C-terminal Hypervariable Region Research Articles

Experimental variables determine the outcome of RAS-RAS interactions

RAS clustering at the cell membrane is critical to activate signaling in cells, but whether this clustering is mediated exclusively by its c-terminal hypervariable region, receives contributions from the G-domain of RAS, and/or is influenced by secondary effectors has been intensely debated. Reports that G-domain mutations do not modulate RAS-RAS interactions, have led some to question the validity of previous experiments that indicate the G-domain plays a role in RAS clustering/interactions. Here we reconcile these findings by clarifying the impact of experimental variables, such as protein expression levels, cellular context, RAS zygosity, and secondary effector interactions on RAS clustering. Lack of control over these variables impact the results using G-domain mutations across various assay systems and can lead to unsound conclusions.

RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis.

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.

Conserved allosteric perturbation of the GTPase domains by region 1 of Ras hypervariable regions

Ras proteins are important intracellular signaling hubs that can interact with numerous downstream effectors and upstream regulators through their GTPase domains (G-domains) anchored to plasma membranes by the C-terminal hypervariable regions (HVRs). The biological functions of Ras were proposed to be regulated at multiple levels including the intramolecular G-domain-HVR interactions, of which the exact mechanism and specificity are still controversial. Here, we demonstrate that the HVRs, instead of having direct contacts, can weakly perturb the G-domains via an allosteric interaction that is restricted to a ∼20 Å range and highly conserved in the tested Ras isoforms (HRas and KRas4B) and nucleotide-bound states. The origin of this allosteric perturbation has been localized to a short segment (residues 167–171) coinciding with region 1 of HVRs, which exhibits moderate to weak α-helical propensities. A charge-reversal mutation (E168K) of KRas4B in region 1, previously described in the Catalog of Somatic Mutations in Cancer database, was found to induce similar chemical shift perturbations as truncation of the HVR does. Further membrane paramagnetic relaxation enhancement (mPRE) data show that this region 1 mutation alters the membrane orientations of KRas4B and moderately increases the relative population of the signaling-compatible state.

Regulation of RAS palmitoyltransferases by accessory proteins and palmitoylation.

Palmitoylation of cysteine residues at the C-terminal hypervariable regions in human HRAS and NRAS, which is necessary for RAS signaling, is catalyzed by the acyltransferase DHHC9 in complex with its accessory protein GCP16. The molecular basis for the acyltransferase activity and the regulation of DHHC9 by GCP16 is not clear. Here we report the cryo-electron microscopy structures of the human DHHC9-GCP16 complex and its yeast counterpart-the Erf2-Erf4 complex, demonstrating that GCP16 and Erf4 are not directly involved in the catalytic process but stabilize the architecture of DHHC9 and Erf2, respectively. We found that a phospholipid binding to an arginine-rich region of DHHC9 and palmitoylation on three residues (C24, C25 and C288) were essential for the catalytic activity of the DHHC9-GCP16 complex. Moreover, we showed that GCP16 also formed complexes with DHHC14 and DHHC18 to catalyze RAS palmitoylation. These findings provide insights into the regulatory mechanism of RAS palmitoyltransferases.

Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-Cell 19F-NMR Spectroscopy.

Ras acts as a molecular switch to control intracellular signaling on the plasma membrane (PM). Elucidating how Ras associates with PM in the native cellular environment is crucial for understanding its control mechanism. Here, we used in-cell nuclear magnetic resonance (NMR) spectroscopy combined with site-specific 19F-labeling to explore the membrane-associated states of H-Ras in living cells. The site-specific incorporation of p-trifluoromethoxyphenylalanine (OCF3Phe) at three different sites of H-Ras, i.e., Tyr32 in switch I, Tyr96 interacting with switch II, and Tyr157 on helix α5, allowed the characterization of their conformational states depending on the nucleotide-bound states and an oncogenic mutational state. Exogenously delivered 19F-labeled H-Ras protein containing a C-terminal hypervariable region was assimilated via endogenous membrane-trafficking, enabling proper association with the cell membrane compartments. Despite poor sensitivity of the in-cell NMR spectra of membrane-associated H-Ras, the Bayesian spectral deconvolution identified distinct signal components on three 19F-labeled sites, thus offering the conformational multiplicity of H-Ras on the PM. Our study may be helpful in elucidating the atomic-scale picture of membrane-associated proteins in living cells.

Markov State Models and Molecular Dynamics Simulations Reveal the Conformational Transition of the Intrinsically Disordered Hypervariable Region of K-Ras4B to the Ordered Conformation.

