably, compared to the unliganded ROS1 structure, ligand bound ROS1 reveals a substantial repositioning of its three N-terminal domains: the CATCH domain and the first two FNIII domains. Upon ligand engagement, the N-terminus transitions from a bent-over (Fig. 1i) to an extended, upright conformation (Fig. 1j). Specifically, these domains rotate as a rigid body by approximately 130 degrees relative to the fixed YWTD-A domain (Fig. 1j). This ligand-induced conformational switch is unique to mammalian ROS1 and is not observed upon BOSS binding to Sevenless.

NELL2 recognition by ROS1 involves a multi-domain interaction

To improve resolution and focus on the ligand-binding interface, we designed a truncated ROS1 construct ending at FNIII-4, encompassing the N-terminal domains required for NELL2 recognition. The resulting structure of the ROS1[N-term]-NELL2 complex was determined at higher resolution (3.21 Å, Supplementary Fig. 7), enabling detailed visualization of the binding interface. The NELL2 and ROS1 complex structure involves three distinct sites of interaction (Fig. 2a). Site 1 involves the interaction between the second VWC domain (VWC-2) of NELL2 and ROS1’s YWTD-A β-propeller. This is a largely hydrophobic interaction mediated by surface exposed hydrophobic residues on ROS1’s YWTD-A (V320, I539, L544 and I547) interacting with NELL2’s VWC-2 surface hydrophobic residues (V349, V355, V357, Y359 and L368) (Fig. 2b). Site 2 involves an interaction of NELL2’s first EGF domain (EGF-1) binding to ROS1’s second FNIII domain (FNIII-2) (Fig. 2c). The prominent beta-hairpin of NELL2’s EGF domain interacts with the surface of the FNIII-2. Side chains R414, D418 and R419 make hydrogen bonds to the mainchain of FNIII-2. Site 3 involves an interaction between NELL2’s second EGF domain (EGF-2) with ROS1’s first FNIII domain (FNIII-1) (Fig. 2d). Unlike site 2, this interface involves the N- and C-termini of NELL2’s EGF-2 and not the beta-hairpin.

Mutation of the interacting residues from either NELL2 (Fig. 2e) or ROS1 (Fig. 2f) weaken the affinity of binding as measured by BLI. Individual mutations of Site 1 residues (NELL2: V357R, Y359R; ROS1: V320E) show the most significant impairment of binding, and concurrently no longer show a response in cell signaling assays (Fig. 2e-2h). Individual mutations of Site 2 or 3 (NELL2: L416E, Y450A, D476R; ROS1: L236E) retain some binding affinity (Fig. 2e-2f), yet also demonstrate abrogated signaling (Fig. 2g-2h). Together, the structural and mutational studies point to a multi-domain interaction where binding at each site is necessary for controlling ROS1 signaling.

Decoupling the Juxtamembrane leg from the ligand binding region

In the unliganded state, ROS1’s extracellular region adopts a bent-over conformation held together by multiple contact points between the N-terminal CATCH domain, several FNIII domains, and the second β-propeller (YWTD-B) (Fig. 1e-1f,3a). Ligand binding induces a roughly 130-degree upward rotation of this ‘arm’ region about the first β-propeller ‘shoulder’ (Fig. 3a-3b). While Figure 1 shows a ligand-bound structure that appears mostly intact, and with the same leg orientation as the unliganded, this represents only about 20% of the particles (Fig. 3b). In fact, a significant fraction of particles – a second 20% – show the ligand bound N-terminal domains as a separate entity, with no visible connection to the rest of the receptor (Fig. 3c). Another 20% of particles capture the complementary portion, the C-terminal "leg" region, as an independent structure (FNIII-3 down through the remaining domains) (Fig. 3d). Finally, a third class of particles includes the N-terminal region plus a partial portion of the central domains, but not the full receptor (Fig. 3e). The N-terminal ligand bound structures ending at first β-propeller or FNIII-4 represent the release of these ‘bent-over’ restraints and subsequent decoupling the N-terminal ligand-binding portion from the rest of the receptor.

Together, these observations are consistent with a ligand-induced conformational switch resulting in a highly dynamic and modular architecture, where the previously constrained domains become physically separated and capable of independent movement. This dynamic structural landscape underscores that the fully intact conformation (Fig. 1g) is just one snapshot, and the receptor primarily exists as more flexible, and decoupled upon ligand binding.

Modeling of Full-Length Trimeric NELL2 and Its Interaction with ROS1

To visualize how full-length NELL2 engages ROS1, we generated a structural model of trimeric human NELL2 using AlphaFold3. The predicted architecture demonstrates high model confidence across the majority of the fold (light blue; pLDDT > 70), with lower-confidence regions primarily restricted to flexible loops (yellow) (Supplementary Fig. 8a-8b). Across five independently generated AlphaFold3 predictions, all models converged on the same overall trimeric arrangement with a central coiled-coil spine, indicating that the global organization of the trimer is robustly defined. In contrast, the three C-terminal VWC domains within the head region did not converge to a single orientation across models, suggesting that the packing interactions of these terminal domains are not precisely predicted. The overall model reveals a compact “head” region formed by the N-terminal laminin-G domain together with the C-terminal EGF (5-6) and VWC (3-4) domains (Supplementary Fig. 8b, 8d). Together these domains assemble into an extensive three-way interface that appears to stabilize trimer formation. Extending from this head is a central, elongated coiled-coil (CC) trimer, reinforced by inter-protomer disulfide bonds at both its N-terminal and C-terminal ends, consistent with a covalently stapled, rigid trimeric spine (Supplementary Fig. 8e-8f). Surrounding this central rod are EGF domains 3 and 4, which arc back toward the head region in the predicted structure (Supplementary Fig. 8c) . Notably, the ROS1-interacting modules (VWC 1-2 and EGF domains 1-2) reside at the end opposite the head, corresponding to the “bottom” in our structural representations. Because our cryo-EM reconstruction resolves only these ROS1-binding domains, we oriented a full ROS1 ECD onto each of the NELL2 binding sites. This composite model supports a structural organization in which the NELL2 head region nucleates symmetric trimerization, the coiled-coil acts as a rigid spacer, and the ROS1-binding domains are displayed at the distal tips of each protomer, poised for simultaneous or sequential engagement of ROS1 ectodomains.

Model of ROS1 Activation

Our finding that mutations that disrupt NELL2 binding to the FNIII domains (site 2) substantially impair signaling (Fig. 2e-2h), indicate that receptor clustering alone is insufficient and that the conformational switch is required for activation. To investigate this requirement, we modeled an encounter complex (site 1 only) in which ROS1 adopts its rigid bent-over conformation and is docked onto all three binding sites of an AlphaFold-modeled NELL2 trimer. In this configuration, the extracellular juxtamembrane regions remain widely spaced, suggesting that ligand-mediated clustering of bent-over ROS1 does not position the kinase domains close enough for productive signaling (Fig. 3f). This observation led us to hypothesize that the bent-over conformation corresponds to an auto-inhibited state. In this model, the N-terminal “arm” region – composed of the CATCH domain and the first two FNIII domains – engages the long distal leg region. This stabilizes both the leg and arm regions together into a relatively rigid structure. Subsequent binding of NELL2 to ROS1’s arm, FNIII domains (site 2), breaks these interactions. Without the restraints between the arm and leg, the leg region becomes decoupled from the ligand binding domains. While we do not observe a defined new conformation for the released leg, its increased mobility may allow the transmembrane helices to reposition into closer proximity, facilitating activation of the intracellular kinase domains.

A NELL2 epitope blocking antibody inhibits ROS1 signaling

Despite ROS1’s relevance to multiple human cancers, no biologics have been developed that target its large ECR. Indeed, there are no commercially available antibodies that recognize ROS1’s ECR. Therefore, we used phage display to pan for Fabs that bind to ROS1. From this screen we identified a distinct Fab that binds with high affinity (K 36.8 nM): Fab-RX5 (Supplementary Fig. 10a). We determined the cryo-EM structure of Fab-RX5 in complex with N-terminal ROS1 to a resolution of 3.06 Å with 166,992 particles for final map reconstruction (Fig. 4a-4b, Supplementary Fig. 9 and Table S1).

The structure shows that Fab-RX5 binds to ROS1’s YWTD-A domain at an epitope that overlaps with that of NELL2 at Site 1 (Fig. 4c-4d). Both Fab-RX5 and NELL2 interact with the 6th blade of ROS1’s YWTD-A domain. The Fab-RX5 interaction is largely mediated by the hydrophobic residues WGYY of CDR-H3 (complementary determining region, heavy chain hypervariable loop 3). These residues engage with surface hydrophobics on YWTD-A: L506, V507, F528, F535 and F550 (Fig. 4e). While not interacting, the variable light chains of RX5 sit atop ROS1’s YWTD-A in a position that covers the NELL2 binding epitope (Fig. 4f). As they share a similar binding footprint, we predict that binding of Fab-RX5 to ROS1 would sterically block NELL2 binding to site 1.

To determine how Fab-RX5 impacts NELL2 binding, we used surface plasmon resonance (SPR, Biacore) to monitor ROS1’s ability to bind ligand in the presence of Fab. In the absence of Fab-RX5, ROS1 binds readily to a surface covalently coupled with NELL2 (Fig. 4g). However, when ROS1 is preincubated with an excess of Fab-RX5 (1:10), ROS1 is prevented from binding to the NELL2 coated surface (Fig. 4g). Thus, RX5 binding to Site 1 on ROS1 is sufficient to block the receptor’s ability to engage ligand – similar to the impact of Site 1 mutations.

