To perform a DMS in TPR-MET, we generated a site saturation mutagenesis DNA library of the MET KD (Figure 1A). Our library also included the final alpha-helix of the JM region (⍺JM), which is resolved in most crystal structures, and is just upstream of the KD. Our variant library carried >99% of all possible missense mutations from positions 1059-1345 of human MET, including two internal controls: a WT-synonymous substitution at each position and premature stop codons every eleven positions (24 total). These controls allow us to use deep sequencing to estimate the fitness of WT and null variants, respectively. The library was cloned into the TPR fusion background containing the remaining JM (aa 963-1058), upstream of the ⍺JM and KD, and the complete C-terminal tail (aa 1346-1390) downstream of the KD (Figure 1A, B). We transduced the library into Ba/F3 cells using retrovirus (Figure 1A-C). Cells were grown in the presence and absence of IL-3 and samples were deep sequenced at distinct timepoints to identify variant frequencies (Figure 1C-D). We then measured variant fitness scores using Enrich2 (Rubin et al., 2017) for each selection condition (Figure 2A).

As expected, we observed no selection in the IL-3 control condition and a low correlation was observed across replicates (Pearson’s R=0.30). In this condition, all variants, including premature stop codons, displayed near WT-like fitness (Supplemental Data Figure 1A-C). In contrast, for the condition where IL-3 was withdrawn, we observed strong evidence of functional selection. There are large differences between the fitness distributions for WT-synonymous, missense, and nonsense mutations. Nonsense mutations are strongly loss-of-function (LOF) (Supplemental Data Figure 1B,C). Very few missense mutations are more fit than the average synonymous (WT) variants, which indicates that gain-of-function (GOF) mutations are rare. In addition fitness estimates for specific mutations across replicates of this treatment are strongly correlated (Pearson’s R=0.96) (Figure 2A, Supplemental Data Figure 1B-C).

MET contains a canonical tyrosine kinase domain, sharing structural and conformational hallmarks with other protein kinases (Linossi et al., 2021; Schiering et al., 2003; Wang et al., 2005). Kinase domains have a conserved hydrophobic core with several motifs that are important for folding, stability, conformational transitions, and catalysis. The conformational transition between an active and inactive state relies on the hinge-like closure of the N- and C-“lobes” around an ATP-bound catalytic site. The N-lobe is a dynamic unit comprised of five β-sheets and the ⍺C helix, while the C-lobe is a more rigid unit composed of seven ⍺-helices connected by loops (Kornev et al, 2006; Kornev et al, 2008). Two hydrophobic “spines” (the R- and C-spine) assemble across the lobes as the kinase transitions to an active state (Taylor & Kornev, 2011; Hu et al., 2014; Taylor et al., 2015). The transitions from inactive to active states of MET are typical for a tyrosine kinase: the ⍺C helix moves to an “in” position, positioning E1127 to form a salt-bridge with K1110. In addition, the flip of the DFG (residues 1222-1224) motif aligns F1223 with the rest of the R-spine and permits hydrogen bonds between D1222 and ATP (Kornev et al, 2006).

Our DMS results highlight that the conserved regulatory and catalytic motifs in MET are highly sensitive to mutation (Figure 2A-D). Catalytic site residues involved in either the chemical step of phosphate transfer or the conformational adaptation to ATP binding, such as K1110, E1127, D1222, and G1224, are intolerant of amino acid substitutions (Figure 2E). Residues surrounding the kinase “hinge”, which are involved in coordinating the adenosine potion of ATP (Azam et al., 2008; Dar and Shokat, 2011), are more tolerant to mutation (Figure 2F). This tolerance is especially prominent in residues that make backbone interactions with ATP. Variants in R-spine residues are strongly enriched in loss-of-function (LOF) fitness values. This result speaks to the importance of residue identity, not just physicochemical characteristics in the function of MET. R-spine residues F1223 and H1202 are part of the catalytic DFG and HRD motifs and do not tolerate any mutations (Figure 2G-H). For R-spine residues M1131 and L1142 positions, only a small number of polar uncharged substitutions show WT-like fitness (Figure 2G-H). Surprisingly, the MET C-spine was only moderately sensitive to mutations, with most hydrophobic and polar uncharged amino acid substitutions showing WT-like fitness (Figure 2G). We performed a sequence alignment of human RTK kinase domains for both the R- and C-spines, referenced to the MET kinase domain sequence (Supplemental Data Figure 2) to explore whether there was greater evolutionary conservation for the R-spine than the C-spine for MET. Consistent with our results, the C-spine was less conserved, but is enriched for hydrophobic residues (Supplemental Data Figure 2). These results indicated that key catalytic and regulatory motifs display varying sensitivity. The R-spine and catalytic residues are more highly constrained, whereas C-spine and hinge residues can tolerate a greater number of semi-conservative mutations.