K-Ras4B, the most frequently mutated Ras isoform in human tumors, plays a vital part in cell growth, differentiation, and survival. Its tail, the C-terminal hypervariable region (HVR), is involved in anchoring K-Ras4B at the cellular plasma membrane and in isoform-specific protein-protein interactions and signaling. In the inactive guanosine diphosphate-bound state, the intrinsically disordered HVR interacts with the catalytic domain at the effector-binding region, rendering K-Ras4B in its autoinhibited state. Activation releases the HVR from the catalytic domain, with its ensemble favoring an ordered α-helical structure. The large-scale conformational transition of the HVR from the intrinsically disordered to the ordered conformation remains poorly understood. Here, we deploy a computational scheme that integrates a transition path-generation algorithm, extensive molecular dynamics simulation, and Markov state model analysis to investigate the conformational landscape of the HVR transition pathway. Our findings reveal a stepwise pathway for the HVR transition and uncover several key conformational substates along the transition pathway. Importantly, key interactions between the HVR and the catalytic domain are unraveled, highlighting the pathogenesis of K-Ras4B mild mutations in several congenital developmental anomaly syndromes. Together, these findings provide a deeper understanding of the HVR transition mechanism and the regulation of K-Ras4B activity at an atomic level.

Facile Synthesis of Boc-Protected Selenocystine and its Compatibility with Late-Stage Farnesylation at Cysteine Site.

The unique hypervariable C-terminal region (HVR) of K-Ras4B, one of the most frequently mutated proteins in many powerful cancers, contains a C-terminal farnesylated and methylated Cys and a poly-lysine motif, which decides the association of K-Ras4B to the inner leaflet of plasma membrane for activating the downstream signaling activity. In our previous work, we inserted an additional Cys in K-Ras4B HVR peptide synthesis for NCL in the semi-synthesis of K-Ras4b protein, but it is not suitable for application in protein dimerization research. The recently developed selenocysteine (Sec, U) mediated native chemical ligation reaction followed by selective deselenization, which can help to broaden the scope of protein synthesis, requires the generation of the peptide fragment with an N-terminal Sec. To synthesize K-Ras4B HVR peptide containing both N-terminal Sec and C-terminal farnesylated and methylated Cys to achieve traceless protein semi-synthesis. We have developed a facile synthesis approach for producing Boc-Sec)2-OH using economic Se powder, which can facilitate scaling up preparation of peptides containing Sec at the N-terminus. Furthermore, we synthesized K-Ras4B HVR peptide containing selenocystine by utilization of Boc-Sec)2-OH. Finally, we took K-Ras4B HVR peptide as an example to test the compatibility of farnesylation reaction at Cys with the N-terminal Sec)2, and the farnesyl group was successfully added to the thiol group of Cys.

Genetic Validation of the Palmitoylation/Depalmitoylation Cycle As a Drug Target in NRAS Mutant Hematologic Malignancies In Vivo