To further test if Fab-RX5 can inhibit ROS1 cellular signaling, we pre-incubated ROS1 cells with Fab-RX5 prior to stimulation by NELL 2. In this context, Fab-RX5 was able to inhibit ROS1 signaling by NELL2 in a concentration dependent manner (Fig. 4h) Additionally, a full antibody based on Fab-RX5 (IgG-RX5) robustly inhibits ROS1 signaling at low nM concentrations (Fig. 4i). We next asked whether the bivalent IgG-RX5 is capable of clustering ROS1 receptors using SEC–MALS. Addition of ROS1 at a concentration sufficient to fully saturate IgG-RX5 yielded a predominant 1:2 (IgG-RX5:ROS1) complex, with an apparent molecular weight of ~537.2 kDa (Supplementary Fig. 10b). These data indicate that an antibody recognizing the ligand-binding epitope (site 1) can cluster ROS1 in a manner analogous to NELL2. Taken together, these results demonstrate that an antibody that blocks ligand binding can prevent ligand induced ROS1 activation. And importantly, that simply cross-linking receptors via a bivalent antibody is insufficient to induce receptor activation – consistent with the need for a conformational change to overcome auto-inhibition (Supplementary Fig. 10c).

An antibody that blocks ROS1’s conformational transition prevents ROS1 signaling

From the phage display screen we identified a second Fab (designated Fab-CT4) which also binds to ROS1 with high affinity (K 28.2 nM) (Supplementary Fig. 12a). We determined the Cryo-EM complex structure between ROS1 and Fab-CT4 to a resolution of 2.99 Å (Fig. 5a, Supplementary Fig. 11 and Table S1). The structure reveals that Fab-CT4 sits in a cleft formed between ROS1’s YWTD-A domain and the second FNIII domain (Fig. 5b). Fab-CT4 interacts with ROS1 primarily through an “anti-parallel” beta-strand augmentation between CDR-H2 of Fab-CT4 and ROS1’s FNIII-2 domain (residues 246-251) (Fig. 5c). Unusually, this includes hydrogen bonds from the antibody beyond the CDR including Y86 and D88 which interact with N246 of ROS1 (Fig. 5d). CDR-H2 simultaneously makes an interaction to H498 of the YWTD-A domain, as does CDRH3 W126. The interaction is further stabilized by a series of hydrophobic interactions between CDR loops of Fab-CT4 and ROS1’s YWTD-A domain and FNIII-2 domain (Supplementary Fig. 12b-12c). Thus, Fab-CT4 makes bonds to both FNIII-2 and YWTD-A of ROS1, stabilizing the bent-over conformation.

When superposing the Fab-CT4 complex structure onto the NELL2-ROS1 complex structure, there are no clashes between the antibody and NELL2 (Fig. 5e). Additionally, their binding epitopes on ROS1’s YWTD-A domain do not overlap (Fig. 5f). Despite not blocking NELL2 directly, Fab-CT4 binding may impact ROS1 activity by stabilizing the bent-over conformation. To determine the impact of Fab-CT4, we performed an SPR competition assay with the same concentrations used in the Fab-RX5 SPR experiment (Fig. 4g). The results show that the ROS1-NELL2 interaction is only partially diminished when ROS1 is preincubated with an excess of Fab-CT4 (1:10) (Fig. 5g). This suggests that antibody Fab-CT4 does not fully block ligand binding, but weakens the receptor’s ability to engage ligand. This is consistent with each site for NELL2 being accessible on ROS1 in the presence of Fab-CT4, but that NELL2 can not engage all sites at the same time.

We next reversed the order of addition to test whether ROS1 prebound with ligand could still engage Fab-CT4. Based on our structural model, we predicted that Fab-CT4 would not bind, because NELL2 engagement drives ROS1 into an open, extended conformation that eliminates the CT4 binding cleft (Supplementary Fig. 12d-12e). We tested this prediction using biolayer interferometry (BLI) (Supplementary Fig. 12f). Consistent with our model, Fab-CT4 showed no measurable binding to ROS1, when ROS1 was prebound to NELL2, indicating that ligand binding abolishes the Fab-CT4 interaction.

To assess the possible impact of Fab-CT4 on ROS1 cellular signaling, we generated a full antibody based on Fab-CT4 (IgG-CT4). IgG-CT4 was able to inhibit ROS1 signaling by NELL2 at low nanomolar concentrations (Fig. 5h). These results demonstrate that an antibody that stabilizes ROS1 in the bent-over conformation can block ligand induced activation, despite the receptor still being able to engage ligand. Additionally, just as with IgG-RX5, IgG-CT4 does stimulate ROS1 despite being bivalent, further highlighting the bent-over conformation as inactive.

Truncation of ROS1’s N-terminal arm bypasses the requirement for conformational switching

Collectively, our data suggest a two-tiered mechanism for ROS1 activation governed by NELL2. While trimeric NELL2 induces necessary receptor clustering, the spatial organization alone is insufficient for activation. The ligand must also drive a conformational switch that decouples the C-terminal leg region from the ligand-binding domain, relieving the inhibitory restraints imposed by the N-terminal arm.

To validate this dual structural requirement, we generated stable cells expressing an N-terminally truncated ROS1 lacking the auto-inhibitory arm region (ROS1arm). As predicted, this construct exhibited elevated basal phosphorylation (Supplementary Fig.13a), confirming the arm’s autoinhibitory role. Notably, the bivalent antibody RX5, which fails to activate full-length ROS1 despite inducing clustering (Fig. 4i, Supplementary Fig. 10b) is able to robustly stimulate the truncated receptor (Supplementary Fig. 13b). Similarly, our site-2-deficient NELL2 mutant, which cannot stabilize the conformational switch or activate the wild-type receptor (Fig. 2e, 2g), is capable of stimulating ROS1arm stable cells (Supplementary Fig. 13c). These findings demonstrate that removal of the N-terminal constraint (arm) bypasses the requirement for a NELL2-induced conformational shift, thereby confirming that physiological ROS1 activation requires both oligomerization and the specific release of auto-inhibition. When the auto inhibition constraint is removed then clustering alone is sufficient to stimulate ROS1 activity.

Methods
Recombinant protein expression and purification

cDNA of ROS1 was purchased from Origene. ROS1 was cloned into a modified vector with a N-terminal hexahistidine tag followed by a Factor Xa cut site and C-terminal twin-strep tag. ROS1[Nterm]was cloned into the vector with a N-terminal hexahistidine tag followed by a Factor Xa cut site. DNA encoding full-length NELL2, with a C-terminal 10 × histidine-tag was purchased from SinoBiological. Mutations of ROS1 and NELL2 constructs were introduced by site directed mutagenesis using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The proteins were purified from Expi 293 cells for ROS1[N-term] and ROS1 . Cells were transient transfected with ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific) at the density of 3 × 106 cells per mL. Conditioned medium was harvested 4 days post transfection by centrifuge at 4000 x g for 30 min and loaded onto Ni-Penta Agarose-Base Resin (Marvelgent Biosciences) by gravity at 4 ℃. After extensive washing with 15 mM Imidazole (pH 8.0), 100 mM NaCl, the bound proteins were eluted with 500 mM Imidazole (pH 8.0), 100 mM NaCl. The proteins were further purified by size-exclusion chromatography with a Hiload 26/600 Superdex 200pg column (Cytiva Life Sciences) equilibrated in 20 mM HEPES (pH 7.5) with 100 mM NaCl. Fractions were pooled and concentrated with an Amicon 10-kDa centrifugal filter (Millipore). The his-tag of ROS1 and its mutants was further cleaved by Factor Xa protease (New England Biolabs) and flowed through Ni-Penta Agarose-Base Resin again. The un-tagged proteins were collected and further purified by Superdex 200 Increase 10/300 GL size exclusion column (Cytiva Life Sciences) in 20 mM HEPES (pH 7.5) with 100 mM NaCl. Conditioned medium containing ROS1 was harvested 7 days post transfection and loaded onto Strep-Tactin XT 4Flow Resin (IBA Lifesciences). After extensive wash with PBS, the bound proteins were eluted with 50 mM biotin in 20 mM HEPES (pH 7.5) with 100 mM NaCl. For purification of NELL2 and the mutants, the proteins were buffer exchanged into 25 mM HEPES, pH 7.5, containing 50 mM NaCl after elution from Ni-Penta Agarose-Base resin and loaded onto a Mono Q column (Cytiva Life Sciences). The bound proteins were eluted using a salt gradient from 50 mM to 1 M NaCl in 25 mM HEPES, pH 7.5. Fractions containing target proteins were pooled, concentrated and purified further with Superose 6 10/300 GL column (Cytiva Life Sciences) equilibrated in 20 mM HEPES (pH 7.5) with 100 mM NaCl. Protein purity was assessed by Coomassie-stained SDS-PAGE. The heavy chains of IgG-RX5 and IgG-CT4 were cloned into pFUSE2ss-CHIg-mG1 vector (Invivogen). The light chains of IgG-RX5 and IgG-CT4 were cloned into pFUSE2ss-CLIg-mk vector (Invivogen) for expression. IgG-RX5 and IgG-CT4 antibodies were expressed in Expi 293 cells. The conditioned medium was harvested 6 days post transfection and loaded onto protein A Sepharose Fast Flow resin (Cytiva Life Sciences) for 1 hour at 4 ℃. After extensive washing with cold DPBS (Gibco), the bound antibodies were eluted with 50 mM NaH PO4, 100 mM H PO , 140 mM NaCl, pH 2.8 and neutralized by adding 1 M Tris (pH 11.0) to a final concentration of 100 mM. The antibodies were further purified by size exclusion chromatography with a Superdex 200 Increase 10/300 GL column (Cytiva Life Sciences) equilibrated in 20 mM HEPES (pH 7.5) with 100 mM NaCl.