On balance, most solvent exposed and loop regions were more permissive to mutations (Figure 2C-D). However, certain key regulatory elements stand out, with stronger mutational sensitivity than might be expected based on structural features alone. For example, the ⍺C helix-β4 loop which is important in ⍺C helix modulation and regulation in many kinases (Chen et al., 2007; Yeung et al., 2020), showed LOF effects in agreement with the previously described “ΦxHxNΦΦx” motif (Yeung et al., 2020). Similarly, the glycine residues in the “GxGxxG” motif of the P-loop, which gates ATP entry into the active site, were intolerant to substitutions. Two other relatively immutable sites included Y1234 and Y1235, in the activation loop (A-loop) of MET. The phosphorylation of these residues is required for stabilizing the A-loop in an extended conformation that enables substrate binding and efficient catalysis (Ferracini et al, 1991; Naldini et al, 1991). Collectively, this deep mutational scan confirms the importance of canonical kinase features and provides a reference point for discovering previously unexplored features of the MET kinase.

Given the importance of juxtamembrane (JM) regions in controlling the activation of many RTKs (Hubbard et al 2004; Zhang et al., 2006; Jura et al., 2009; Cabail et al., 2015; Wybenga-Groot et al., 2001; Wiesner et al., 2006) and the prevalence of exon 14 skipping within the JM of MET in cancer (Lu et al., 2017), we were interested in how patterns of mutational sensitivity from the DMS could relate the JM to the activation mechanism of MET. The MET JM (aa 956-1075) is predicted to be largely unstructured, but a small region (aa 1059-1070) folds into an alpha helix (⍺JM) that packs on top of the ⍺C helix of the kinase domain, forming a hydrophobic interface (Figure 3B). Since we included ⍺JM in the DMS library, we examined the pattern of substitutions for this region and the ⍺C helix. We observed a strong hydrophobic preference along both the ⍺JM and ⍺C helix residues comprising this interface (Figure 3A). The adjacent ⍺JM residue 1071 also strongly prefers hydrophobic residues. This residue does not interact with ⍺C helix, but evidently plays an important role in maintaining interactions with a hydrophobic patch in the N-lobe that includes residues L1076, L1097, and V1158. This result indicates the importance of burying the hydrophobic surfaces of the N-lobe, and ⍺C helix in particular, for maintaining an active kinase in the TPR-MET background.

Since the ⍺C helix conformation is a strong predictor of activity in kinases (Ung et al., 2018; Modi and Dunbrack, 2019), and is often modulated by protein-protein interactions or autoregulatory domains (Huse & Kuriyan, 2002), we hypothesized that these hydrophobic contacts might form preferentially in active MET kinase. The precedence for this idea comes from structural studies of RTK ICDs in the EGF, PDGF, EPH, and IR families (Zhang et al., 2006; Jura et al., 2009; Griffith et al., 2004; Wybenga-Groot et al., 2001; Wiesner et al., 2006; Li et al, 2003; Cabail et al., 2015). For example, in EGFR the JM stabilizes an active, asymmetric head-to-tail dimer (Zhang et al., 2006; Jura et al., 2009). In IR, the ⍺JM engages with ⍺C helix to maintain an inactive state, until ⍺JM-⍺C helix interactions are released and swapped to stabilize an active kinase dimer (Cabail et al., 2015). In contrast, in FLT3 the JM packs against the catalytic cleft to stabilize an inactive KD confirmation (Griffith et al., 2004). To test how MET kinase activation is linked to JM conformation, we compared the 93 crystal structures of the MET kinase domain with a modeled portion of its JM, as a MET pseudo-ensemble (Figure 3B). We aligned these structures on the N-lobe to eliminate the effect of the changes in relative N- and C-lobe alignment that occurs with activation. Despite large changes in the relative position of the C-lobe, corresponding to active and inactive structural hallmarks, there was little ⍺JM conformational variability or dependence on active/inactive state (3A,C). Rather, ⍺JM consistently packed against ⍺C helix and maintained the hydrophobic interface across all structures (Figure 3A-C).

To compare the MET ⍺JM-⍺C helix to other RTK JM-KD interactions, we compiled a set of human RTK kinase domain crystal structures that contain a modeled ⍺JM, and have both active and inactive structures available in the PDB. Within this set, IR and KIT displayed large conformational variability of the ⍺JM between active and inactive states, but MET stood out again in low conformational variability independent of the kinase activation state (Figure 3D,E). Indeed, when we examined JMs and kinase domains in a protein sequence alignment of all RTK, we observed that while ⍺C helix is conserved across RTKs, only MET (MET, RON), TAM (TYRO3, AXL, MER), and the RYK pseudokinase harbor an ⍺JM with a hydrophobic sequence pattern (Figure 3F, G). Together, the mutational sensitivity and structural conservation of MET’s ⍺JM-⍺C helix interface point to a model, that may be shared with RON and TAM family RTKs, where ⍺JM-⍺C helix contacts are maintained in both the active and inactive state and are important for TPR-MET stability and activity.

Although most regions of high sensitivity from the DMS impinge on well-described aspects of kinase activation, P1153 stood out as a previously unremarked upon position with extremely low tolerance to mutation in our DMS (Figure 4A). Only proline was tolerated in this position. P1153 is located in the β4-β5 region, which plays a role in ⍺C helix coordination and R-spine support (McClendon et al., 2014; Meharena et al., 2013; Taylor and Kornev, 2011). The β4 strand is connected to ⍺C helix and influences activity through regulatory mechanisms such as the “molecular break” in RTKs like FGFR, or through cis-and trans-protein interactions (Chen et al., 2007; Yeung et al., 2020). The β5 strand engages with ⍺C helix, the hinge, and harbors the disease-associated “gatekeeper” position (Azam et al., 2008). The immutability phenotype was especially notable because P1153 is not conserved across kinases (Supplemental Data Figure 3A).

