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1.
Protein Sci ; 33(6): e5016, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38747381

RESUMO

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.


Assuntos
Proteínas 14-3-3 , Nanoestruturas , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas p21(ras) , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Humanos , Nanoestruturas/química , Multimerização Proteica , Ligação Proteica , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo
2.
Eur J Cell Biol ; 103(2): 151414, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38640594

RESUMO

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K.

4.
Protein Expr Purif ; 212: 106361, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37652393

RESUMO

DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50-60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack of understanding of the biochemical and biophysical properties of the protein, likely due to its instability and poor solubility. To overcome this solubility issue, we engineered a DiRAS3 variant (C75S/C80S), which significantly improved soluble protein expression in E. coli. Recombinant DiRAS3 was purified by Ni-NTA and size exclusion chromatography (SEC). Concentration dependence of the SEC chromatogram indicated that DiRAS3 exists in monomer-dimer equilibrium. We then produced truncations of the N-terminal (ΔN) and both (ΔNC) extensions to the GTPase domain. Unlike full-length DiRAS3, the SEC profiles showed that ΔNC is monomeric while ΔN was monomeric with aggregation, suggesting that the N and/or C-terminal tail(s) contribute to dimerization and aggregation. The 1H-15N HSQC NMR spectrum of ΔNC construct displayed well-dispersed peaks similar to spectra of other GTPase domains, which enabled us to demonstrate that DiRAS3 has a GTPase domain that can bind GDP and GTP. Taken together, we conclude that, despite the substitutions in the G-box motifs, DiRAS3 can switch between nucleotide-bound states and that the N- and C-terminal extensions interact transiently with the GTPase domain in intra- and inter-molecular fashions, mediating weak multimerization of this unique small GTPase.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas ras , Escherichia coli/genética , Aminoácidos , Biofísica
5.
Angew Chem Int Ed Engl ; 62(18): e202218698, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-36883374

RESUMO

KRAS is a peripheral membrane protein that regulates multiple signaling pathways, and is mutated in ≈30 % of cancers. Transient self-association of KRAS is essential for activation of the downstream effector RAF and oncogenicity. The presence of anionic phosphatidylserine (PS) lipids in the membrane was shown to promote KRAS self-assembly, however, the structural mechanisms remain elusive. Here, we employed nanodisc bilayers of defined lipid compositions, and probed the impact of PS concentration on KRAS self-association. Paramagnetic NMR experiments demonstrated the existence of two transient dimer conformations involving alternate electrostatic contacts between R135 and either D153 or E168 on the "α4/5-α4/5" interface, and revealed that lipid composition and salt modulate their dynamic equilibrium. These dimer interfaces were validated by charge-reversal mutants. This plasticity demonstrates how the dynamic KRAS dimerization interface responds to the environment, and likely extends to the assembly of other signaling complexes on the membrane.


Assuntos
Bicamadas Lipídicas , Proteínas Proto-Oncogênicas p21(ras) , Bicamadas Lipídicas/química , Eletricidade Estática , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas/metabolismo , Conformação Molecular
6.
Nat Chem Biol ; 18(6): 578-579, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35314815
7.
J Mol Biol ; 434(9): 167527, 2022 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-35257782

RESUMO

Ral Guanine Nucleotide Dissociation Stimulator Like 1 (RGL1) is a RAS effector protein that activates Ral GTPase by stimulating nucleotide exchange. Most structures of RAS-effector complexes are for the HRAS isoform; relatively few KRAS-effector structures have been solved, even though KRAS mutations are more frequent in human cancers. We determined crystal structures of KRAS/RGL1-RAS-association (RA) domain complexes and characterized the interaction in solution using nuclear magnetic resonance spectroscopy, size-exclusion chromatography combined with multi-angle light scattering and biolayer interferometry. We report structures of wild-type KRAS and the oncogenic G12V mutant in complex with the RA domain of RGL1 at < 2 Å resolution. KRASWT/RGL1-RA crystallized as a 1:1 heterodimer, whilst KRASG12V/RGL1-RA crystallized as a heterotetrameric structure in which RGL1-RA dimerized via domain-swapping the C-terminal beta-strand. Solution data indicated that KRASWT and KRASG12V in complex with RGL1-RA both exist predominantly as 1:1 dimers, while tetramerization occurs through very slow association. Through detailed structural analyses, the distance and angle between RAS α1 helix and RBD/RA α1 helix were found to differ significantly among RAS and RBD/RA complexes. The KRAS/RGL1-RA structures possess some of the largest α1RAS/α1Effector distances (21.7-22.2 Å), whereas the corresponding distances in previously reported RAS/RAF complexes are significantly shorter (15.2-17.7 Å). Contact map analysis identified unique structural signatures involving contacts between the ß1-ß2 loop of RA and the α1 helix of RAS, clearly distinguishing the KRAS/RGL1-RA (and other RAS/RA complexes) from RAS/RBD complexes. These results demonstrate that RAS effectors employ an assortment of finely-tuned docking surfaces to achieve optimal interactions with RAS.


