Functional and structural insights into RAS effector proteins

Abstract

RAS proteins are conserved guanosine triphosphate (GTP) hydrolases (GTPases) that act as molecular binary switches and play vital roles in numerous cellular processes. Upon GTP binding, RAS GTPases adopt an active conformation and interact with specific proteins termed RAS effectors that contain a conserved ubiquitin-like domain, thereby facilitating downstream signaling. Over 50 effector proteins have been identified in the human proteome, and many have been studied as potential mediators of RAS-dependent signaling pathways. Biochemical and structural analyses have provided mechanistic insights into these effectors, and studies using model organisms have complemented our understanding of their role in physiology and disease. Yet, many critical aspects regarding the dynamics and biological function of RAS-effector complexes remain to be elucidated. In this review, we discuss the mechanisms and functions of known RAS effector proteins, provide structural perspectives on RAS-effector interactions, evaluate their significance in RAS-mediated signaling, and explore their potential as therapeutic targets.

Keywords: GTPase; RAS; RAS-binding domain; RBD; effector.

Figure 1.. The fundamentals of RAS-effector signaling

(A) Representative overview of the RAS binary switch. The effector binding domain (highlighted in blue) undergoes extensive conformational changes upon nucleotide exchange facilitating effector complex formation (red). PDB 6MBT (KRAS-GDP), 6MBQ (KRAS-GppNHp), and 6XHB (KRAS and CRAF RBD-CRD). (B) Potential factors affecting the competitive nature of RAS effectors (different effectors depicted as colored shapes). Dissociation constant (K_D) is the main driver of effector affinity, and some studies have systematically assessed the interaction (K_D) between RAS proteins and effectors. For instance, HRAS^GTP K_D (μM) for CRAF (0.09) < RASSF5 (0.24) < RALGDS (2.5) < PLCε (3.7) < PI3Kα (84.3). The dynamics of the interaction are also affected by association (k_on) and dissociation (k_off) rate constants, affecting the interaction between RAS and different effectors even if their affinity at equilibrium is similar. Local concentrations (or subcellular localization) and post-translational modifications of effectors can also modify their apparent affinity. For instance, RAP1 S11 phosphorylation and H/K/NRAS K147 mono-ubiquitination have been suggested to modify RAF interaction^, (C) Dendrogram of the RAS family of GTPases highlights their phylogenetic relationships and main clades, representing important subfamilies like classical RAS proteins (in blue). (D) Protein sequence alignment in the effector binding domain of different RAS GTPases demonstrates similarities and differences in critical residues that contribute to effector specificity. Amino acids are highlighted as follows: small polar (orange), hydrophobic (green), polar (magenta), negatively charged (red), and positively charged (blue). Red font indicates similar properties to the KRAS amino acid, but distinct amino acid (synonymous change). Amino acid sequences include KRAS (32–40), RRAS (58–66), RALA (43–51), RAP2A (32–40), RIT1 (50–58), ERAS (70–78), RHEB (35–43), RHES (48–56), DIRAS1 (36–44) and GEM (106–114). (E) Structural overlay of RAS protein in RAS-effector complexes, highlighting the conformational similarities of key interacting residues in the effector binding domain.

Figure 2.. Proposed human RAS effectors

There are over 50 human proteins in the human proteome that contain a prototypical RBD/RA domain in their amino acid sequence and have been proposed as RAS effectors. The figure shows a dendrogram resulting from the amino acid alignment of the RBD/RA domains of the indicated potential RAS effectors. Heat maps represents the protein expression levels of these effectors in the selected tissues (PRIDE Dataset). The values represent protein levels normalized to the highest expressing protein across each tissue (a) and to the highest espressing tissue (b) (and given a value of 100).

Figure 3.. Structural overview of RAS-effector complexes.

(A) Domain organization of human effector proteins, whose structures have been solved in complex with RAS proteins, is depicted. The effector domain(s), which are part of the RAS-effector complex, are shown in color, while the remaining domains are in gray. (B) The structures of RAS-effector complexes solved so far are shown in cartoon representation. These structures were aligned using RAS proteins, which are depicted in a grey cartoon with switch-I and switch-II regions colored purple and blue, respectively. Nucleotides and Mg ions are represented as sticks and spheres and are colored gray and green, respectively. RBD and adjacent domains are color-coded following the same scheme as in panel A. The structures of CRAF (PDB: 6XI7), PI3Kγ (PDB: 1HE8), GRB14 (PDB: 4K81), and SIN1 (PDB: 7LC1) in complex with H/KRAS were solved with RBD and adjacent domains, whereas the structures of RALGDS (PDB: 1LFD), RGL1 (PDB: 7SCW), RASSF5 (PDB: 3DDC), AFDN (PDB: 6AMB), and PLCε (PDB: 2C5L) in complex with HRAS were primarily solved using RBD/RA domains.

Figure 4.. Signaling pathways of RAS-effector complexes in model organisms and humans.

(A) Biochemical pathways regulated by RAS effectors in model organisms, including fission and baker’s yeast, slime mold, roundworms and fruit flies. Note the conserved modes of signal transduction, despite the diverse phenotypic outputs. (B) Overview of some of the different RAS effector signaling pathways that have been characterized in humans. Although many of these have been studied in the context of oncogenic KRAS signaling, they might represent pathways physiologically regulated by non-classical RAS GTPases.