To test the importance of this residue in a more physiological membrane-bound context, we expressed WT and P11553L variants of the full-length (FL) MET receptor in HEK293 cells. Based on the DMS in Ba/F3 cells, we expected that P1153 mutations would not signal or express poorly in this context. Indeed, relative to the WT MET receptor, P1153L expressed equivalently, but dramatically reduced signals for phosphorylation of the MET activation loop tyrosines, a marker of active MET signaling (Figure 4B). To test whether this importance extended to another MET family member, RON receptor, which also has proline residue at the equivalent position, we tested and observed similar results. Notably, in RON the equivalent proline mutation also significantly compromised receptor expression (Supplemental Data Figure 3B). These experiments provided initial validation that the results from the TPR-MET construct would translate to a full-length MET receptor context, and indicated the family-specific functional importance of proline in this position, of the β4-β5 loop.

Next we analyzed the structural environment of P1153. It packs in a hydrophobic cluster with the resistance-associated β3-⍺C helix “keymaster” position (L1112 in MET) (Persky et al., 2020) and F1124 of ⍺C helix. The hydrophobic packing around P1153 by F1124 and L1112 changes across MET active and inactive structures (Figure 4C). As ⍺C helix adopts an active “in” or inactive “out” conformation, L1112 rotates inward toward the core of the N-lobe to replace F1124 and maintain the hydrophobic environment around P1153 (Figure 4C). The maintenance of hydrophobic contacts across these conformational changes led us to re-examine the relative lack of sequence conservation of P1153 (Supplemental Data Figure 3A). We compiled a set of representative kinase domain structures from each RTK family, as well as those that have had DMS studies performed previously, and analyzed the Ramachandran angles of the analogous residue in the β4-β5 loop. Although most kinases have a non-proline amino acid at that position, the analogous residues cluster around or inside the proline-permissive contour of Ramachandran space (Figure 4D). Interestingly, while no other RTK family aside from MET and TAM family kinases have a proline at the β4-β5 loop position, most do have a proline exactly one position upstream. This shift occurs both in sequence and in structure: the upstream proline does not participate in the hydrophobic pivot observed here for P1153 in MET (Figure 4D). This analysis suggests that the MET and TAM families represent a subset of RTKs that have evolved the surrounding sequence to accommodate only proline in this structurally restrained region of the β4-β5 loop, explaining the immutability in our DMS experiment.

Exon 14 skipping, which maintains the ECD and transmembrane region while truncating the ICD at the JM, is one of the most common driver mutations observed in MET(Fernandes et al., 2021; Lu et al., 2017). The oncogenic effect is thought to, at least partially, result from removal of the docking site for Cbl, a ubiquitin ligase responsible for MET lysosomal degradation (Mak et al., 2007; Peschard et al., 2001; Petrelli et a., 2003; Abella et al., 2005; Kong-Beltran et al., 2006). Based on previous findings showing a TPR-METΔEx14 growth advantage over the MET receptor and METΔEx14 receptor, and TPR-MET in anchorage-independent assays of AALE cells (Lu et al., 2017) and fibroblasts (Vigna et al.,1999), we tested whether there was also a growth advantage in Ba/F3 cells (Figure 5A). Counter to expectations, we did not observe a discernible growth differential for TPR-METΔEx14 relative to TPR-MET (Figure 5B, Supplemental Data Figure 5A). It is important to note that exon 14 skipping normally occurs in the context of the full length protein, not in fusions such as TPR-MET. Therefore, the lack of a growth rate difference may be due to the need for Cbl regulation to occur at the plasma membrane (Mak et al., 2007) or due to other aspects of the Ba/F3 system. Although the lack of a growth differential means an exon 14-specific mechanism cannot be fully addressed in this experimental format, KD mutational responses are still representative and comparable.

We performed a parallel DMS of the MET KD in the background of an exon 14-skipped ICD (TPR-METΔEx14) (Figure 5C). Most regions implicated in the TPR-MET DMS were similarly sensitive in the TPR-METΔEx14 (Figure 5C,D; Supplemental Data Figure 5B). For example, the P1153 β4-β5 loop site was also intolerant to mutations in TPR-METΔEx14 (Supplemental Data Figure 5C). Similarly, the R- and C-spine were sensitive to physicochemical changes outside of hydrophobic and polar uncharged substitutions (Supplemental Data Figure 5D). To identify specific mutations of the KD that are uniquely sensitive to the presence or absence of exon 14, we filtered variants with the largest differences in activity scores between TPR-MET and TPR-METΔEx14 (Figure 5D, Supplemental Data Figure 5E).