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Proteínas Proto-Oncogênicas p21(ras) , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Mutação , Domínios Proteicos , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética
8.
Mol Cell ; 82(5): 950-968.e14, 2022 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-35202574

RESUMO

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.


Assuntos
GTP Fosfo-Hidrolases , Transdução de Sinais , Animais , Cristalografia por Raios X , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mamíferos/metabolismo , Nucleotídeos , Proteínas
9.
J Thorac Oncol ; 17(2): 277-288, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34648945

RESUMO

INTRODUCTION: Mutations in BRAF occur in 2% to 4% of patients with lung adenocarcinoma. Combination dabrafenib and trametinib, or single-agent vemurafenib is approved only for patients with cancers driven by the V600E BRAF mutation. Targeted therapy is not currently available for patients harboring non-V600 BRAF mutations. METHODS: A lung adenocarcinoma patient-derived xenograft model (PHLC12) with wild-type and nonamplified EGFR was tested for response to EGFR tyrosine kinase inhibitors (TKIs). A cell line derived from this model (X12CL) was also used to evaluate drug sensitivity and to identify potential drivers by small interfering RNA knockdown. Kinase assays were used to test direct targeting of the candidate driver by the EGFR TKIs. Structural modeling including, molecular dynamics simulations, and binding assays were conducted to explore the mechanism of off-target inhibition by EGFR TKIs on the model 12 driver. RESULTS: Both patient-derived xenograft PHLC12 and the X12CL cell line were sensitive to multiple EGFR TKIs. The BRAFG469V mutation was found to be the only known oncogenic mutation in this model. Small interfering RNA knockdown of BRAF, but not the EGFR, killed X12CL, confirming BRAFG469V as the oncogenic driver. Kinase activity of the BRAF protein isolated from X12CL was inhibited by treatment with the EGFR TKIs gefitinib and osimertinib, and expression of BRAFG469V in non-EGFR-expressing NR6 cells promoted growth in low serum condition, which was also sensitive to EGFR TKIs. Structural modeling, molecular dynamic simulations, and in vitro binding assays support BRAFG469V being a direct target of the TKIs. CONCLUSIONS: Clinically approved EGFR TKIs can be repurposed to treat patients with non-small cell lung cancer harboring the BRAFG469V mutation.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética
10.
J Cell Sci ; 134(24)2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34851405

RESUMO

Cadherin-mediated cell adhesion requires anchoring via the ß-catenin-α-catenin complex to the actin cytoskeleton, yet, α-catenin only binds F-actin weakly. A covalent fusion of VE-cadherin to α-catenin enhances actin anchorage in endothelial cells and strongly stabilizes endothelial junctions in vivo, blocking inflammatory responses. Here, we have analyzed the underlying mechanism. We found that VE-cadherin-α-catenin constitutively recruits the actin adaptor vinculin. However, removal of the vinculin-binding region of α-catenin did not impair the ability of VE-cadherin-α-catenin to enhance junction integrity. Searching for an alternative explanation for the junction-stabilizing mechanism, we found that an antibody-defined epitope, normally buried in a short α1-helix of the actin-binding domain (ABD) of α-catenin, is openly displayed in junctional VE-cadherin-α-catenin chimera. We found that this epitope became exposed in normal α-catenin upon triggering thrombin-induced tension across the VE-cadherin complex. These results suggest that the VE-cadherin-α-catenin chimera stabilizes endothelial junctions due to conformational changes in the ABD of α-catenin that support constitutive strong binding to actin.


Assuntos
Caderinas , Células Endoteliais , Citoesqueleto de Actina , Actinas/genética , Caderinas/genética , Junções Intercelulares , Vinculina , alfa Catenina/genética
11.
Nat Commun ; 12(1): 6274, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34725361

RESUMO

Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.