Generation of stable cell lines and cell stimulation assay

The chimeric ROS1 receptor consisting of an HaloTag, the extracellular domain of ROS1 and the transmembrane with intracellular domain of ALK was constructed into pcDNA3.1(−)-HaloGRD plasmid using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs)38. The ROS1 mutants and ROS1arm construct were generated by PCR amplification using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). For stable expression of individual chimeric wild-type, mutated ROS1 or ROS1arm construct cell lines, the recombinant plasmids of ROS1 wild-type, mutants or ROS1arm were transfected into NIH/3T3 cells (ATCC CRL-1658, mouse embryo fibroblast cell) using Lipofectamine 2000 (Thermo Fisher Scientific). The stable cell lines were selected with Opti-MEM I reduced serum medium (Gibco) containing 10 % HI FBS, 1 % penicillin–streptomycin (Sigma Aldrich), and 400 μg mL−1 G-418 (Santa Cruz Biotechnology).

For cell stimulation studies, stable cells that reached 90 % confluency were serum starved overnight prior to stimulation with 4 nM NELL2 wild type or mutants at 37 ℃ for 10 min. The effects of Fab-RX5 or Fab-CT4 were evaluated by adding the indicated concentrations of Fab-RX5 or Fab-CT4 for 30 min before NELL2 stimulation. ROS1arm stable cells were stimulated with 10 nM IgG-RX5 or NELL2 L416E variant at 37℃ for 10 min to evaluate the effect of removing the autoinhibitory arm of ROS1.

Western blot analysis

After stimulation, cells were washed twice with ice-cold phosphate buffered saline (PBS) and lysed on ice for 30 min in lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 % NP-40) with 1× protease/phosphatase inhibitor cocktail (Cell Signaling Technology no. 5872). Cell lysates were then clarified by centrifugation at 16250 x g at 4 ℃ for 15min and the supernatants were collected and boiled in 1× Laemmli sample buffer (Bio-Rad). Samples were separated on 4-12 % Tris-Glycine gradient gel, then transferred to PVDF membrane and immunoblotted with either anti-ALK antibody (1:1000, Cell Signaling Technology, no. 3633), anti-phospho ALK (Y1507) antibody (1:1000, Cell Signaling Technology, no. 14678), anti-ERK antibody (1:1000, Cell Signaling Technology, no. 9102), or anti-phospho ERK antibody (1:1000 Cell Signaling Technology, no.4370). HRP-linked anti-rabbit IgG (Cell Signaling Technology, no. 7074S) was used at a dilution of 1:4000. Blots were developed with Amersham ECL Prime western blotting detection reagent (Cytiva Life Sciences) and imaged using a ChemiDoc (Bio-Rad).

Cryo-EM sample preparation and data collection

Purified ROS1 at 0.6 mg/mL was applied to glow-discharged Quantifoil holey carbon grids (Cu R1.2/1.3 300 mesh). ROS1 -NELL2 complex at 0.3 mg/mL with a molar ratio of 3:1 was applied to glow-discharged Quantifoil holey carbon grids (Au R1.2/1.3 300 mesh).ROS1[N-term]-NELL2 complex was prepared by mixing purified ROS1[N-term]and NELL2 with a 3:1 molar ratio. The ROS1-Fab-RX5 complex and the ROS1-Fab-CT4 complex were prepared by mixing ROS1[N-term] and purified Fabs with a 1:1.2 molar ratio, respectively. 3 µL of the mixture at approximately 0.3 mg mL-1 was applied to glow-discharged Quantifoil holey carbon grids (Cu R1.2/1.3 300 mesh). Grids were then blotted for 3 s under -1 force at 100 % humidity and 4℃ before plunging into liquid ethane using a Vitrobot Mark IV (FEI). Cryo-EM micrographs were collected on a Titan Krios microscope equipped with a K3 summit direct electron detector (Gatan) and a GIF quantum energy filter (15 eV). ROS1 and ROS1 -NELL2 complex images were recorded using SerialEM software in super-resolution mode at a nominal magnification of 105,000×, corresponding to a calibrated super-resolution pixel size of 0.416 Å with a preset defocus range from -0.8 to -2.0 µm. The dose rate was set to 19.03 electrons per physical pixel per second. The exposure time of each movie was 1.964 s, leading to a total dose of 50 electron per Å2, fractionated into 50 frames. Images of the complexes of ROS1[N-term]-NELL2, ROS1[N-term]-Fab-RX5 and ROS1[N-term]-Fab-CT4 were recorded using EPU (Thermo Fisher Scientific) in super-resolution mode at a nominal magnification of 105,000×, corresponding to a calibrated super-resolution pixel size of 0.413 Å with a preset defocus range from -0.8 to -2.0 µm. The dose rate was set to 15 electrons per physical pixel per second. The exposure time of each movie was 2.3 s, leading to a total dose of 50 electron per Å2, fractionated into 40 frames. For ROS1 and ROS1 -NELL2 structure, a total of 8320 and 14568 images were collected, respectively. For ROS1[N-term]-NELL2 complex, a total of 17000 images were collected. For the ROS1[N-term]-Fab-RX5 complex, a total of 8548 images were collected. For the ROS1[N-term]-Fab-CT4 complex, a total of 7936 images were collected.

Cryo-EM data processing

The datasets were processed with cryoSPARC v4.3.050. Motion-correction and CTF estimation were first carried out for all images. Micrographs with a CTF estimation resolution worse than 6.0 Å were excluded. Next, blob pick was used for the first round of particle picking. After several rounds of 2D classification, the selected good 2D classes were then used as templates for the second round of template picking from all images. Several rounds of 2D classification were performed, and the well-aligned 2D classes showing clear 3D projections were chosen for 3D reconstruction. Ab initio model reconstruction was used to generate initial 3D models. Particles corresponding to different classes were selected and optimized through iterative rounds of heterogeneous refinement in cryoSPARC. The best models were refined using non-uniform51 refinement and local refinement. The overall resolutions were estimated based on the gold-standard Fourier shell correlation (FSC) = 0.143 criteria. Local resolution was estimated from two half data maps under cryoSPARC.

Model building, refinement and validation

AlphaFold prediction52 models of ROS1’s ECR, NELL2, Fab-RX5 and Fab-CT4 were used as starting templates for the model building. The corresponding initial models were fitted into the cryo-EM maps using UCSF ChimeraX53 and manually rebuilt using Coot54. Model refinement was performed using phenix.real_space_refine in PHENIX55,56. Structural figures were generated in PyMOL (http://pymol.org/2/), UCSF Chimera57 and UCSF ChimeraX53. The refined model was further manually adjusted in Coot54. Detailed parameters and statistics for the model building and refinement process are provided in Table S1.

Size Exclusion Chromatography-Multiangle Light Scattering

SEC-MALS data were carried out on a Dawn Heleos-II Multi-Angle static Light Scattering (MALS) detector (Wyatt) coupled to a Zenix-C SEC-300 column (Sepax Technologies) on an Agilent 1260 Infinity HPLC system. For NELL2-ROS1 analysis, 90 µL of 1µM NELL2 (trimer concentration), 1µM ROS1 or a trimeric NELL2-ROS1 complexes at 1:1, 1:2 or 1:3 molar ratios were injected. For IgG-RX5-ROS1 analysis, 90 µL of 1µM IgG-RX5, 1µM ROS1 or a IgG-RX5-ROS1 complexes at 1:1 or 1:2 molar ratios were injected. All measurements were performed at a flow rate of 0.3 mL min-1 at room temperature. Data were processed using ASTRA 7.1.3 software (Wyatt Technology) with a dn/dc value of 0.185.

Bio-layer interferometry

Using an Octet RED96e instrument (FortéBio), HIS1K biosensors (FortéBio) were first loaded with NELL2 wild-type or mutants to a response of about 1 nm followed by quenching in 10 ng ml−1 biocytin (Sigma-Aldrich) for 30 s and equilibrated in the working buffer (20 mM HEPES, pH 7.5, 100 mM NaCl) for 30 s. The sensors were then dipped into indicated concentrations of ROS1 wild type or ROS1 mutants for 60 s, then dissociated in the working buffer for 60 s. The interactions between Fab and ROS1 were analyzed in the same way. Equilibrium responses were plotted against concentrations and one-site specific binding model was used to fit the data. Relative binding was determined as the response divided by the B value of NELL2 binds to ROS1. Data represent mean ± S.E.M. of three independent measurements. Data analysis was performed using Data Analysis software 10.0 (FortéBio) and binding curves were exported to GraphPad Prism 9.2.0 (Graphpad Software) for plotting of curves.