As an example of the differences between the two backgrounds, we focused on the ⍺JM-⍺C helix interface. This region displayed relatively high sensitivity to non-hydrophobic substitutions in TPR-MET (Figure 3B), but it was more tolerant to mutations in the TPR-METΔEx14 background (Figure 5C, Supplemental Data Figure 5F). To test whether this ⍺JM and ⍺C helix sensitivity difference translated to the full-length receptor, we introduced L1062D, a mutation at the ⍺JM interface, and S1122Q, an ⍺C helix surface mutation, into FL-MET and FL-METΔEx14 backgrounds in HeLa cells lacking endogenous MET (Figure 5E, F). Consistent with the results from the DMS screen, in unstimulated cells, we observed that a marker of MET activation, A-loop phosphorylation, was dramatically reduced in MET receptor for L1062D, but less so in FL-METΔEx14, relative to WT controls (Figure 5F). Furthermore, upon HGF treatment, L1062D in the FL-METΔEx14 background exhibited a high degree of activation, but L1062D FL-MET did not. An additional marker of active MET signally, phosphorylation on p44/42 MAPK (ERK), was similarly responsive (Figure 5F). In contrast, S1122Q was expected to have a gain-of-function effect in the MET receptor and a loss-of-function effect in the METΔEx14 receptor. For this mutation we observed no difference relative to WT in A-loop phosphorylation at baseline or upon HGF stimulation (Figure 5F).

These results suggest that the correlation between DMS fitness in TPR-MET and acute A-loop phosphorylation in the MET receptor is not absolute. Since KD activation at a single time point was the only parameter we explored with the membrane-associated receptor, other aspects such as sustained signaling, recruitment of specific signaling adapters, or changes in MET regulation may be more consistent with the proliferative readout of the DMS. In addition, the inconsistency in the S1122Q result may be a specific feature of the dimerised, cytoplasmic TPR-MET signaling that is sensitive to interactions with the portion of the JM deleted by exon 14. Collectively, these results highlight that the ⍺JM-⍺C helix interface is sensitive to mutation and the presence or absence of exon 14 can alter this sensitivity. While the experimental parameters of this screen may limit how translatable some mutations are in a membrane-associated receptor or in non-proliferative conditions, the consistency of most residues between backgrounds provides increased confidence in the atlas of mutational effects on MET activation.

To assess the ability of the DMS to classify driver, passenger, and resistance mutations, we first gathered all MET kinase domain mutations reported from clinical observations in cBioPortal (Cerami et al., 2012; Gao et al., 2013) (Figure 6A). Relative to the distribution of all missense mutations, the distribution of clinically observed mutations is strongly shifted to higher fitness values for both TPR-MET and TPR-METΔEx14 screens (Figure 6B). Most of these mutations have near WT-fitness levels, with a small number having GOF fitness effects (Figure 6B). Next we further subdivided our observations based on annotations in cBioPortal as either “clinical, validated” or ”clinical, not validated”. Notably, mutations in the “clinical, not validated” category were outliers with lower fitness values, indicating that these are likely passenger mutations (Figure 6C,D). These results validate that the DMS recapitulates known oncogenic MET kinase variants and suggest that the screen can be used to help classify driver vs. passenger mutations.

Interestingly, there are some missense mutations that are strong GOF variants in the DMS that have not been reported clinically, potentially indicative of novel activating mutations. We classify these mutations as “not clinically observed, GOF.” We hypothesized that these variants are more difficult to observe clinically because of the constraints of the genetic code. To test this idea, we calculated the Hamming distance between the WT MET codon and mutant codon for each position within our dataset (Figure 6C-E). We found that most “clinical, validated” mutations had Hamming distances of 1-2 nucleotide substitutions from WT (Figure 6C,D). However, the most common Hamming distances for “not clinically observed, GOF’’ codons shifted 2-3 changes away from WT (Figure 6C, D). These GOF mutations are dispersed throughout the structure of MET kinase and have distinct patterns depending on the presence or absence of exon 14 (Figure 6E). These results suggest that the DMS can identify GOF mutations that require a larger genetic “leap” than what is observed in natural populations. Furthermore, the relative paucity of “not clinically observed, GOF ’’ mutations at Hamming distance of 1 suggests that clinical observations have nearly identified all possible activating MET mutations that require only a single nucleotide change. Collectively, this suggests that our atlas will be of particular use for deciding on driver status for rare mutations that require multiple nucleotide changes.

Finally, we assessed the distribution of fitness effects for clinically observed resistance mutations (Duplaquet et al., 2018; Fernandes et al., 2021; Saraon et al., 2021; Lu et al., 2017; Fujino et al., 2019). Resistance mutations, which are clustered around the active site, are also enriched towards higher fitness values than the background missense mutation distribution (Figure 6F, G). This result suggests that most of these mutations can pre-exist in the population even in the absence of selective pressure from an inhibitor. GOF resistance mutations may indicate an effect on the equilibrium of kinase activation, whereas LOF resistance mutations likely affect inhibitor-protein interactions directly. For instance, Y1230C is a recurrent resistance mutation (Bardelli et al., 1998) that interrupts Pi interactions that stabilize inhibitors at the active site, but in the absence of an inhibitor is unfavorable in METΔEx14 (Figure 6F, Supplemental Data Figure 6). These results indicate that DMS has potential to interpret the effects of resistance mutations, an area of active concern for patients being treated with MET inhibitors in the clinic.