The C-terminal hypervariable region (HVR) of Ras proteins specifies post-translational modifications required for their subcellular localization and biologic function. The HVR of all four Ras isoforms terminates with a CAAX motif that is prenylated by farnesyltransferase, producing a weak membrane binding affinity that is further stabilized by a second signal motif. For K-Ras4b, this signal is provided by a polybasic lysine domain that allows direct localization of the protein at the plasma membrane (PM). By contrast, H-Ras, N-Ras, and K-Ras4a PM localization requires palmitoylation at cysteine(s) adjacent to the CAAX motif and is maintained through a dynamic cycle regulated by depalmitoylation and repalmitoylation. Importantly, these differences raise the provocative possibility that inhibiting palmitoylation could selectively target NRAS mutant cancer cells without toxicity to normal cells that retain intact K-Ras4b function.Indeed, Nras oncogenes with a cysteine-to-serine second site amino acid substitution at the palmitoylation site (C181S) exhibited defective transforming activity in hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo (Cuiffo et al., Blood 2010; Xu et al., Blood 2012). However, these studies relied on retroviral promoters to express oncogenic N-RasG12D that resulted in supra-physiological protein levels in HSPCs. To overcome this limitation, we retargeted embryonic stem cells that were used to generate Lox-STOP-Lox (LSL) NrasG12D/+ knock in mice ( Haigis et al., Nat Genet 2008 ) to create a NrasG12D,C181S allele (hereafter called NrasC181S). We then performed intercrosses to generate Mx1-Cre, NrasC181S/C181S mice as well as cohorts of congenic Mx1-Cre, NrasWT/WT and Mx1-Cre, NrasG12D/G12D control animals. As expected (Li et al ., Blood 2011; Wang et al ., Cell Cycle 2011), Mx1-Cre, NrasG12D/G12D mice that were injected with poly(I:C) at weaning had a mean white blood cell (WBC) count of 58.3 x 103/µL at six months of age (Fig.1 A) and died from hematologic disease after 225 days (range 119-267 days, n= 14, Fig. 1B). By contrast, Mx1-Cre, NrasC181S/C181S mice had normal WBC counts at the same time point (mean: 11.34x103 WBC/µL) and remained well until 19-20 months of age (n=12). The difference in survival between these groups was highly significant (p=3x10-6 by Kaplan-Meier analysis). Detailed analysis of the hematopoietic compartment at six months of age (n=5 per genotype) revealed profound splenomegaly (mean weight: 1.272g) in NrasG12D/G12D mice, but normal spleen weights in the NrasWT/WT and NrasC181S/C181S animals (Fig. 1C). The expansion of granulocyte-monocyte progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and more primitive CD48+ Lin- c-Kit+ Sca1+ (LKS) cells previously observed in NrasG12D/G12D mice was also rescued by the C181S second site mutation. Furthermore, in vitro culture of HSPC-enriched bone marrow cells over a range of granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations revealed normal cytokine sensitivity of NrasC181S/C181S cells, while NrasG12D/G12D cells were characterized by cytokine-independent growth and GM-CSF hypersensitivity as previously described. Importantly, Ras-GTP levels were similarly elevated in bone marrow cells from both NrasG12D/G12D and NrasC181S/C181S mice compared to wild-type cells. This indicates that the C181S mutation abrogates the cytokine hypersensitivity and increased cell proliferation that results from oncogenic N-RasG12D expression without reverting the canonical biochemical consequence of the oncogenic G12D substitution. This observation, in turn, suggests that abnormal subcellular localization and trafficking of oncogenic N-Ras potently suppresses myeloid transformation. Taken together, these in vivo studies directly validate the palmitoylation/depalmitoylation cycle as a therapeutic target in hematologic malignancies and potentially in other cancers characterized by oncogenic NRAS mutations. [Display omitted] DisclosuresFirestone:Calico Life Sciences: Employment.

Genome-wide dissection reveals diverse pathogenic roles of bacterial Tc toxins.

Tc toxins were originally identified in entomopathogenic bacteria, which are important as biological pest control agents. Tc toxins are heteromeric exotoxins composed of three subunit types, TcA, TcB, and TcC. The C-terminal portion of the TcC protein encodes the actual toxic domain, which is translocated into host cells by an injectosome nanomachine comprising the other subunits. Currently the pathogenic roles and distribution of Tc toxins among different bacterial genera remain unclear. Here we have performed a comprehensive genome-wide analysis, and established a database that includes 1,608 identified Tc loci containing 2,528 TcC proteins in 1,421 Gram-negative and positive bacterial genomes. Our findings indicate that TcCs conform to the architecture of typical polymorphic toxins, with C-terminal hypervariable regions (HVR) encoding more than 100 different classes of putative toxic domains, most of which have not been previously recognized. Based on further analysis of Tc loci in the genomes of all Salmonella and Yersinia strains in EnteroBase, a “two-level” evolutionary dynamics scenario is proposed for TcC homologues. This scenario implies that the conserved TcC RHS core domain plays a critical role in the taxonomical specific distribution of TcC HVRs. This study provides an extensive resource for the future development of Tc toxins as valuable agrochemical tools. It furthermore implies that Tc proteins, which are encoded by a wide range of pathogens, represent an important versatile toxin superfamily with diverse pathogenic mechanisms.