Phage display for fragment antigen-binding (Fab) selection

Phage display was performed to select ROS1 specific antibody as described previously58,59. In brief, the Fab library (library F)59 was first added to a 96-well plate Nunc MaxiSorp (Thermo Fisher Scientific) coated with streptavidin and incubated at room temperature for 2 h to reduce non-specific phage binders. The phage solution was then transferred into another Nunc MaxiSorp plate coated with ROS1 protein and incubated at room temperature for 2 h. The wells were washed with phosphate-buffered saline (PBS, pH 7.4) containing 0.05 % Tween-20. Phages were eluted with 100 mM HCl and neutralized with 1 M Tris (pH 11.0), then eluted phages were amplified and subjected to a second round of selection. Five rounds of phage display were performed. The phages from round three to five were diluted and used for bacteria omnimax infection. Single colonies were picked and cultured overnight. The media containing phages were directly used in subsequent enzyme-linked immunosorbent assay (ELISA).

ELISAs

For the ELISA, a 384-well Nunc MaxiSorp plate (Thermo Fisher Scientific) was coated with ROS1, streptavidin as a negative control, and anti-Flag M2 antibody (Sigma-Aldrich) as a positive control overnight at 4 ℃. The phage media were then diluted and split into the coated wells. ELISA signals were measured by absorbance at 450 nm with a microplate spectrophotometer reader (Agilent Technologies). ELISA signals (signal of ROS1 divided by the signal of streptavidin) over 10-fold were considered as positive hits. Eighty positive hits out of 96 screened clones were obtained. The corresponding clones were subjected to DNA sequencing analysis. Colonies with unique sequences were cloned into RH2.2 p441 vector for Fab expression60. Fab-RX5 was expressed in Escherichia coli (E. coli) BL21 cells, purified with protein A Sepharose Fast Flow resin (Cytiva Life Sciences). The bound protein was eluted with 50 mM NaH PO4, 100 mM H PO , 140 mM NaCl, pH 2.8 and neutralized by adding 1 M Tris (pH 11.0) to a final concentration of 100 mM. The Fab were further purified by size exclusion chromatography with a Superdex 200 Increase 10/300 GL column (Cytiva Life Sciences) equilibrated in 20 mM HEPES (pH 7.5) with 100 mM NaCl.

Surface plasmon resonance

Fab competition assay was performed using a Biacore 3000 instrument (GE Healthcare) at room temperature. NELL2 was immobilized on a CM5 sensor chip (Cytiva Life Sciences) using amide coupling, to a final level of approximately 5,200 response units. Purified 100 nM ROS1 or 1 𝜇M Fab-RX5 or Fab-CT4 or their combination with ROS1 were injected over the sensor chip surface at 10 𝜇L min-1 for 2 min in 25 mM HEPES (pH 7.5) buffer containing 100 mM NaCl. The dissociation was performed in the same buffer for 2.5 min. After each injection, the sensor chip was regenerated with 5 𝜇L10 mM sodium acetate (pH 5.5) containing 1 M NaCl. The response unit was corrected by subtracting the background signal from a control surface. The relative response is normalized to the ROS1 binding response unit. Data represents three independent measurements.