Ba/F3 cells were purchased from DSMZ and maintained in 90% RPMI (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), and 10ng/ml IL-3 (Fisher), and incubated at 37°C with 5% CO2. Ba/F3 cells were passaged at or below 1.0E6 cells/ml in order to avoid acquired IL-3 resistance, and regularly checked for IL-3 addiction by performing 3x PBS (Gibco) washes and outgrowth in the absence of IL-3 to confirm cell death in the parental, empty cell line.

Plat-E cells stably expressing retroviral envelope and packaging plasmids were originally gifted by Dr. Wendell Lim. Plat-E cells were maintained in 90% DMEM+HEPES (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), 10ug/ml blasticidin, 1ug/ml puromycin, and incubated at 37°C with 5% CO2. Plat-E cells were maintained under blasticidin and puromycin antibiotic pressure unless being transfected.

HEK293 cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C in 5% CO2.

Human MET knockout HeLa cells were purchased from Abcam and maintained in 90% DMEM+HEPES (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), and incubated at 37°C with 5% CO2

Both pUC19 (Cat. 50005) and MSCV (Cat. 68469) were ordered from Addgene. To ensure unique cut sites within the vectors for introduction and shuttling of the variant library, a new multiple cloning site was introduced into each plasmid. Wild type TPR-MET-IRES-mCherry and TPR-METΔEx14-IRES-eGFP genes were cloned into pUC19 as the parental constructs for library generation and all site-directed mutagenesis. The full-length MET, METΔEx14, and RON receptor cDNAs were subcloned into pcDNA3.1 vector by Gibson assembly and incorporated a C-terminal single Flag tag sequence. All mutations were introduced by quick change mutagenesis.

The MET kinase domain variant library was designed to span amino acid positions 1059-1345, which contained the full kinase domain (aa 1071-1345) and a portion of the juxtamembrane (aa 1059-1070). The library was synthesized by Twist Bioscience with one mammalian high-usage codon per amino acid to prevent over-representation of specific residues. The library was received in 96-well arrays of lyophilized DNA at 50 ng per well, where each well contained all variants (missense and WT-synonymous) per position of the kinase domain. The lyophilized library was resuspended in 100uL of 1X TE buffer, and 5ng of DNA from each well was amplified with low cycle PCR to increase the starting material using the following the NEB Q5 High-Fidelity recipe per well: 10μL 5X Q5 buffer, 5ng template DNA, 2.5μL 10μM forward primer, 2.5μL 10μM reverse primer, 1μL 10mM dNTPs (2.5μM each), 0.5μL Q5 Polymerase, nuclease free water to a final volume of 50μL. The following thermocycler parameters were then applied: initial denaturation at 98°C for 30s, followed by 10x cycles of denaturation at 98°C for 10s, annealing at 62°C for 30s, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. A 1% agarose, 1X TBE diagnostic gel was run with 2μL of each sample to confirm amplification of all positions, then the samples were PCR cleaned using the Zymo 96-well DNA clean and concentrate kit, eluted in 10μL nuclease free water, pooled together in a low DNA-bind tube, and then DNA cleaned (Zymo) once more to further concentrate the pooled library.

The kinase domain variant library was digested with PstI-HF (NEB) and NdeI-HF (NEB) and cleaned up with the Zymo DNA clean and concentrate kit. Next, the two cloning vectors (pUC19_kozak-TPR-METΔEx14-IRES-eGFP and pUC19_kozak-TPR-MET-IRES-mCherry), were digested with PstI-HF(NEB) and NdeI-HF (NEB), phosphatase treated with rSAP (NEB), gel extracted to isolate the backbone, and DNA cleaned (Zymo). The variant library was ligated into each vector with a 1:3 (insert: vector) T4 ligation at 16°C overnight (NEB). Ligations were DNA cleaned (Zymo), eluted in 10uL of nuclease free water, and electroporated into 50μL MegaX 10 beta cells (Invitrogen). Transformations were then recovered in 1mL of SOC for 1hr at 37°C. Post recovery, 10μL cells were collected, serial diluted, plated at varying dilutions (1:100, 1:1k, 1:10k, 1:100k, 1:1M) to evaluate transformation efficiencies. The remainder of the transformation then propagated in 50mL LB and Carbyinacillin at 37°C to an OD of 0.5, and then midi-prepped (Zymo).