Characterizing KRAS Membrane Structures by Data-Driven Molecular Docking

KRAS is a GTPase that plays an important role in cell growth and signaling pathways. of the different RAS isoforms, KRAS also has the highest prevalence of mutations related to human cancers, making it an attractive therapeutic target in these cases. Once attached to the membrane, KRAS in the active (GTP) form is capable to bind effector proteins, like RAF kinase. However, certain molecular details concerning KRAS conformation and orientational changes when interacting with the membrane and binding partners are not fully understood. To provide new insights, we used a variety of biophysical approaches to characterize KRAS structure and dynamics. Here, we focus on our results utilizing data-driven computational docking to investigate both KRAS and KRAS/RAF1-RBD (RAS Binding Domain) complex at the membrane. with the HADDOCK program, we incorporated experimental restraints derived from our NMR paramagnetic relaxation enhancement (PRE) and neutron reflectivity (NR) measurements to dock these KRAS forms to a 70:30 POPC:POPS lipid membrane surface. Using NMR-PRE restraints alone, we performed one series of docking runs with the KRAS G-domain directly interacting with the membrane to discern membrane-proximal states. Based on our experimental evidence, and particularly from NR, a highly populated membrane-distal state also exists, where the G-domain does not directly contact the membrane but KRAS remains tethered via the C-terminal hypervariable region (HVR). Therefore, we also conducted a second series of docking runs that incorporated both NMR-PRE and NR restraints to better elucidate the conformations in this state. From these results, we were able to generate atomistic models for KRAS and KRAS/RAF1-RBD with averaged 1-D profiles closely matching the respective NR profiles. Overall, the findings should assist in elucidating the role of KRAS structural dynamics in recruiting effectors, like RAF kinase, to the membrane for activation.

Cloning and Bioinformatics Analysis of the GlROP6 gene in Glehnia littoralis

Rho-related GTPase from plants (ROP) proteins play an essential role in plant stress resistance. In this study, the full-length <i>GlROP6</i> gene was cloned based on <i>G. littoralis</i> transcriptome sequencing data acquired in response to salt stress. The protein sequence, conserved domains, secondary structure, three-dimensional structure, phylogenetic relationships, and expression pattern of the <i>GlROP6</i> gene were systematically analysed. Our results showed that the full-length <i>GlROP6</i> gene had an open reading frame of 606 bp, which encoded 201 amino acid residues with a relative molecular weight of 22.23463 kDa and a theoretical isoelectric point of 9.06. Amino acid sequence analyses indicated that the structure of the GlROP6 protein was conserved, and included five G-box motifs (G1–G5), an effector binding region, a Rho insert region and a C-terminal hypervariable region. According to our phylogenetic analysis, the GlROP6 protein was closely related to the ROP protein of <i>Daucus carota</i> subsp. <i>Sativus</i>. Our quantitative real-time PCR results revealed that <i>GlROP6</i> was highly expressed in flower, and <i>GlROP6</i> expression was significantly upregulated in <i>G. littoralis</i> roots treated with NaCl. This study will facilitate investigations into the function of <i>GlROP</i> genes in response to salt stress in <i>G. littoralis</i>.

Membrane Tethering Potency of Rab-Family Small GTPases Is Defined by the C-Terminal Hypervariable Regions.

Membrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to enhance their intrinsic tethering potency, exhibiting 5- to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. Reconstituted membrane tethering assays with the C-terminally-truncated mutants of Rab4a further uncovered that the N-terminal residues in the HVR linker, located adjacent to the G-domain, are critical for regulating the intrinsic tethering activity. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

Abstract IA07: KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions

Abstract RAS proteins are GTP-dependent switches that control and regulate signaling pathways involved in cell fate and are frequently mutated in cancer. RAS association with the plasma membrane, or with certain endomembrane compartments, is a required step for its activity. Why precisely this is so remains an open question. One possibility is that RAS is merely required for recruitment of RAF and other effectors to the membrane where it can associate with key regulatory molecules that lead to signaling activation. However, four isoforms exist in humans (HRAS, NRAS, and two splice variants, KRAS4b and KRAS4a) and their differences lie within 22 amino acids in the C-terminal hypervariable region (HVR, aa 167-189). These differences in the domain responsible for membrane association result in the recruitment and organization of RAS into distinct membrane nanodomains, and it is thought that this results in differential signaling behavior from RAS isoforms. Isoform-specific RAS nanodomains are thought to both be highly dynamic, differentially composed of a variety of lipids and proteins, and supported by interactions with cytoskeletal structures. How these domains are regulated, and how they influence isoform-specific RAS signaling, remains unclear. Here, we report the molecular mobility of RAS variants in the plasma membrane of living cancer cells using single-molecule tracking methods. Detailed analysis of tracks revealed that KRAS4b molecules exhibit confined mobility with three diffusive states in the active plasma membrane of living cells. This diffusion characteristic was unique to KRAS4b and influenced by both the hypervariable region and globular domain of the protein, compared to all the other Ras isoforms. Importantly, the occupancy of each diffusive states was altered for the oncogenic mutant of KRAS4b, suggesting that the diffusive states we observe are related to signaling events. Our working hypothesis is that the HVR of KRAS4b may be directly involved in assembling the membrane lipid and protein nanodomain necessary for KRAS4b signaling activity. In addition, using two-color single-molecule tracking studies, we are beginning to characterize the interactions of KRAS4b with its major effector RAF. From these studies, we are learning about the kinetics of RAS/RAF interactions in the membrane of living cells. Understanding the underlying principle of KRAS4b functionality on cell membranes is useful for developing novel therapeutic strategies for targeting oncogenic KRAS4b. Citation Format: Debanjan Goswami, De Chen, John Columbus, Yue Yang, Felice Lightstone, Thomas Turbyville. KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA07.

