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 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 differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for kinase domain activation 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.

Rosace: a robust deep mutational scanning analysis framework employing position and mean-variance shrinkage

“…To test the performance of , we generate simulated data using from two distinctive growth-based assays: the transporter OCT1 data where LOF variants are positively selected [4] and the kinase MET data where LOF variants are negatively selected [5]. The OCT1 DMS screen measures the impact of variants on cytotoxic drug SM73 uptake mediated by the transporter OCT1.…”

“…[4] as an example of positive selection and the MET dataset by Estevam et al . [5] as an example of negative selection. Specifically, we use replicate 2 of the cytotoxicity selection screen in the OCT1 dataset for both score distribution and raw count dispersion.…”

“…The relevant phenotype for a membrane transporter might be folding and trafficking or substrate transport [4]. These phenotypes are often captured by either growth-based [2, 4, 5, 6, 7, 8] or fluorescence-based assays [3, 4, 9]. Those two experiments are inherently differently designed and merit separate analysis frameworks.…”

“…However, as presented in Faure et al 2020 [10], this ratio-based method only applies to the DMS screen with one round of selection, while more recent DMS screens might have more than two rounds of selection (i.e. sampling at multiple time points) [4, 5]. Alternatively, EMPIRIC fits a Bayesian model that infers each variant separately with non-informative uniform priors to all parameters and thus does not shrink the estimates to robustly correct the variance in estimates due to the small sample size.…”

MET variants with activating N-lobe mutations identified in hereditary papillary renal cell carcinomas still require ligand stimulation

“…H1086 was mapped on the apo human MET kinase domain crystal structure (2G15) and on a representative “active” structure (3R7O) 58,59 (Figure 8D). MET has a canonical kinase domain structure, and H1086 is located in a structurally conserved helix belonging to the juxtamembrane sequence (⍺JM-helix) and packing on top of the ⍺C-helix 58,60 . The ⍺JM-helix and ⍺C-helix form a sensitive interface that is maintained by hydrophobic interactions between residues V1084/ I1147, L1080/L1143, and L1076/V1139 (Figure 8E).…”