Amino acid variants were successfully synthesized by Twist Biosciences for all positions with the exception of 1194 and 1278. In addition, premature stop codons were not included in the synthesized Twist library. To include these missing positions and early stop control, we generated a “fill-in” library. For positions 1194 and 1278, a forward primer for each amino acid mutation and a single reverse primer was designed for inverse PCR. An early stop codon “fill-in” library was also generated to introduce one stop codon every 33 bases, evenly spaced throughout the gene. This resulted in one stop codon every 11 positions, or 24 total premature stops. Again, a single forward and reverse primer was designed for each stop codon mutation using inverse PCR. Mutations were introduced into wild type pUC19_kozak-TPR-METΔEx14-IRES-eGFP and pUC19_kozak-TPR-MET-IRES-mCherry with the following NEB Q5 High-Fidelity conditions per reaction: 10μL 5X Q5 buffer, 5ng template DNA, 2.5μL 10μM forward primer, 2.5μL 10μM reverse primer, 1μL 10mM dNTPs (2.5μM each), 0.5μL Q5 Polymerase, nuclease free water to a final volume of 50μL. The following thermocycler parameters were then applied: initial denaturation at 98°C for 30s, 10x cycles of denaturation at 98°C for 10s, annealing at 62°C for 30s, extension at 72°C for 4.4 min, and a final extension at 72°C for 10 min. A 1% agarose diagnostic gel was run with 2μL of each reaction to conform amplification. Then all PCR samples were pooled, DNA cleaned (Zymo) and eluted in 50μL nuclease free water, DPN1 digested to remove template (NEB), DNA cleaned again and eluted in 12μL nuclease free water, PNK treated (NEB), and blunt-end ligated at 16°C overnight with T4 ligase. Ligations were DNA cleaned the next morning (Zymo) and electroporated into MegaX 10 beta cells (Invitrogen). Transformations were recovered in 1mL of SOC for 1hr at 37°C, plated at varying dilutions to estimate transformation efficiencies, propagated in 50mL LB and Carbyinacillin at 37°C to an OD of 0.5, and then midi-prepped (Zymo). The Twist-synthesized library was then pooled together with the 1278, 1194, and premature stop “fill-in” libraries at equimolar concentrations to a total of 1ug of DNA.

7μg of each pooled library was restriction digested with MluI-HF (NEB) and MfeI-HF (NEB) to cut out variant libraries in the kozak-TPR-MET-IRES-mCherry and kozak-TPR-METΔEx14-IRES-eGFP backgrounds. Digests were gel extracted from pUC19 and DNA cleaned (Zymo). The empty, Puromycin resistant, retroviral expression vector MSCV (addgene) was also cut MluI-HF (NEB) and MfeI-HF (NEB), phosphatase treated with rSAP (NEB), and DNA cleaned. Isolated libraries were then ligated 1:1 (insert to vector) into the MSCV retroviral vector at 16°C overnight with T4 ligase. Ligations were then DNA cleaned (Zymo) and electroporated into ElectroMAX Stbl4 Competent Cells (Thermo Fisher). Transformations were recovered in 1mL of SOC for 1hr at 37°C and after recovery 10μL was serial diluted and plated to estimate transformation efficiencies, while the remainder was plated on bioassay dishes. Colonies were scraped from the bioassay dishes and midi-prepped for transfections (Zymo).

The MSCV_kozak-TPR-MET-IRES-mCherry and MSCV_kozak-TPR-METΔEx14-IRES-eGFP variant libraries were transfected into Plat-E cells for retroviral packaging using Lipofectamine3000 (Invitrogen). Two T-175 flasks of Plat-E cells were prepared for each library in the absence of blasticidin and puromycin 24hr prior to transfection such that they would be at 70-80% confluency at the time of transfection. On the day of transfection, each flask of Plat-E cells was gently washed with PBS to remove the culturing media, and replaced with 35mL Opti-MEM. For the transfection, Opti-MEM was brought to room temperature and two pairs of DNA Lo-bind 5mL tubes were prepared following manufacturer’s instructions for Lipofectamine3000 scaled to a T-175 format. A total of 46μg DNA was used to transfect the libraries and package virus in parallel: MSCV_TPR-MET-IRES-eGFP, MSCV_TPR-METΔEx14-IRES-mCherry. Each flask was incubated with the transfection reagents for 5hr at 37°C, 5% CO2; the transfection media was then replaced with 50mL OptiMEM, 5% FBS, 1x GlutaMax, and 2% Sodium Pyruvate (Gibco) for viral packaging. After 48 hr post-transfection, the viral supernatant was harvested, passed through a 0.45 μm filter to remove cell debris, then precipitated overnight with 1:4 Retro-X concentrator (TakaraBio) at 4°C, then pelleted at 380xg for 45 min at 4C, and resuspended in 5mL of sterile, cold PBS and stored at 4°C in 1mL aliquots until transduced into Ba/F3 cells.

The concentrated virus was titiered in Ba/F3 cells in a 6-well plate format. Cells were seeded at 1.0E5 cells/ml with 10 ng/ml IL-3 and 8μg/ml polybrene (Sigma-Aldrich). Virus was added to wells at 0, 10x, 20x, 40x dilutions to determine the proper volume for a transduction MOI of 0.1-0.3. Cells were spinfected at 250xg for 60 minutes at room temperature, then incubated for 48 hr. The viral titer was calculated from the percent of fluorescent cells and viral dilution.

For the DMS viral transduction, 6 million cells were spinfected at an MOI of 0.1, in triplicate for a total of 3 biological replicates for each library, and incubated post spinfection in a 15cm dish with 30mL Ba/F3 media and 10ng/L IL-3 for 48hr. Infected cells were then selected with 1μg/ml puromycin for a total of 4 days with fluorescence and cell counts tracked each day.

After puromycin selection, all three biological replicates for both libraries, TPR-MET and TPR-METΔEx14, were washed free of puromycin and IL-3 with 3x PBS washes. A total of 6 million cells from each replicate was harvested and pelleted at 250xg to serve as the “time point 0” pre-selection sample (T0).

