Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain

Abstract

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase αC-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

Figure 1.. MET domain boundaries and DMS experimental workflow.

(A) Domain boundaries of the full-length MET receptor (MET) and TPR-MET fusion (TPR-MET). Extracellular domain (ECD) and intracellular domain (ICD) are distinguished with important phosphorylation sites highlighted in red. Juxtamembrane domain (JM) boundaries are sectioned to annotate the exon 14 coding region and the remainder of the JM (JM2), which includes an αJM-helix (αJM). (B) Schematic of the full-length, membrane-associated MET receptor with posited MET ECD dimerization upon hepatocyte growth factor (HGF) binding; schematic of the cytoplasmically expressed, constitutively dimerized TPR-MET construct. The DMS mutagenesis region of the kinase domain (KD) is bordered in red. (C) Experimental screen workflow applied to generate and express kinase domain variants prior to selection, beginning with virus generation in Plat-E cells, transduction into Ba/F3 cells at a 0.1 multiplicity of infection (MOI), puromycin selection to enrich for positively infected cells, followed by the IL-3 selection process and time point collection for deep sequencing. (D) Post-selection method for analyzing and validating mutation scores based on observed variant frequencies at each time point, measured as a slope which can then be plotted as a distribution.

Figure 2.. Measured effect of MET kinase domain variants across 287 amino acid positions in the context of the full-length juxtamembrane.

(A) Heatmap of MET kinase domain mutation scores. WT-synonymous substitutions are outlined in green. (B) Active structure of the MET kinase domain (PDB 3R7O), and two representative inactive structures (PDB 2G15, 5HTI) with motif details highlighted. (C) Surface representation of average mutational scores mapped on an active structure (PDB 3R7O). Synonymous and nonsense mutations were left out of the averaging and surface representation. Residues at the N- and C-term that were not screened, but modeled in the crystal structure are in white and not considered in the averaging and mapping. (D) Comparison of surface and core residues scores distributions. A vertical dashed line in both graphs represents the mean score of WT-synonymous mutations. (E) Catalytic site and key residues involved in ATP binding and stabilization. Average score of variants mapped onto an active structure (PDB 3R7O). (F) Hinge region residues involved in ATP binding and stabilization. Average score of variants mapped onto an active structure (PDB 3R7O), and overlaid with the ATP molecule of the ATP-bound MET structure (PDB 3DKC). (G) Mutation scores and physiochemistry of variants shown for each residue position of the R- and C- spine of MET. (H) R-spine (blue) and C-spine (green) residues highlighted on an active structure (PDB 3R7O) overlaid with the ATP molecule of the ATP-bound MET structure (PDB 3DKC).

Figure 3.. Essential αJM and αC interactions revealed through variant and structural analysis.

(A) Ensemble of 93 MET kinase domain crystal structures available in the PDB. All structures, independent of conformation, were locally aligned to JM residues 1059–1070 (all resolved JM and αJM-helix residues in orange), and αC-helix residues 1117–1134 (teal). In solid gray is a representative active structure (PDB 3R7O). Residues involved in the αJM-helix and αC-helix interface. (B) Heatmap sections of the αJM-helix and αC-helix from the MET ICD screen. (C) Distribution of alpha carbon distances for residues in the αJM-helix and αC-helix interface, shown for 63 MET crystal structures in the ensemble with residues modeled for positions 1058, 1062, 1066, 1121, 1125, 1129. Distances are independent of conformation. (D) Global alignment of inactive and active RTK kinase domain structures with resolved JM regions. (E) Residue-by-residue, backbone RMSD comparisons of inactive and active structures of MET, AXL, IR, EPHA3, KIT, and RET. (F) MuscleWS alignment of human MET and TAM family juxtamembrane helix sequences. (G) Crystal structures of MET (PDB 3R7O), RON (PDB 3PLS), and AXL (PDB 5U6B) kinase domains, with αJM-helix (orange) and αC-helix (teal) highlighted. The inactive conformation of AXL shows an αJM-helix and αC-helix hydrophobic interaction similar to MET, but unlike MET, these interactions are slightly pivoted by an αJM-helix turn in its active conformation.

Figure 4.. β5 Proline motif is a structural pivot for the MET kinase domain.

(A) Respective sections of the MET ICD heatmap. (B) FLAG-IP western blot of the P1153L mutation post 24hr expression in HEK293 cells. (C) Residues of the MET P1153 N-lobe network displayed in an active (PDB 3R7O) and inactive structure (PDB 2G15). Surface representation of residues involved in the P1153 network. (D) Ramachandran plot and structural position of P1153 in MET and one representative kinase domain of each RTK subfamily. (E) Structural representation of the RTK Pro shift of the β4–5 loop. One representative RTK kinase domain from each sub-family is locally aligned to β4–5 of MET.

Figure 5.. Comparative measurement of MET kinase domain variants across 287 amino acid positions in the absence (TPR-METΔEx14) and presence of exon 14 (TPR-MET).

(A) Domain boundaries and schematics of the TPR-MET ICD and TPR-METΔEx14 ICD constructs. (B) Proliferation assay of parental TPR-MET, TPR-METΔEx14, and MSCV empty vectors expressed in Ba/F3 under IL-3 withdrawal and IL-3 conditions. Cell viability was normalized to day 0. (C) Heatmap of METΔEx14 kinase domain variant scores. WT-synonymous substitutions are outlined in green. (D) Scatter plot of TPR-METΔEx14 versus TPR-MET scores for each variant with distributions displayed on the margins. Dashed lines represent the WT synonymous average score for METΔEx14 versus MET. (E) Schematic of the kinase domain (PDB 2G15, 3R7O) in a full-length receptor and TPR-METΔEx14 context. (F) Western blot of endogenous MET KO HeLa cells transiently transfected with L1062D and S112Q mutants in the MET and METΔEx14 receptor, with and without HGF stimulation (50ng/ml, 15 min stimulation, 37°C).

Figure 6.. Mutations with greater proliferative effects than cancer-associated mutations, and differential sensitivities between MET and METΔEx14 identified.

(A) Lollipop diagram of MET kinase domain mutations and frequencies annotated in cBioPortal. (B) Distributions of clinically-associated mutations (green, right y-axis scale) overlaid with all missense mutations (gray, left y-axis scale). (C-D) Distributions of categorized cancer-associated mutations. Hamming distance distributions of clinical, validated MET cancer mutations and clinically unobserved, GOF mutations detected in the screen for both ICD backgrounds. (E) Cancer-associated mutations mapped onto a MET kinase domain structure, colored according to MET and METΔEx14 backgrounds, with Hamming distance represented by the ribbon thickness at each position (PDB 3R7O). (F) Reported resistance mutation distributions (teal, right y-axis scale) for MET and METΔEx14, overlaid with their respective missense distributions (gray, left y-axis scale). (G) Inhibitor resistance mutation positions shown on an active MET kinase domain structure in teal (PDB 3R7O).