Comprehensive Mutational Analysis of the MET Receptor Tyrosine Kinase Domain Reveals Critical Regulatory Motifs and Potential Activating Mutations

Core Concepts

The deep mutational scanning of the MET kinase domain has revealed critical regulatory motifs, including a conserved hydrophobic interaction between the juxtamembrane segment and the kinase α-C helix, as well as a structural pivot role for a proline residue in the β4-β5 loop. The study also identified previously unknown activating mutations in the MET kinase domain, which can aid in annotating driver, passenger, and drug resistance mutations.

Abstract

This study performed a comprehensive deep mutational scanning (DMS) of the MET receptor tyrosine kinase (RTK) domain to understand the impact of mutations on kinase function and regulation. The key findings are:

Regulatory and catalytic motifs in the MET kinase domain are highly sensitive to mutations, with the R-spine and catalytic residues being the most constrained.

The juxtamembrane (JM) region of MET forms a critical hydrophobic interface with the kinase α-C helix, which is important for maintaining kinase activity. This interaction is differentially sensitive to mutations in the presence or absence of the exon 14 coding region.

A proline residue (P1153) in the β4-β5 loop acts as a structural pivot for the kinase N-lobe, and is highly intolerant to mutations, likely due to co-evolution of the surrounding hydrophobic network.

The DMS identified several previously unknown activating mutations in the MET kinase domain, which can help classify driver, passenger, and drug resistance mutations.

Comparison of the mutational landscapes between wild-type MET and the exon 14-skipped METΔEx14 variant revealed some differential sensitivities, particularly at the JM-kinase interface, providing insights into the mechanism of action of this common oncogenic lesion.

Overall, this comprehensive mutational analysis of the MET kinase domain provides valuable insights into its regulation and identifies novel activating mutations, which can aid in understanding MET-driven cancers and developing targeted therapies.

Stats

MET kinase domain positions that are intolerant to mutations include the catalytic site residues K1110, E1127, D1222, and G1224.
The R-spine residues F1223 and H1202 do not tolerate any mutations.
The C-spine was more tolerant to mutations, with most hydrophobic and polar uncharged amino acid substitutions showing wild-type-like fitness.
The glycine residues in the "GxGxxG" P-loop motif were intolerant to substitutions.
Phosphorylation sites Y1234 and Y1235 in the activation loop were also highly constrained.

Quotes

"The deep mutational scanning of the MET kinase domain has revealed critical regulatory motifs, including a conserved hydrophobic interaction between the juxtamembrane segment and the kinase α-C helix, as well as a structural pivot role for a proline residue in the β4-β5 loop."
"The DMS identified several previously unknown activating mutations in the MET kinase domain, which can help classify driver, passenger, and drug resistance mutations."

Key Insights Distilled From

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

by Estevam,G. O... at www.biorxiv.org 08-03-2023

https://www.biorxiv.org/content/10.1101/2023.08.03.551866v3

Deeper Inquiries

What are the potential therapeutic implications of the identified critical regulatory motifs and novel activating mutations in the MET kinase domain?

The identified critical regulatory motifs and novel activating mutations in the MET kinase domain have significant therapeutic implications. Firstly, the critical hydrophobic interaction between the juxtamembrane segment and the kinase ⍺C-helix suggests a potential target for allosteric inhibitors. By disrupting this interaction, it may be possible to modulate MET activity and potentially inhibit aberrant signaling in MET-driven cancers. Additionally, the β5 motif identified as a structural pivot for the MET kinase domain N-lobe could be targeted for the design of novel inhibitors. By understanding the structural importance of this region, researchers can develop allosteric inhibitors that specifically target this pivot point, potentially leading to more effective therapeutic strategies for MET-related cancers.
Furthermore, the novel activating mutations identified through the deep mutational scanning provide valuable insights into potential driver mutations in MET. These mutations, which have not been clinically observed, may represent new targets for therapeutic intervention. By understanding the functional impact of these mutations on MET activity, researchers can develop targeted therapies that specifically inhibit these activating variants, offering personalized treatment options for patients with MET-driven cancers.

How might the differential sensitivities observed between wild-type MET and the METΔEx14 variant inform the development of targeted therapies for MET-driven cancers with exon 14 skipping?

The differential sensitivities observed between wild-type MET and the METΔEx14 variant provide important insights into the development of targeted therapies for MET-driven cancers with exon 14 skipping. The unique mutational landscape of the METΔEx14 variant, particularly in the juxtamembrane and kinase domains, highlights potential vulnerabilities and regulatory mechanisms specific to this oncogenic lesion. By understanding the differential sensitivities of key residues between wild-type MET and METΔEx14, researchers can design targeted therapies that specifically exploit the altered signaling pathways in cancers with exon 14 skipping.
For example, the increased sensitivity of certain residues in the juxtamembrane and kinase domains of METΔEx14 may indicate potential targets for therapeutic intervention. By developing inhibitors that selectively target these sensitive residues, it may be possible to disrupt the oncogenic signaling pathways activated by exon 14 skipping. Additionally, the identification of novel activating mutations in the METΔEx14 variant provides opportunities for the development of targeted therapies that specifically inhibit these driver mutations, offering more effective treatment options for patients with MET-driven cancers.

Could the structural pivot role of the proline residue in the β4-β5 loop be exploited for the design of allosteric inhibitors that avoid resistance mutations?

The structural pivot role of the proline residue in the β4-β5 loop presents a promising opportunity for the design of allosteric inhibitors that can avoid resistance mutations. The immutability of this proline residue to mutations suggests that it plays a critical role in maintaining the structural integrity of the MET kinase domain. By targeting this structural pivot point with allosteric inhibitors, researchers may be able to modulate MET activity in a way that circumvents the development of resistance mutations.
Allosteric inhibitors designed to interact with the proline residue in the β4-β5 loop could potentially stabilize specific conformations of the MET kinase domain, leading to the inhibition of aberrant signaling pathways associated with MET-driven cancers. By exploiting the unique structural properties of this proline residue, researchers can develop novel therapeutic strategies that target key regulatory motifs in the MET kinase domain, offering new avenues for the treatment of MET-related cancers while minimizing the risk of resistance development.