To begin selection of each replicate for each library, two sets of 15cm dishes were prepared with 2.0E5 cells/ml in 30mL 90% RPMI, 10% HI-FBS, 1% penicillin/streptomycin. One plate was kept free of IL-3 as the experimental IL-3 withdrawal condition, while the other plate was supplemented with 10ng/mL IL-3 to provide the control condition. Three time points post T0 were collected for each library replicate and condition for a total of four time points (T0, T1, T2, T3). Time points were harvested every 48hr across 7 days; 6 million cells were harvested for each condition and pelleted at 250xg for 5min; 2.0E5 cells/ml were split at every time point and maintained either in IL-3 or IL-3 withdrawal conditions.

The gDNA of each time point sample was isolated with the TakaraBio NucleoSpin Blood QuickPure kit the same day the cells were harvested. gDNA was eluted in a 50μl elution buffer using the high concentration and high yield elution manufacturer’s protocol. Immediately after gDNA was isolated, 5μg of gDNA was used for PCR amplification of the target MET KD gene to achieve the proper variant coverage. A 150μl PCR master mix was prepared for each sample using the TakaraBio PrimeStar GXL system according to the following recipe: 30μl 5X PrimeStar GXL buffer, 4.5μl 10μM forward primer (0.3uM final), 4.5μl 10μM reverse primer (0.3uM final), 5μg gDNA, 12μl 10mM dNTPs (2.5mM each NTP), 6μl GXL polymerase, nuclease free water to a final reaction volume of 150uL. The PCR master mix was split into three PCR tubes with 50μl volumes and amplified with the following thermocycler parameters: initial denaturation at 98°C for 30 s, followed by 24x cycles of denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 68°C for 14 s, and a final extension at 68°C for 1 min.

After all time points were selected, harvested, and PCR amplified, the target gene amplicon was isolated from gDNA by gel purification. The entire 150μl PCR reaction for each sample was mixed with 1X NEB Purple Loading Dye (6X stock) and run on a 0.8% agarose, 1X TBE gel, at 100 mA until there was clear ladder and band separation. The target amplicons were gel excised and purified with the Zymo Gel DNA Recovery kit. To remove excess agarose contamination, each sample was then further cleaned using the Zymo DNA Clean and Concentrator-5 kit. Amplicon DNA concentrations were then determined by Qubit dsDNA HS assay (Invitrogen).

Libraries were then prepared for deep sequencing using the Nextera XT DNA Library Prep kit in a 96-well plate format (Illumina). Manufacturer’s instructions were followed for each step: tagmentation, indexing and amplification, and clean up. Libraries were indexed using the IDT for Nextera Unique Dual Indexes Set C (Illumina). Then, indexed libraries were quantified using the Agilent TapeStation with HS D5000 screen tape (Agilent) and reagents (Agilent). DNA concentrations were further confirmed with a Qubit dsDNA HS assay. All samples were manually normalized and pooled at 10nM for MET and METΔEx14. The libraries were then paired-end sequenced (SP300) on two lanes of a NovaSeq6000.

Demultiplexed paired-end reads were received from the sequencing core and processed further using a snakemake-based pipeline previously developed (Macdonald et al., 2022; Mölder et al., 2021). Initial QC was performed via FastQC (Andrews and Others, 2010), and continued via aggregation of intermediate output statistics with MultiQC (Ewels et al., 2016). First, any remaining adapter sequences or contaminant sequences were removed with BBDuk. Next, overlap-based error-correction was employed with BBMerge, before being mapped to the reference sequence with BBMap (Bushnell, 2014). Variant counts from each mapped BAM file were then made with the GATK AnalyzeSaturationMutagenesis tool (Van der Auwera and O’Connor, 2020). The output of this tool was processed using a script to remove variants that were not in our initial library design and to prepare output in a format for further processing with Enrich2 using weighted-least squares with wild type normalization (Rubin et al., 2017). It was noted that Enrich2 produces unexpected scores when some variants are unobserved across replicates or go to zero over a single time course: to avoid this, our script also detects this and removes them in advance.

Raw Enrich2 scores were used for all comparative “activity score” measurements. gain-of-function and loss-of-function missense mutations were classified and calculated as ±2 SD from the mean activity score of the WT-synonymous distributions for MET and METΔEx14. For comparative analysis, propagation of error was calculated from the delta activity score and delta SE of each variant for MET and METΔEx14, and only variants with a SE difference lower than the activity score difference were used.

HEK293 cells were transiently transfected with Lipofectamine3000 (Invitrogen) according to the manufacturer’s protocols. Cells were harvested 24 hours post transfection, lysed in buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 1 % w/v Triton X-100 supplemented with protease inhibitor tablets (Roche), 1 mM sodium fluoride and 1 mM sodium vanadate). Clarified lysates were incubated with G1 affinity resin (Genscript) overnight at 4°C. Resin was washed with lysis buffer (without inhibitors) and proteins eluted by addition of Laemmli sample reducing buffer. Proteins were separated by SDS-PAGE on a 4-15% gradient gel (BioRad) and transferred to PVDF membrane (Millipore). Membranes were probed with Flag (2368), MET pY1234/5 (3077) (Cell Signaling Technologies).