Statistics & Reproducibility

All experiments were performed with three independent biological repeats. GraphPad Prism 9.2.0 (Graphpad Software) was used for generation of graphs, plotting of curves, and for statistical analysis. Results represent mean ± S.E.M. of three independent measurements. For western blots, a representative blot is presented.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Klein and colleagues used cryoEM to determine the structures of human full-length ROS1, both in its unbound “apo” form and when bound to the ligand NELL2, aiming to clarify how ROS1 is regulated at the molecular level. NELL2 is thought to function as a homotrimer—three identical units held together by coiled-coil interactions—which could cluster three ROS1 molecules and trigger their activation. The apo ROS1 structure demonstrates that its N-terminal ligand-binding domains adopt a \"bend over,\" closed shape, which prevents NELL2 from binding, while its C-terminal or \"leg\" domains are extended and rigid. Surprisingly, the complex structure reveals a single NELL2 molecule (rather than a trimer) interacting with the N- terminal domains of just one ROS1 molecule. In this structure, NELL2 binding induces a large conformational change in the N-terminal ligand binding domain but the position of leg region remains unaffected. The authors conclude that the “bend over” conformation adopted by the N-terminal ligand binding domains of ROS1 restricts the movement of the extended \"leg\" regions, thereby keeping them separated and preventing ligand-independent dimerization of ROS1. Ligand engagement with these N-terminal domains lifts the structural constraint, conferring greater mobility in the leg regions. This flexibility might support new interactions between transmembrane helices or leg regions, possibly allowing transphosphorylation of intracellular kinase domains. The model of ROS1 regulation proposed by the authors presents a novel perspective and, if fully proven, would significantly advance our understanding of receptor tyrosine kinase signaling.\nMajor comments:\n1) Based on this proposed model, disrupting contacts that maintain the bend over conformation should relieve structural constraints on the “leg” regions, resulting in some degree of ligand-independent receptor activation; however, this premise has not been experimentally tested by the authors. In lines 390-393, the authors cite Jone et al., 2024, to support their model. Yet, data in Figures 1 and 2 of the referenced publication indicate that truncating the ROS1 N-terminus up to FNIII-5 does not enhance ligand-independent activity. Only more extensive truncations confer oncogenic activity to ROS1. According to the structure of Apo ROS1, the CATCH domain plays a crucial role in maintaining ROS1 in an inhibited state by interacting with the YWTD-B domain in cis. Thus, removal of the CATCH domain may increase basal ROS1 activity or, at a minimum, heighten sensitivity to NELL2. While it is understandable that abrogating the structural constraints imposed on the leg region by the N-terminal domains alone, without trimerization, is insufficient for full activation of ROS1, it remains reasonable to predict that such modifications could sensitize ROS1 to NELL2 binding and potentially elevate its basal activity.\n2) Additional robust experimental evidence is required to substantiate the proposed model. Have the authors considered employing MALS or mass photometry to measure the size of ROS1 complexed with wild-type NELL2 and NELL2 variants lacking the coiled-coil domain? Such assays may also be adopted to dissect the interactions between the “legs” of ROS1 monomers within the putative 1:3 NELL2-ROS1 trimeric complex. Furthermore, could the length of this “leg” be shortened without compromising ROS1’s activity?\n3) In panel d of Figure 1, the authors present cell-based experiments demonstrating that NELL2 must trimerize through its coiled-coil domain to oligomerize and activate ROS1. This observation begs the question: might a monomeric variant of NELL2, lacking the coiled-coil domain, function as an antagonist to wild-type NELL2? Has this hypothesis been explored by the authors or are there published literature regarding this.\n4) According to the ROS1-NELL2 complex structure (Figure 1), residues beyond the EGF-2 repeat in NELL2 appear disordered, implying that they may not participate in binding to ROS1 or be essential for NELL2 function. Have the authors conducted experiments to assess this possibility? If the disordered region is found to be necessary, what potential roles might it play in ROS1 signaling?\n5) The authors propose that simply forming bivalent dimers of ROS1 does not lead to activation, based on the fact that their intact IgG-RX5 and IgG-CT4 antibodies do not activate ROS1. However, have they confirmed that these antibodies actually bind bivalently to the ROS1 ectodomain? This could be easily checked using mass photometry or MALS.\n6) Fab-CT4 stabilizes ROS1 in its inhibited \"bent-over\" form by interacting with both FNIII-2 and YWTD-A regions. When NELL2 binds to ROS1, it causes ROS1 to adopt an open, extended conformation. This suggests Fab-CT4 may no longer be able to bind ROS1 after NELL2 is attached. Has this possibility been addressed by the authors?\n7) The analogy between EGFR and ROS1 activation mechanisms is largely superficial. In the case of EGFR, ligand binding induces a conformational change that relieves autoinhibition and exposes the dimerization arm, directly facilitating receptor dimerization—a well-established connection exists between autoinhibition relief and dimer formation. While ROS1 also undergoes a conformational shift upon NELL2 binding similar to EGF-induced changes in EGFR, this does not lead to direct receptor-receptor contact. This critical distinction should be recognized, and the manuscript revised accordingly to address it.\nMinor comments:\n1) The Introduction should briefly outline the existing structural studies of Sevenless, the Drosophila counterpart to human ROS1, since its N-terminal \"bend-over\" formation is similar to what’s shown for ROS1 in this work.\n2) Please provide the resolution of the structures/maps in the relevant figures.\n3) At line 33, please correct the misspelling of \"devlopment\".\n4) Line 326: It may be advisable to exclude the section titled “NELL1 is not a ROS1 ligand,” as it is not directly relevant to the main focus of the manuscript and could potentially distract from its central message.\n5) Lines 631-635: The legends for panels b and c are reversed and should be corrected.", "reviewer_2": "This study provides a significant advancement in our understanding of ROS1 activation mechanisms and the development of targeted therapeutics. The authors demonstrate that NELL2 functions as a high-affinity ligand for ROS1, inducing its phosphorylation in a dose-dependent manner. Structural analyses reveal that unliganded ROS1 adopts an auto-inhibited bent conformation, which is reversed upon NELL2 binding via an approximately 130° rotation of the N-terminal domains and decoupling of the \"leg\" domain, ultimately leading to kinase activation. Functional assays confirm that NELL2 engages ROS1 at three distinct sites, with mutations at any of these sites disrupting downstream signaling. The authors propose a plausible model for NELL2-mediated ROS1 activation, which holds broader interest for the RTK protein family. From a therapeutic perspective, two monoclonal antibodies were developed. Evolutionary comparisons highlight key divergences between mammalian ROS1 and Drosophila Sevenless, underscoring differences in regulatory mechanisms. Additionally, NELL1—a homolog lacking a conserved VWC-2 domain—fails to activate ROS1, emphasizing ligand specificity.\nOverall, this work offers a solid and timely elucidation of the NELL2-induced activation mechanism of ROS1, which will considerably propel the field forward. I support its publication in Nature Communications. Below are several suggestions that could further enhance the manuscript's rigor and accessibility, especially for non-specialist readers:\n1. In Figure 1h, clearly label the EGF-1 and EGF-2 domains of NELL2, similar to the labeling of VWC-1 and VWC-2.\n2. Consistently refer to the YWTD β-propeller domain using a single terminology throughout the manuscript to improve clarity.\n3. Comment on the observation that phosphorylation signals of ERK and ROS1 appear stronger at 10 minutes than at 30 minutes post-stimulation (Extended Data Fig. 1d). Potential explanations, such as rapid signal attenuation via endocytosis, could be discussed.\n4. Provide additional validation details for the AlphaFold-predicted model of NELL2-induced ROS1 oligomers (Fig. 3f), such as pLDDT score distributions and consistency across prediction replicates.\n5. Include SDS-PAGE and gel filtration results in the supplementary information to demonstrate the purity and stoichiometry of the protein complexes used.\n6. Label residues D418 and R419 in NELL2 and D27 and K28 in EGF in Extended Data Fig. 7 to facilitate comparison of the structural alignment.\n7. Ensure the side chain of EGF Y13 is properly positioned and visible in the final panel of Extended Data Fig. 7.\n8. Further refine the ROS1–Fab–CT4 structure to reduce the currently high rotamer outlier rate (4.51%) to an acceptable level.\n9. Carefully proofread Table S2 to correct typographical errors; for example, the MolProbity score for the ROS1(N-term)– NELL2 complex should be 1.95, not 195.\n10. Correct the figure panel labeling in line 144 to \"Figure 1g\" to align with the journal’s formatting style.", "reviewer_3": "Li et al. report structural and biochemical studies of the murine receptor tyrosine kinase ROS1 and its ligand NELL2. Key results include: (i) a cryo-EM structure of the ROS1 ECR both alone and with NELL2 bound (and observation of a large interdomain movement when ligand is bound), (ii) demonstration of high-affinity binding of NELL2 to ROS1 and activation of a ROS1/Alk chimera by NELL2, (iii) creation of monoclonal antibodies that bind ROS1 and block activation—either by blocking ligand binding or the ROS1 conformational change—and determination of cryo-EM structure of each Fab bound to ROS1.\nThese studies reveal key and novel features of interactions between ROS1 and its ligand as well as lay the groundwork for ROS1-targeting monoclonal antibody therapeutics, and I recommend publication in Nature Communications.\nThat said, I found several aspects of the presentation puzzling and offer the following comments for the authors’ consideration:\n1. The authors make no mention in the introduction of the recently published structures of the ECR of the Drosophila ROS1 homolog Sevenless, in one case bound to the ECR of its ligand Boss. I think it would help the general reader to be made aware of these structures along with brief comments about what they revealed. Later comments in the text about these structure would make more sense. I also think a comparison of the vertebrate and Drosophila structures—is the domain arrangement the same?—would be helpful, perhaps as a panel in figure 1 with comment in the results and/or discussion. Do the authors think Sevenless undergoes the same conformational change as ROS1 during its signaling cycle?\n2. Lines 92-93. The authors state “A significant challenge for studying ROS1 activity in cells has been the absence of cell lines stably expressing exogenous ROS1” As a rationale for creating the ROS1/Alk chimera. I found this rationale confusing and can only guess that the authors mean “no existing cell lines that express active ROS1 are available, and transfection of ROS1 into heterologous cells fails to result in expression of active protein”—or something like that.\n3. Figure 1b and 1c mixed up in the legend.\n4. Line 127. Figure 4d should be Extended Figure 4d.