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Issue 34, 2020
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Structural impact of GTP binding on downstream KRAS signaling

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Abstract

Oncogenic RAS proteins, involved in ∼30% of human tumors, are molecular switches of various signal transduction pathways. Here we apply a new protocol for the NMR study of KRAS in its (inactive) GDP- and (activated) GTP-bound form, allowing a comprehensive analysis of the backbone dynamics of its WT-, G12C- and G12D variants. We found that Tyr32 shows opposite mobility with respect to the backbone of its surroundings: it is more flexible in the GDP-bound form while more rigid in GTP-complexes (especially in WT- and G12D-GTP). Using the G12C/Y32F double mutant, we showed that the presence of the hydroxyl group of Tyr32 has a marked effect on the G12C-KRAS-GTP system as well. Molecular dynamics simulations indicate that Tyr32 is linked to the γ-phosphate of GTP in the activated states – an arrangement shown, using QM/MM calculations, to support catalysis. Anchoring Tyr32 to the γ-phosphate contributes to the capture of the catalytic waters participating in the intrinsic hydrolysis of GTP and supports a simultaneous triple proton transfer step (catalytic water → assisting water → Tyr32 → O1G of the γ-phosphate) leading to straightforward product formation. The coupled flip of negatively charged residues of switch I toward the inside of the effector binding pocket potentiates ligand recognition, while positioning of Thr35 to enter the coordination sphere of the Mg2+ widens the pocket. Position 12 mutations do not disturb the capture of Tyr32 by the γ-phosphate, but (partially) displace Gln61, which opens up the catalytic pocket and destabilizes catalytic water molecules thus impairing intrinsic hydrolysis.

Graphical abstract: Structural impact of GTP binding on downstream KRAS signaling

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Supplementary files

Article information


Submitted
19 Jun 2020
Accepted
10 Aug 2020
First published
19 Aug 2020

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2020,11, 9272-9289
Article type
Edge Article

Structural impact of GTP binding on downstream KRAS signaling

D. K. Menyhárd, G. Pálfy, Z. Orgován, I. Vida, G. M. Keserű and A. Perczel, Chem. Sci., 2020, 11, 9272
DOI: 10.1039/D0SC03441J

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