Dissociation of the Signaling Protein K‐Ras4B from Lipid Membranes Induced by a Molecular Tweezer

Abstract

Oncogenic Ras mutationso ccur in more than 30 %o fh uman cancers. K-Ras4Bi st he most frequently mutatedisoform of Ras proteins. Developmentofeffective K-Ras4Bi nhibitors has been challenging, hence new approaches to inhibitt his oncogenic protein are urgently required.T he polybasic domain of K-Ras4Bw ith its stretch of lysine residues is essential for its plasma membrane targeting and localization. Employing CD and fluorescence spectroscopy,c onfocal fluorescence, and atomic force microscopy we showt hat the molecular tweezerC LR01 is able to efficiently bind to the lysine stretchi nt he polybasic domain of K-Ras4B, resulting in dissociation of the K-Ras4B protein from the lipid membranea nd disintegration of K-Ras4B nanoclusters in thel ipid bilayer.T hese results suggest that targeting of the polybasicd omain of K-Ras4B by properly designed tweezers mightr epresent an effective strategy for inactivation of K-Ras4Bsignaling.Ras proteins are oncoproteinsa nd play am ajor role in human cancers where they failt os witch off the signal for cell growth. More than 30 %o fh uman cancers are relatedt om utations of Ras proteins with K-Ras4Bb eing the most frequently mutated isoform. [1,2] They are plasma membrane localizedm olecular switches that function by shuttling between inactiveG DPbound and active GTP-bound forms. Signalingo fK -Ras4B strongly dependso ni ts correct localization in the plasma membrane. The polybasic domain (including 8lysine residues altogether) of K-Ras4Ba cts in combination with aC AAX motif to target this protein to the membrane. [3] For stable insertion, K-Ras4Bhas to be farnesylated in the C-terminal CAAX cysteine[a] L.Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles

“…Other tweezer conjugates have shown K D values mostly in the lower μM range (4–6 × 10 –6 M) against protein targets, such as K-Ras4B or survivin, including also multivalent tweezer conjugates targeting Taspase 1 . Binding in the upper nanomolar region (0.4 × 10 –6 M), similar to the tweezer NP conjugates of this study (0.7–1.2 × 10 –6 M), have previously only been observed for a conjugate of a tweezer with a high-affinity peptide ligand targeting 14-3-3 .…”

Exploring the Binding Mechanism of a Supramolecular Tweezer CLR01 to 14-3-3σ Protein via Well-Tempered Metadynamics

“…It can specifically form more stable inclusion complex with lysine rather than arginine ( Fokkens et al, 2005 ; Talbiersky et al, 2008 ; Schrader et al, 2016 ). It has been widely applied to modulate protein functions ( Bier et al, 2013 ; Schrader et al, 2016 ; Trusch et al, 2016 ; Vopel et al, 2017 ; Wilch et al, 2017 ; Mittal et al, 2018 ; Li et al, 2019 ; Bengoa-Vergniory et al, 2020 ; Weil et al, 2020 ; Brenner et al, 2021 ). Additionally, the optimization of CLR01 has gained considerable attention ( Dutt et al, 2013a ; Dutt et al, 2013b ; Klarner and Schrader, 2013 ; Heid et al, 2018 ; Herrera-Vaquero et al, 2019 ; Guillory et al, 2021 ; Li et al, 2021 ; Meiners et al, 2021 ).…”