\n5. Lines 187-201. Not sure if the comparison of the mode of EGF domain binding ROS1 with EGF binding to EGFR is meaningful. EGF, which is small, is almost fully engulfed when bound to EGFR, and almost any binding mode would overlap. I also don’t see any insight that is gained by the comparison.\n6. Line 235 and following. I don’t understand the statement “Our finding that mutations that disrupt NELL2 binding to the FNIII domains (site 2) substantially impair signaling (Fig. 2e-2h), indicate that receptor clustering alone is insufficient and that the conformational switch is required for activation.” If the NELL2 mutants disrupt binding, how could they still cluster ROS1?\n7. Lines 243-246. I also find the statement “In this model, the N-terminal “arm” region – composed of the CATCH domain and the first two FNIII domains – engages the long distal leg region. This stabilizes both the leg and arm regions together into a relatively rigid structure” confusing. The previous Sev structures display flexibility between FNIII4 and FNIII5, i.e. the leg flexibility occurs between these domains. The “arm” region doesn’t to FNIII5 or below and doesn’t seem able to stabilize the leg."}, "author_response": "Response to Reviewer #1\nWe thank the reviewer for their comments including “The model of ROS1 regulation proposed\nby the authors presents a novel perspective and, if fully proven, would significantly advance our\nunderstanding of receptor tyrosine kinase signaling.” We sincerely thank the reviewer for their\nexpert suggestions and comments, which we believe has significantly improved our manuscript.\nMajor comments:\n1) Based on this proposed model, disrupting contacts that maintain the bend over conformation\nshould relieve structural constraints on the “leg” regions, resulting in some degree of ligand-\nindependent receptor activation; however, this premise has not been experimentally tested by the\nauthors. In lines 390-393, the authors cite Jone et al., 2024, to support their model. Yet, data in\nFigures 1 and 2 of the referenced publication indicate that truncating the ROS1 N-terminus up to\nFNIII-5 does not enhance ligand-independent activity. Only more extensive truncations confer\noncogenic activity to ROS1. According to the structure of Apo ROS1, the CATCH domain plays\na crucial role in maintaining ROS1 in an inhibited state by interacting with the YWTD-B domain\nin cis. Thus, removal of the CATCH domain may increase basal ROS1 activity or, at a minimum,\nheighten sensitivity to NELL2. While it is understandable that abrogating the structural\nconstraints imposed on the leg region by the N-terminal domains alone, without trimerization, is\ninsufficient for full activation of ROS1, it remains reasonable to predict that such modifications\ncould sensitize ROS1 to NELL2 binding and potentially elevate its basal activity.\nWe appreciate the reviewer’s insightful comment. Our model proposes that disrupting\ninteractions that stabilize the bent-over conformation relieves structural constraints on the leg\nregions and can influence basal ROS1 activity. In our structure, the CATCH domain together\nwith the first two FNIII domains (the “arm”) buttress the leg region and stabilize the bent-over\nconformation. To test this hypothesis experimentally, we generated a stable cell line expressing a\ntruncated ROS1 construct in which the CATCH domain and FNIII 1-2 domains were removed.\nCompared with full-length ROS1, this arm-deleted variant showed increased basal activity\n(Extended Data Fig. 13a), consistent with a role for the CATCH/arm region in maintaining the\nauto-inhibited state.\nWe next asked whether relieving this arm restraint sensitizes ROS1 to clustering. Using the same\nstable cell line, we clustered the arm-deleted receptor either with IgG-RX5 (Extended Data Fig.\n13b) or with a NELL2 mutant that engages only site 1 (Extended Data Fig. 13c). In contrast to\nfull-length ROS1, clustering of the arm-deleted receptor by either reagent was sufficient to\nactivate ROS1. Together, these experiments demonstrate the auto-inhibitory nature of the N-\nterminal arm and show that removing this constraint both increases basal activity and lowers the\nthreshold for clustering-dependent activation, in line with our conformational switch model for\nROS1 activation.\n2) Additional robust experimental evidence is required to substantiate the proposed model. Have\nthe authors considered employing MALS or mass photometry to measure the size of ROS1\ncomplexed with wild-type NELL2 and NELL2 variants lacking the coiled-coil domain? Such\nassays may also be adopted to dissect the interactions between the “legs” of ROS1 monomers\nwithin the putative 1:3 NELL2-ROS1 trimeric complex. Furthermore, could the length of this\n“leg” be shortened without compromising ROS1’s activity?\nWe appreciate the reviewer’s thoughtful suggestions regarding additional biophysical approaches\nto further evaluate the proposed activation model. We agree that MALS or mass photometry\ncould, in principle, provide complementary information on the stoichiometry of the ROS1-\nNELL2 complex. As suggested, we performed SEC-MALS analysis for the ROS1-NELL2\ncomplex.\nAs we now show in Extended Data Fig. 3b, NELL2 alone eluted at 282.5 kDa, consistent with\nits expected trimeric mass (276.9 kDa), and ROS1 (N-Term) eluted at 196.5 kDa. The\npreincubated 1:1 mixture yielded a species with an apparent molecular weight of ~476.4 kDa,\nconsistent with formation of a 1:1 complex. Increasing ROS1 to a (1 NELL2 trimer : 2 ROS1)\nmolar ratio produced a species of ~676.6 kDa, indicative of a 1:2 complex. Further increasing\nROS1 to a ratio of 3 resulted in a species of ~711.8 kDa, a complex smaller than the predicted\n1:3 species, but larger than the 1:2. This is likely attributable to a weaker dissociation constant\n(Kd) associated with incorporation of the third ROS1 molecule into the NELL2 complex.\nWe agree that the functional relevance of the ROS1 “leg” length is an interesting question. In\nprinciple, shortening the leg could alter the spatial relationship between the extracellular and\nintracellular regions, potentially affecting both clustering geometry and kinase trans-\nautophosphorylation. Systematically testing a series of leg truncations would therefore require a\ndedicated structure-function study that we feel is beyond the scope of the present work, which is\nfocused on defining the NELL2-ROS1 activation mechanism. We have added this as an\ninteresting avenue for future investigation.\n3) In panel d of Figure 1, the authors present cell-based experiments demonstrating that NELL2\nmust trimerize through its coiled-coil domain to oligomerize and activate ROS1. This\nobservation begs the question: might a monomeric variant of NELL2, lacking the coiled-coil\ndomain, function as an antagonist to wild-type NELL2? Has this hypothesis been explored by the\nauthors or are there published literature regarding this.\nWe thank the reviewer for this insightful question. Based on our structural and biochemical\nanalyses, the coiled-coil–mediated trimerization of NELL2 is essential for productive ROS1\noligomerization and activation (Figure 1). However, a monomeric variant lacking the coiled-coil\ndomain would likely be ineffective or inefficient in competing with wild-type NELL2 in vivo, as\nthe high avidity and cooperative binding of the NELL2 trimer would make it difficult for a\nmonomer to outcompete the trimer on the cell surface. Our delta coiled-coil construct produces\npoorly compared to the WT, and we would not be able to achieve the very high concentrations\nthat we would predict to need to overcome the wildtype activation. We used our antibody\ndiscovery platform to generate inhibitors that can function essentially in the proposed manner by\nbinding to and competing for the same epitope as NELL2 (RX5).\n4) According to the ROS1-NELL2 complex structure (Figure 1), residues beyond the EGF-2\nrepeat in NELL2 appear disordered, implying that they may not participate in binding to ROS1\nor be essential for NELL2 function. Have the authors conducted experiments to assess this\npossibility? If the disordered region is found to be necessary, what potential roles might it play in\nROS1 signaling?\nWe appreciate the reviewer’s thoughtful question regarding the potential role of the structurally\nunresolved region of NELL2. In our ROS1-NELL2 complex structure, residues beyond the EGF-\n2 repeat are not resolved, indicating that they are flexible and unlikely to contribute directly to\nthe ROS1-binding interface. Based on AlphaFold predictions, as updated in Extended Data Fig.\n8, the N-terminal Laminin G domain appears to interact with the C-terminal regions beyond\nEGF-2, suggesting potential intra- and inter- molecular contacts. We believe this critically\nstabilizes the complex and supports the folding and appropriate disulfide bonding of the coiled-\ncoil region. Indeed, we attempted to generate NELL2 truncation constructs starting immediately\nafter the Laminin G domain. These failed to produce, implying that these regions together with\ndomains after EGF-2 might be important for protein stability or proper folding. Although the\nstructural data indicate that these region does not directly engage ROS1, we cannot rule out\nadditional functional roles that may mediate interactions with cofactors or extracellular\ncomponents that modulate ROS1 signaling.\n5) The authors propose that simply forming bivalent dimers of ROS1 does not lead to activation,\nbased on the fact that their intact IgG-RX5 and IgG-CT4 antibodies do not activate ROS1.\nHowever, have they confirmed that these antibodies actually bind bivalently to the ROS1\nectodomain? This could be easily checked using mass photometry or MALS.\nWe thank the reviewer for the insightful comment regarding the stoichiometry of the ROS1-\nantibody interaction. To address this, we performed SEC-MALS analysis for IgG-RX5, which\ntargets the YWTD-A domain of ROS1 (same epitope, site 1 as NELL2), using samples\npreincubated at either a 1:1 or 1:2 molar ratio of IgG-RX5 to ROS1. The result showed that the\nantibody can bind bivalently to two ROS1 molecules simultaneously. We now included this\nSEC-MALS result as Extended Data Fig. 10b in our revised manuscript.\n6) Fab-CT4 stabilizes ROS1 in its inhibited \"bent-over\" form by interacting with both FNIII-2\nand YWTD-A regions. When NELL2 binds to ROS1, it causes ROS1 to adopt an open, extended\nconformation. This suggests Fab-CT4 may no longer be able to bind ROS1 after NELL2 is\nattached. Has this possibility been addressed by the authors?\nWe appreciate the reviewer’s comment regarding the potential possibility of Fab-CT4 interaction\nwith ROS1 in the presence of NELL2. To address this, we performed biolayer interferometry\n(BLI) analysis. Fab-CT4 was loaded onto the sensor and dipped into ROS1 alone, NELL2 alone\nor the ROS1-NELL2 complex. The results showed that Fab-CT4 bound robustly to ROS1 alone\nand showed no detectable binding to NELL2 alone or the ROS1-NELL2 complex (Extended\nData Fig 12f). In fact, our Fab-CT4-ROS1 cryo-EM structure reveal that Fab-CT4 engages\nunliganded ROS1 primarily through an “anti-parallel” beta-strand augmentation with ROS1’s\nFNIII-2 domain (residues 246-251) (Fig. 5b, 5c and Extended Data Fig 12d). Upon NELL2\nbinding, ROS1 undergoes a conformational transition to an open, extended state that disrupts the\nbeta-strand augmentation and repositions the interface involved in this interaction (Extended\nData Fig 12e). These results indicate that Fab-CT4 specifically recognize ROS1 only in its\nunliganded state, and its binding epitope becomes inaccessible once ROS1 is bound by NELL2.\nWe have now included this BLI result, together with a superimposed structural comparison of\nFab-CT4 bound unliganded ROS1 and liganded ROS1, as Extended Data Fig. 12 in the revised\nmanuscript.