Human MET knockout HeLa cells were transiently transfected with Lipofectamine3000 (Invitrogen) according to the manufacturer’s protocols in a 6-well plate format. Post transfection (24hr), cells were washed with PBS (Gibco) (3x washes) to remove serum and transfection media, and replaced with DMEM (Gibco) in the absence of any additives. Cells were serum starved for 4 hr, then stimulated with 50 ng/ml HGF (PeproTech) for 15 min at 37°C, then immediately washed with cold PBS (3x washes), and maintained on ice. Cells were then lysed in buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 1 % w/v Triton X-100 supplemented with protease inhibitor tablets (Roche), 1 mM sodium fluoride, and 1 mM sodium vanadate) on ice. Clarified whole cell lysates were run on a 8-16% SDS-PAGE gel (BioRad) and transferred to a nitrocellulose membrane (BioRad). Membranes were probed with MET pY1234/5 (Cat# 3077), Met (8198), P-p44/42 MAPK Erk1/2 (T202/Y204) (4376), p44/42 MAPK Erk1/2 (4695), and β-Actin (4970) (Cell Signaling Technologies).

Ba/F3 cells stably expressing TPR-MET, TPR-METΔEx14, and empty MSCV constructs were seeded at 2.5E4 cells/ml in triplicate in a 94-well, round bottom plate for each time point in the presence and absence of 10ng/ml IL-3. CellTiter-Glo reagent (Promega) was mixed at a 1:1 ratio with cells and luminescence was measured on a Veritas luminometer at 0, 48, and 96 hrs post-seeding. In this study, we use a modified nomenclature, where we refer to TPR-MET as the TPR-fusion of MET with the full-length juxtamembrane sequence and TPR-METΔEx14 as the TPR-fusion lacking exon 14. Cell numbers were determined from a Ba/F3 cell and ATP standard curve generated according to the manufacturer’s instructions. Data are presented as cell viability normalized to the fold change from the 0hr time point.

For IL-3 titrations, Ba/F3 cells stably expressing TPR-MET, TPR-METΔEx14, and empty MSCV constructs were 3x PBS washed and 5000 cells were seeded in a 94-well, round bottom plate. IL-3 was added to wells at 0-10ng/ml (0, 0.078, 0.16, 0.31, 1.3, 2.5, 5, 10 ng/ml). CellTiter-Glo reagent (Promega) was mixed at a 1:1 ratio with cells and luminescence was measured on a Veritas luminometer at 0, 24, and 48 hrs after seeding and IL-3 addition.

Cell numbers for all proliferation assays were determined from a Ba/F3 and ATP CellTiter-Glo standard curve generated according to the manufacturer’s instructions. Data are presented as cell viability normalized to the fold change from the 0hr time point.

Structural visualization, mapping, and analysis was completed using PyMOL unless otherwise stated. All human MET (UniProtKB accession: P08581) kinase domain crystal structures currently available were downloaded from the PDB. All PDB structures were loaded and globally aligned to generate the kinase domain ensemble. Residue distances were calculated from alpha-carbon x,y,z coordinates and computationally analyzed. Raw PDB files were used to categorize structure features: resolution, construct boundaries, conformation, sequence features, mutations, and apo/holo states.

To choose representative active and inactive structures for score mapping and visualization, we generated an ensemble of 88 human MET kinase domain structures currently deposited in the PDB, and classified activity states based on alpha-carbon distances between catalytic site residues K1110, E1127, and F1223 (Modi and Dunbrack, 2019), with the majority of the MET KD structures in a “BLBplus” or “SRC-like” inactive conformation (Modi and Dunbrack, 2019). In study we refer to 3R7O, 3Q6W, 4IWD as as our representative “active” structures because they display classical active confirmation hallmarks (⍺C helix-in, K1110-E1127 salt bridge, DFG-in, solvent exposed A-loop despite being inhibited. Within the ensemble, there is only one ATP-bound structure (PDB 3DKC), which harbors A-loop Y1234F and Y1235D stabilizing mutations, and also displays an inactive conformation. Within the group of inactive structures, there are two main conformational species based on DFG/⍺C helix positioning and A-loop conformation: “BLBplus” and “BBAminus” (PDB 2G15 and PDB 5HTI represent the two species) (Modi and Dunbrack, 2019).

Crystal structures of active and inactive human IR (PDB 4XLV, 4IBM), KIT (PDB 1PKG, 1T45), EPHA3 (PDB 2QO9), RET (PDB 2IVT, 2IVS), MET (PDB 3R7O, 2G15), and AXL (PDB 5UAB) were obtained through the PDB. RMSD was calculated and plotted with the Bio3D package in R for each kinase using the inactive structure as the reference.

For β5 positional comparison and Ramachandran analysis, PDB files were obtained for each kinase, and analyzed with the Bio3D package in R to attain the Phi and Psi angles of each residue. The general and proline contour data was obtained from Lovell et al., 2003 and plotted as an overlay with the specific kinase β5 residues aforementioned.

Cancer-associated missense mutations for the MET kinase domain was obtained from cBioPortal (NCBI ID: NM_000245). Resistance mutations were obtained from literature references (Duplaquet et al., 2018; Fernandes et al 2021; Saraon et al., 2021; Lu et al., 2017; Fujino et al., 2019).

All human RTK protein sequences used in alignments were acquired from UniProt. Unless otherwise stated, alignments were done with MuscleWS using default parameters through JalView, and amino acids were colored according to physicochemical properties, or percent sequence identity where noted.