The Hypervariable Region of K-Ras4B Governs Molecular Recognition and Function.

The flexible C-terminal hypervariable region distinguishes K-Ras4B, an important proto-oncogenic GTPase, from other Ras GTPases. This unique lysine-rich portion of the protein harbors sites for post-translational modification, including cysteine prenylation, carboxymethylation, phosphorylation, and likely many others. The functions of the hypervariable region are diverse, ranging from anchoring K-Ras4B at the plasma membrane to sampling potentially auto-inhibitory binding sites in its GTPase domain and participating in isoform-specific protein–protein interactions and signaling. Despite much research, there are still many questions about the hypervariable region of K-Ras4B. For example, mechanistic details of its interaction with plasma membrane lipids and with the GTPase domain require further clarification. The roles of the hypervariable region in K-Ras4B-specific protein–protein interactions and signaling are incompletely defined. It is also unclear why post-translational modifications frequently found in protein polylysine domains, such as acetylation, glycation, and carbamoylation, have not been observed in K-Ras4B. Expanding knowledge of the hypervariable region will likely drive the development of novel highly-efficient and selective inhibitors of K-Ras4B that are urgently needed by cancer patients.

Organization of Farnesylated, Carboxymethylated KRAS4B on Membranes

Mutations of the Ras proteins HRAS, KRAS4A, KRAS4B, and NRAS are associated with a high percentage of all human cancers. The proteins are composed of highly homologous N-terminal catalytic or globular domains, plus C-terminal hypervariable regions (HVRs). Post-translational modifications of all RAS HVRs helps target RAS proteins to cellular membrane locations where they perform their signaling functions. For the predominant KRAS4 isoform, KRAS4B, post-translational farnesylation and carboxymethylation, along with a patch of HVR basic residues help foster membrane binding. Recent investigations implicate membrane-bound RAS dimers, oligomers, and nanoclusters as landing pads for effector proteins that relay RAS signals. The details of these RAS signaling platforms have not been elucidated completely, in part due to the difficulties in preparing modified proteins. We have employed properly farnesylated and carboxymethylated KRAS4B in lipid monolayer incubations to examine how the proteins assemble on membranes. Our results reveal novel insights into to how KRAS4B may organize on membranes.

Abstract 2763: Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface

Abstract Ras proteins are classical members of small GTPases, controlling signal transduction pathways and promoting cell proliferation. Ras consists of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and activation is terminated by GTPase-activating proteins that induce the GTP hydrolysis. Ras has multiple partners, signals through several key pathways and fulfills critical functions in the cell life. Mutations in Ras are common in a variety of cancers; yet it is still undruggable. KRas4B is frequently mutated in cancer. Elucidation of Ras conformational ensembles at the signaling platforms in the plasma membrane including the ligand-bound conformations, membrane orientations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, and transition states are essential for deciphering Ras functions from its conformational landscapes. In the MAPK pathway, KRas4B preferentially recruits Raf-1 and activates it. Raf kinases contain Ras binding domain (RBD) and cysteine-rich domain (CRD) at conserved region 1 (CR1), and kinase domain at CR3. The high-affinity interaction of Raf RBD with Ras was solved in the absence of CRD. It is known that Raf CRD play a key role in the membrane anchoring mechanism. Recently, pivotal roles of CRD in the membrane interactions of KRas4B-Raf-1 RBD-CRD complex were reported. Here, we employ all-atom molecular dynamics (MD) simulations to investigate dimeric KRas4B-Raf-1 complex at the anionic lipid bilayer composed of DOPC and DOPS (4:1). Active KRas4B dimer modulates Raf-1 activation, promoting dimerization of Raf-1’s kinase domain. In the dimeric complex of KRas4B-Raf-1, Raf-1 CRD stably binds anionic lipid bilayers inserting its positively-charged loop into the amphipathic interface. The key basic residues at the loop are responsible for the membrane association, suggesting that CRD is an intrinsic membrane binding domain of Raf kinase. Our simulations suggest that Raf-1 CRD not only offers an additional membrane anchor for the KRas4B-Raf-1 complex, but also reduces the fluctuations of Ras-RBD, enhancing the high affinity interaction between Ras and Raf. Raf-1 CRD supports the active-state Ras orientation, promoting Ras dimerization through the allosteric lobe helical interface at the membrane. The enhanced stability of the complex at the membrane rendered by CRD promotes Raf dimerization in the MAPK signaling, which is the key Ras proliferative pathway. Here we suggest these significant roles of CRD at the membrane in the Raf activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Ruth Nussinov. Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2763.

Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

Rap1 proteins are members of the Ras subfamily of small GTPases involved in many biological responses, including adhesion, cell proliferation, and differentiation. Like all small GTPases, they work as molecular allosteric units that are active in signaling only when associated with the proper membrane compartment. Prenylation, occurring in the cytosol, is an enzymatic posttranslational event that anchors small GTPases at the membrane, and prenyl-binding proteins are needed to mask the cytoplasm-exposed lipid during transit to the target membrane. However, several of these proteins still await discovery. In this study, we report that cyclase-associated protein 1 (CAP1) binds Rap1. We found that this binding is GTP-independent, does not involve Rap1's effector domain, and is fully contained in its C-terminal hypervariable region (HVR). Furthermore, Rap1 prenylation was required for high-affinity interactions with CAP1 in a geranylgeranyl-specific manner. The prenyl binding specifically involved CAP1's C-terminal hydrophobic β-sheet domain. We present a combination of experimental and computational approaches, yielding a model whereby the high-affinity binding between Rap1 and CAP1 involves electrostatic and nonpolar side-chain interactions between Rap1's HVR residues, lipid, and CAP1 β-sheet domain. The binding was stabilized by the lipid insertion into the β-solenoid whose interior was occupied by nonpolar side chains. This model was reminiscent of the recently solved structure of the PDEδ-K-Ras complex; accordingly, disruptors of this complex, e.g. deltarasin, blocked the Rap1-CAP1 interaction. These findings indicate that CAP1 is a geranylgeranyl-binding partner of Rap1.

Ras Signaling: Allostery, Conformation, and Function

Ras is a small GTPase, controlling signal transduction pathways and promoting cell proliferation and survival. KRAS is frequently mutated in cancer. Ras consists of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and inactivation is terminated by GTPase-activating proteins that accelerate the intrinsic GTP hydrolysis rate by orders of magnitude. Ras has multiple partners, signals through several key pathways and fulfills critical functions in the cell life. Mutations in Ras are common in a variety of cancers; yet it is still undruggable. Elucidation of Ras conformational ensembles including the ligand-bound conformations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, transition states, and nonfunctional states are essential for deciphering Ras functions from its conformational landscapes. Our recent works highlight how these may help in elucidating vital mechanistic questions in Ras biology and hopefully contribute to therapeutic strategies. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

Intracellular mature IL-37 suppresses tumor metastasis via inhibiting Rac1 activation.

IL-37, a newly found anti-inflammatory cytokine of the IL-1 family, has both extracellular and intracellular functions. Accumulating evidences indicate that it is also involved in tumor progression. However, the mechanism and its intracellular target are unclear. In this study, clinical data from 84 patients showed that loss or reduced expression of IL-37 in lung adenocarcinoma tissues was significantly associated with tumor metastasis. We further provided evidence that IL-37 inhibited effectively tumor metastasis in vitro and in vivo. Moreover, we uncovered a novel mechanism by which IL-37 suppressed tumor cell migration via its intracellular mature form (amino acids 46-218). Intracellular mature form of IL-37, but not its extracellular form, markedly inhibited migration of multiple kinds of tumor cells through inhibiting Rac1 activation. Mechanistically, intracellular mature IL-37 directly bound to the CAAX motif in the C-terminal hypervariable region of Rac1, and then inhibited Rac1 membrane translocation and subsequent downstream signaling. Our research identifies intracellular mature IL-37 as a novel endogenous inhibitor of Rac1. Given the crucial roles of Rac1 in tumor angiogenesis and metastasis, intracellular mature IL-37 might serve as a potential strategy for the control of Rac1 activity and tumor progression.