\n7) The analogy between EGFR and ROS1 activation mechanisms is largely superficial. In the\ncase of EGFR, ligand binding induces a conformational change that relieves autoinhibition and\nexposes the dimerization arm, directly facilitating receptor dimerization—a well-established\nconnection exists between autoinhibition relief and dimer formation. While ROS1 also\nundergoes a conformational shift upon NELL2 binding similar to EGF-induced changes in\nEGFR, this does not lead to direct receptor-receptor contact. This critical distinction should be\nrecognized, and the manuscript revised accordingly to address it.\nWe thank the reviewer for highlighting this important conceptual point. Our intention in the\noriginal Discussion was precisely to emphasize that, although there are similarities between\nEGF-EGFR and NELL2-ROS1, the activation mechanisms are fundamentally distinct. As we\ndescribe in the Discussion, ROS1 uniquely integrates two regulatory paradigms: (i) ligand-\nmediated clustering via oligomeric NELL2, and (ii) ligand-induced relief of intramolecular\nautoinhibition. In contrast to EGFR, where ligand binding exposes a dimerization arm that\ndirectly mediates receptor-receptor contacts, our structural and functional data indicate that\nNELL2-bound ROS1 does not form an analogous dimer interface and instead relies on the\nNELL2 coiled-coil as the clustering scaffold. We highlight this structurally in Extended Data\nFig. 15 for clarity.\nTo make this distinction more explicit, we have revised the Discussion to state that, while\nNELL2 binding induces a large conformational change in ROS1 reminiscent of that seen in\nEGFR, this does not expose a ROS1 dimerization arm or drive direct receptor–receptor contact;\nrather, conformationally “unlocked” ROS1 molecules are clustered exclusively through\noligomeric NELL2. We hope this clarifies that our analogy to EGFR is limited to the theme of\nligand-induced relief of autoinhibition, while underscoring that the mechanism of clustering and\nactivation is unique to ROS1.\nMinor comments:\n1) The Introduction should briefly outline the existing structural studies of Sevenless, the\nDrosophila counterpart to human ROS1, since its N-terminal \"bend-over\" formation is similar to\nwhat’s shown for ROS1 in this work.\nWe thank the reviewer for this suggestion. We have now revised the Introduction to briefly\nsummarize existing structural studies of Sevenless (including our own work). Specifically, we\nnote that Sevenless adopts an N-terminal “bent-over” conformation, highlighting the structural\nconservation and supporting the relevance of our findings on ROS1. References to Sevenless\nstructural studies have been added to provide appropriate context.\n2) Please provide the resolution of the structures/maps in the relevant figures.\nWe thank the reviewer for this suggestion. We now included the resolutions of the structures in\nthe relevant figure legends.\n3) At line 33, please correct the misspelling of \"devlopment\".\nThank you for pointing out this typographical error. We have corrected it to \"development\" in the\nrevised manuscript.\n4) Line 326: It may be advisable to exclude the section titled “NELL1 is not a ROS1 ligand,” as\nit is not directly relevant to the main focus of the manuscript and could potentially distract from\nits central message.\nThank you for this valuable suggestion. We have now removed this section from the revised\nmanuscript to make it more focused.\n5) Lines 631-635: The legends for panels b and c are reversed and should be corrected.\nWe thank the reviewer for finding the error. We have corrected in the revised manuscript.\nResponse to Reviewer #2\nWe are grateful to the reviewer for their comments, “This study provides a significant\nadvancement in our understanding of ROS1 activation mechanisms and the development of\ntargeted therapeutics. Overall, this work offers a solid and timely elucidation of the NELL2-\ninduced activation mechanism of ROS1, which will considerably propel the field forward. I\nsupport its publication in Nature Communications.” We have addressed each point raised and\nincorporated suggestions to strengthen our manuscript.\n1. Improvements to Figure 1h\nReviewer comment:\n“In Figure 1h, clearly label the EGF-1 and EGF-2 domains of NELL2, similar to the labeling of\nVWC-1 and VWC-2.”\nWe have adjusted the labels of EGF-1 and EGF-2 domains of NELL2 in Figure 1h, ensuring they\nare easily distinguishable.\n2. Consistent terminology of YWTD β-propeller domain\nReviewer comment:\n“Consistently refer to the YWTD β-propeller domain using a single terminology throughout the\nmanuscript to improve clarity.”\nWe thank the reviewer for this suggestion. We have now standardized the terminology\nthroughout the manuscript, consistently referring to this region as the “YWTD” domain to\nimprove clarity.\n3. Discussion on the Stronger Phosphorylation Signals\nReviewer comment:\n“Comment on the observation that phosphorylation signals of ERK and ROS1 appear stronger at\n10 minutes than at 30 minutes post-stimulation (Extended Data Fig. 1d). Potential explanations,\nsuch as rapid signal attenuation via endocytosis, could be discussed.”\nWe thank the reviewer for highlighting the temporal differences in ERK and ROS1\nphosphorylation observed in Extended Data Fig. 1d. Indeed, the phosphorylation signals peak at\n10 minutes post-stimulation and decrease by 30 minutes. This pattern is consistent with well-\ncharacterized rapid attenuation mechanisms in receptor tyrosine kinase (RTK) signaling (e.g.\nEGF-EGFR). One explanation is ligand-induced receptor endocytosis, which can rapidly reduce\nsurface receptor levels and thereby diminish downstream signaling. Additionally, activation of\nnegative feedback regulators such as MAPK phosphatases or ROS1-directed phosphatases may\ncontribute to the decline in ERK and ROS1 phosphorylation at later time points.\n4. Additional validation details for the AlphaFold predicted model\nReviewer comment:\n“Provide additional validation details for the AlphaFold-predicted model of NELL2-induced\nROS1 oligomers (Fig. 3f), such as pLDDT score distributions and consistency across prediction\nreplicates.”\nThank you for this valuable suggestion. We have added the pLDDT score distribution as\nExtended Data Figure 8 and detailed description of the AlphaFold predicted model of NELL2\nand in the revised manuscript.\n5. Include SDS-PAGE and gel filtration in the supplementary information\nReviewer comment:\n“Include SDS-PAGE and gel filtration results in the supplementary information to demonstrate\nthe purity and stoichiometry of the protein complexes used.”\nWe thank the reviewer for this suggestion. We have now included SDS-PAGE and gel filtration\n(SEC) results in the Extended Data Fig.1 to demonstrate the purity of the proteins used in this\nstudy. For protein complex formation, NELL2 and ROS1 were preincubated at a 1 trimer\nNELL2 : 3 ROS1 molar ratio prior to cryo-EM sample preparation.\n6.\nReviewer comment:\nLabel residues D418 and R419 in NELL2 and D27 and K28 in EGF in Extended Data Fig. 7 to\nfacilitate comparison of the structural alignment.\nWe thank the reviewer for these suggestions. While we agree that the residues should be well\nlabeled, we note that one of the reviewers found that this comparison provides limited additional\ninsight. After careful consideration, we have decided to remove this comparison paragraph along\nwith Extended Data Fig. 7 from the revised manuscript.\n7.\nReviewer comment:\nEnsure the side chain of EGF Y13 is properly positioned and visible in the final panel of\nExtended Data Fig. 7.\nWe thank the reviewer for these suggestions. We note that one of the reviewers found that this\ncomparison provides limited additional insight. After careful consideration, we have decided to\nremove this comparison paragraph along with the associated data in Extended Data Fig. 7 from\nthe revised manuscript.\n8. Improvement of ROS1-Fab-CT4 structure\nReviewer comment:\n“Further refine the ROS1–Fab–CT4 structure to reduce the currently high rotamer outlier rate\n(4.51%) to an acceptable level.”\nWe have further refined the ROS1-Fab-CT4 structure to reduce rotamer outlier to 0.0 %.\nThe\ncorresponding parameters have been updated in Table S1.\n9. Correction of typographical error\nReviewer comment:\n“Carefully proofread Table S2 to correct typographical errors; for example, the MolProbity score\nfor the ROS1(N-term)–NELL2 complex should be 1.95, not 195.”\nWe thank the reviewer for finding the error. We have corrected the typo of MolProbity score of\nthe ROS1[N-term]-NELL2 complex structure to “1.95”.\n10. Correction the label of figure panel\nReviewer comment:\nCorrect the figure panel labeling in line 144 to \"Figure 1g\" to align with the journal’s formatting\nstyle.??\nWe thank the reviewer for finding the error. We have corrected to “Figure 1g” to align with the\njournal’s format.\nResponse to Reviewer #3\nWe are grateful to the reviewer for their positive evaluation including “These studies reveal key\nand novel features of interactions between ROS1 and its ligand as well as lay the groundwork for\nROS1-targeting monoclonal antibody therapeutics, and I recommend publication in Nature\nCommunications.” We have addressed each point raised and detailed our responses below.\n1. The authors make no mention in the introduction of the recently published structures of the\nECR of the Drosophila ROS1 homolog Sevenless, in one case bound to the ECR of its ligand\nBoss. I think it would help the general reader to be made aware of these structures along with\nbrief comments about what they revealed. Later comments in the text about these structure\nwould make more sense. I also think a comparison of the vertebrate and Drosophila structures—\nis the domain arrangement the same?—would be helpful, perhaps as a panel in figure 1 with\ncomment in the results and/or discussion. Do the authors think Sevenless undergoes the same\nconformational change as ROS1 during its signaling cycle?\nWe thank the reviewer’s suggestion. We have now revised the Introduction to briefly summarize\nthe recently published structures (including our own) of the extracellular region (ECR) of the\nDrosophila ROS1 homolog Sevenless and highlights the structural conservation and supporting\nthe relevance of our findings on ROS1. Overall, the domain arrangement of ROS1 and Sevenless\nis conserved. However, their activation mechanisms are entirely different. BOSS binds to the\nthird FNIII domain of Sevenless with relatively low affinity and does not induce conformational\nchange in the receptor. The low affinity is likely related to the spatial constraints of both BOSS\nand Sevenless on adjacent membranes. Activation of Sevenless then occurs only after a low pH\ninduced dimerization of the receptor and release of the ligand.\n2. Lines 92-93. The authors state “A significant challenge for studying ROS1 activity in cells has\nbeen the absence of cell lines stably expressing exogenous ROS1” As a rationale for creating the\nROS1/Alk chimera. I found this rationale confusing and can only guess that the authors mean\n“no existing cell lines that express active ROS1 are available, and transfection of ROS1 into\nheterologous cells fails to result in expression of active protein”—or something like that.\nWe are sorry for the confusion. We have revised this section for clarity:\n“A significant challenge for studying ROS1 activity in cells is that no cell lines are known to\nendogenously express full length ROS1. Moreover, repeated attempts to generate stable ROS1-\nexpressing heterologous cell lines by transfection have failed to yield detectable protein.”\n3. Figure 1b and 1c mixed up in the legend.\nWe thank the reviewer for finding the error. We have corrected and updated in the revised\nmanuscript.\n4. Line 127. Figure 4d should be Extended Figure 4d.\nThank you for catching this error. We have corrected \" Fig. 4d \" to \" Extended Data Fig. 4d \" in\nthe manuscript.