Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring

Abstract

We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G­(i)­α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G­(i)­α, for which we synthesized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

“…f FLAG-KRAS WT or mutant, or constitutively active MRAS-Q71L immune precipitates were probed for endogenous RAF1 interaction and pathway activation was measured in lysates. "HTE" denotes substitution of inter-switch residues 43 QVV 45 (KRAS) to HTE (MRAS), and "HTE-NQWAI" was changed from 43 QVV 45 -48 GETCL 52 (KRAS) to HTE-NQWAI (MRAS). Representative of n = 3 independent experiments.…”

“…Protein purification. All RAF1(RBDCRD) proteins were purified following the protocols described in Lakshman et al 42 , whereas KRAS, RAF1(52-131), and MAP2K1 K97R-Avi-TEV-His6 proteins were purified as outlined for KRAS4b (1-169) in Kopra et al 43 Briefly, the expressed proteins of the form His6-MBP-TEV-target were purified from clarified lysates by IMAC, treated with His6-TEV protease to release the target protein, and the target protein separated from other components of the TEV protease reaction by the second round of IMAC. Proteins were further purified by gel-filtration chromatography in a buffer containing 20 mM HEPES, pH 7.3, 150 mM NaCl, 2 mM MgCl 2 (GTPases only), and 1 mM TCEP.…”

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

“…The structures provide detailed insights into the KRAS-CRD interaction interface and show how RBD and CRD are arranged with respect to one another. They also extend the footprint of RAF binding to RAS beyond the classical, highly conserved effector/RBD binding region in the switch I, to residues 23, 24 and 26, the inter-switch region (42)(43)(44)(45) and even residues 149, 153 and 157 in helix a5. The inter-switch region differs significantly amongst RASrelated proteins in the RRAS, RIT and RAP families and may explain why all of these proteins can bind RAF kinases, but only HRAS, KRAS and NRAS proteins activate RAF kinases efficiently.…”

“…The expression culture was grown at 37°C until OD600 reached 6-8, protein expression was induced with 0.5 mM IPTG, the culture was incubated at 16°C for 18-20 h, and the cells were harvested by centrifugation.Protein purification. All RAF1(RBDCRD) proteins were purified following the protocols described in Lakshman et al40 , whereas KRAS, RAF1, and MAP2K1 K97R-Avi-TEV-His6 proteins were purified as outlined for KRAS4b(1-169) in Kopra et al42 . Briefly, the expressed proteins of the form His6-MBP-TEV-target were purified from clarified lysates by IMAC, treated with His6-TEV protease to release the target protein, and the target protein separated from other components of the TEV protease reaction by the second round of IMAC.…”

Nanomolar Protein–Protein Interaction Monitoring with a Label-Free Protein-Probe Technique

“…We have previously studied protein–protein interactions using time-resolved FRET (TR-FRET) and developed a quencher-modulated TR-FRET method enabling simultaneous monitoring of both PLI and PPI reactions. 26 In addition, we have recently introduced an external Eu 3+ -labeled protein probe technique for the detection of protein stability and PLIs at a low nanomolar sensitivity level. 27 In this study, we introduce a further development of the protein probe for the detection of PPIs.…”