\n5. Lines 187-201. Not sure if the comparison of the mode of EGF domain binding ROS1 with EGF\nbinding to EGFR is meaningful. EGF, which is small, is almost fully engulfed when bound to\nEGFR, and almost any binding mode would overlap. I also don’t see any insight that is gained by\nthe comparison.\nWe thank the reviewer’s comment. We have removed the paragraph of comparison of the mode\nwith EGF domain in the revised manuscript.\n6. Line 235 and following. I don’t understand the statement “Our finding that mutations that disrupt\nNELL2 binding to the FNIII domains (site 2) substantially impair signaling (Fig. 2e-2h), indicate\nthat receptor clustering alone is insufficient and that the conformational switch is required for\nactivation.” If the NELL2 mutants disrupt binding, how could they still cluster ROS1?\nWe thank the reviewer for pointing out the potential confusion. The sentence was intended to\nconvey that receptor clustering by NELL2 is necessary but not sufficient for ROS1 activation. In\nour experiments, NELL2 variants with mutations at site 2 can still bind ROS1 but fail to trigger\nsignaling. These data demonstrate that simply bringing ROS1 molecules into proximity\n(clustering) is insufficient for activation; proper engagement at site 2 is required to induce the\nconformational change that drives signaling. Notably, we have added new data Extended Data\nFig 13, that shows if the arm of ROS1 is removed, that site 2 mutants of NELL2 (which still bind\nat site 1) can now stimulate ROS1 signaling through clustering. This demonstrates that in the\nabsence of an auto-inhibitory arm, clustering is sufficient for stimulating ROS1.\n7. Lines 243-246. I also find the statement “In this model, the N-terminal “arm” region –\ncomposed of the CATCH domain and the first two FNIII domains – engages the long distal leg\nregion. This stabilizes both the leg and arm regions together into a relatively rigid structure”\nconfusing. The previous Sev structures display flexibility between FNIII4 and FNIII5, i.e. the leg\nflexibility occurs between these domains. The “arm” region doesn’t to FNIII5 or below and\ndoesn’t seem able to stabilize the leg.\nWe thank the reviewer for this comment. In the previously published Sevenless structures\n(including ours), the major hinge that confers flexibility resides between FNIII4 and FNIII5,\nindicating that the “leg” region can pivot at this junction. Rather, our structural data of ROS1\nindicates that engagement of the arm with the portion of the leg contributes to stabilizing\nthe relative orientation of these domains, thereby constraining part of the leg and reducing\noverall flexibility in this segment. This rigid organization stands in contrast to prior structures of\nROS1’s Drosophila homolog, Sevenless, in which only the N-terminal bent-over portion was\nresolved – while the remainder of the ectodomain distal to that appeared disordered and could\nnot be modeled."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed the majority of my initial comments satisfactorily; however, their responses to comments #1 and #2 prompt further inquiries regarding the specific mechanism of ROS1 activation that warrant careful consideration.\nRegarding comment #1, the authors demonstrated that deletion of the \"arm\" region leads to increased basal activity of ROS1. This finding could be interpreted in two ways: as proposed by the authors, removal of the \"arm\" may relieve structural constraints on the “leg” region, or alternatively, the \"arm\" might serve as a physical barrier restricting interactions between “leg” regions required for receptor dimerization. It is therefore recommended that both biochemical and structural analyses be performed on the ROS1 variant lacking the \"arm\" to clarify its activation mechanism. Based on the cell-based data provided, it is reasonable to expect that this “arm-less” variant could form dimers at higher concentrations, potentially detectable via cryoEM. Furthermore, the authors should examine how mutations at interfaces 1 and 2 (i.e., Extended data Figure 5)—which maintain the “bent over” conformation of ROS1—affect activity. Similar to the “arm-deleted” ROS1, these mutants are expected to show elevated basal activity. Biophysical and structural studies of these mutants would offer valuable insights into ROS1 activation.\nIn response to comment #2, the authors conducted SEC-MALS analysis of NELL2-ROS1 and report that NELL2 interacts with two copies of ROS1 with high affinity, while the binding of a third ROS1 appears comparatively weak, indicating possible negative cooperativity. In addressing comment #3, however, they state that \"the high avidity and cooperative binding of the NELL2 trimer would make it difficult for a monomer to outcompete the trimer on the cell surface.\" The authors are asked to clarify these statements, as they appear inconsistent. Additionally, in comment #2, I requested further explanation regarding the role of the “leg” region in ROS1 activation. It remains unclear whether the “legs” function merely as passive linkers or actively facilitate dimerization/trimerization through direct interaction. Clarifying this point is essential to understanding the mechanisms underlying ROS1 activation.", "reviewer_2": "All of my concerns have been addressed.", "reviewer_3": "The authors responses have adequately addressed my comments."}, "author_response": "Reviewer #1:\nThe authors have addressed the majority of my initial comments satisfactorily; however,\ntheir responses to comments #1 and #2 prompt further inquiries regarding the specific\nmechanism of ROS1 activation that warrant careful consideration.\nRegarding comment #1, the authors demonstrated that deletion of the \"arm\" region\nleads to increased basal activity of ROS1. This finding could be interpreted in two ways:\nas proposed by the authors, removal of the \"arm\" may relieve structural constraints on\nthe “leg” region, or alternatively, the \"arm\" might serve as a physical barrier restricting\ninteractions between “leg” regions required for receptor dimerization. It is therefore\nrecommended that both biochemical and structural analyses be performed on the ROS1\nvariant lacking the \"arm\" to clarify its activation mechanism. Based on the cell-based\ndata provided, it is reasonable to expect that this “arm-less” variant could form dimers at\nhigher concentrations, potentially detectable via cryoEM. Furthermore, the authors\nshould examine how mutations at interfaces 1 and 2 (i.e., Extended data Figure 5)—\nwhich maintain the “bent over” conformation of ROS1—affect activity. Similar to the\n“arm-deleted” ROS1, these mutants are expected to show elevated basal activity.\nBiophysical and structural studies of these mutants would offer valuable insights into\nROS1 activation.\nWe agree that biophysical and structural studies of how mutation and deletion is an\ninteresting avenue for future research. Our cellular studies on the arm-deleted version\n(suggested by the reviewer) was insightful and we thank the reviewer. The fact that we\nobserve some elevated basal activity is noted, but could simply be due to a pool of\nsticky protein or aggregated less stable protein. In RTKs, it is quite common that such\nchanges may lead to a portion of the receptors being trapped in the ER or Golgi and\nleading to higher basal signals. Importantly, there is no particular structure to study that\ngives a basis for such increased signaling.\nWhat we were very excited about from a mechanistic standpoint, is that in our\nstable cell lines that express truncated (arm deleted) ROS1, is that these receptors can\nnow respond to either bivalent antibody or site2/3 mutant ligand. This is a mechanistic\nslam dunk for our model: driving dimerization without the inhibitory arm can signal,\nwhereas simple dimerization when the arm restraint is in place does not lead to\nactivation. Therefore, the arm itself is not a physical restraint, but the arm releases\nconstraints in the leg that keep the TMs and kinase domains far enough apart to be\nnon-productive.\nIn response to comment #2, the authors conducted SEC-MALS analysis of NELL2-\nROS1 and report that NELL2 interacts with two copies of ROS1 with high affinity, while\nthe binding of a third ROS1 appears comparatively weak, indicating possible negative\ncooperativity. In addressing comment #3, however, they state that \"the high avidity and\ncooperative binding of the NELL2 trimer would make it difficult for a monomer to\noutcompete the trimer on the cell surface.\" The authors are asked to clarify these\nstatements, as they appear inconsistent.\nThese statements are not inconsistent; they refer to different features of a\nmultivalent interaction. Our SEC–MALS data indicate that a third ROS1 binding event to\na pre-assembled NELL2 trimer is comparatively weak, which is consistent with negative\ncooperativity or steric/geometric constraints that reduce the incremental affinity of the\nfinal binding step. However, on the cell surface the relevant quantity for competition is\nnot solely the microscopic affinity of each sequential binding step, but the functional\navidity created by multivalency. Once one NELL2 protomer engages a surface ROS1,\nthe trimer is effectively tethered in close proximity to additional receptors, which\nincreases the local effective concentration of its remaining binding sites and strongly\nsuppresses complete dissociation. Even if one ROS1–protomer contact transiently\nbreaks, the ligand can remain surface-associated through the other engagement(s) and\nrapidly rebind a second (or third) receptor. This “rebinding” effect lowers the apparent\noff-rate and yields high overall avidity, despite the possibility that the final binding step is\nweaker.\nBy contrast, a monomeric NELL2 species lacks this multivalent tethering and\ntherefore competes poorly against trimeric NELL2 at the cell surface unless present at\nsubstantially higher concentrations. Thus, weak or negatively cooperative occupancy of\nthe third site is compatible with strong overall binding/retention of the trimer through\navidity.\nFinally, we note that a more physiologically plausible antagonism mechanism is\nreceptor ectodomain shedding: a shed receptor can sequester ligand in solution and\nreduce productive engagement of cell-surface receptors, without requiring unrealistically\nhigh concentrations of monomeric ligand to outcompete a multivalent trimer.\nAdditionally, in comment #2, I requested further explanation regarding the role of\nthe “leg” region in ROS1 activation. It remains unclear whether the “legs” function\nmerely as passive linkers or actively facilitate dimerization/trimerization through direct\ninteraction. Clarifying this point is essential to understanding the mechanisms\nunderlying ROS1 activation.\nOur cryo-EM structures address this point directly. In our reconstructions, the “leg”\nregions do not form discernible leg–leg interfaces, and we do not observe architectural\nfeatures consistent with stable leg-mediated dimerization or higher-order assembly. This\ncontrasts with the invertebrate Sevenless system, where leg–leg contacts have been\nreported; we highlight this distinction explicitly in the final figure.\nThat said, while our data argue against a specific, high-affinity leg–leg interaction\nas a primary driver of activation, we cannot exclude the possibility of very weak,\ntransient contacts in the native 2D membrane environment. At high local receptor\ndensity, short-lived interactions in the micromolar to low-millimolar range could occur\ndue to confinement and proximity effects. Nonetheless, the absence of resolved leg–leg\ncontacts in the cryo-EM data supports the conclusion that the legs are not acting as an\nobligate, structured dimerization module in our system."}]

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