Assuntos
Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Guanosina Trifosfato/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Mutação de Sentido Incorreto , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
12.
Chem Sci ; 12(38): 12827-12837, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34703570

RESUMO

KRAS forms transient dimers and higher-order multimers (nanoclusters) on the plasma membrane, which drive MAPK signaling and cell proliferation. KRAS is a frequently mutated oncogene, and while it is well known that the most prevalent mutation, G12D, impairs GTP hydrolysis, thereby increasing KRAS activation, G12D has also been shown to enhance nanoclustering. Elucidating structures of dynamic KRAS assemblies on a membrane has been challenging, thus we have refined our NMR approach that uses nanodiscs to study KRAS associated with membranes. We incorporated paramagnetic relaxation enhancement (PRE) titrations and interface mutagenesis, which revealed that, in addition to the symmetric 'α-α' dimerization interface shared with wild-type KRAS, the G12D mutant also self-associates through an asymmetric 'α-ß' interface. The 'α-ß' association is dependent on the presence of phosphatidylserine lipids, consistent with previous reports that this lipid promotes KRAS self-assembly on the plasma membrane in cells. Experiments using engineered mutants to spoil each interface, together with PRE probes attached to the membrane or free in solvent, suggest that dimerization through the primary 'α-α' interface releases ß interfaces from the membrane promoting formation of the secondary 'α-ß' interaction, potentially initiating nanoclustering. In addition, the small molecule BI-2852 binds at a ß-ß interface, stabilizing a new dimer configuration that outcompetes native dimerization and blocks the effector-binding site. Our data indicate that KRAS self-association involves a delicately balanced conformational equilibrium between transient states, which is sensitive to disease-associated mutation and small molecule inhibitors. The methods developed here are applicable to biologically important transient interactions involving other membrane-associated proteins.

13.
J Cell Biol ; 220(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34128958

RESUMO

Rab11 GTPase proteins are required for cytokinesis, ciliogenesis, and lumenogenesis. Rab11a is critical for apical delivery of podocalyxin (PODXL) during lumen formation in epithelial cells. SH3BP5 and SH3BP5L are guanine nucleotide exchange factors (GEFs) for Rab11. We show that SH3BP5 and SH3BP5L are required for activation of Rab11a and cyst lumen formation. Using proximity-dependent biotin identification (BioID) interaction proteomics, we have identified SH3BP5 and its paralogue SH3BP5L as new substrates of the poly-ADP-ribose polymerase Tankyrase and the E3 ligase RNF146. We provide data demonstrating that epithelial polarity via cyst lumen formation is governed by Tankyrase, which inhibits Rab11a activation through the suppression of SH3BP5 and SH3BP5L. RNF146 reduces Tankyrase protein abundance and restores Rab11a activation and lumen formation. Thus, Rab11a activation is controlled by a signaling pathway composed of the sequential inhibition of SH3BP5 paralogues by Tankyrase, which is itself suppressed by RNF146.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Sialoglicoproteínas/genética , Ubiquitina-Proteína Ligases/genética , Proteínas rab de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina , Humanos , Ligação Proteica , Transdução de Sinais/genética , Tanquirases/genética
14.
J Cell Sci ; 134(3)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33468626

RESUMO

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.


Assuntos
Sinalização do Cálcio , Proteínas de Neoplasias , Pancreatite Crônica , Molécula 1 de Interação Estromal , Cálcio/metabolismo , Sinalização do Cálcio/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Pancreatite Crônica/genética , Pancreatite Crônica/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
15.
J Biomol NMR ; 74(10-11): 531-554, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32804298

RESUMO

Mutations in RAS oncogenes occur in ~ 30% of human cancers, with KRAS being the most frequently altered isoform. RAS proteins comprise a conserved GTPase domain and a C-terminal lipid-modified tail that is unique to each isoform. The GTPase domain is a 'switch' that regulates multiple signaling cascades that drive cell growth and proliferation when activated by binding GTP, and the signal is terminated by GTP hydrolysis. Oncogenic RAS mutations disrupt the GTPase cycle, leading to accumulation of the activated GTP-bound state and promoting proliferation. RAS is a key target in oncology, however it lacks classic druggable pockets and has been extremely challenging to target. RAS signaling has thus been targeted indirectly, by harnessing key downstream effectors as well as upstream regulators, or disrupting the proper membrane localization required for signaling, by inhibiting either lipid modification or 'carrier' proteins. As a small (20 kDa) protein with multiple conformers in dynamic equilibrium, RAS is an excellent candidate for NMR-driven characterization and screening for direct inhibitors. Several molecules have been discovered that bind RAS and stabilize shallow pockets through conformational selection, and recent compounds have achieved substantial improvements in affinity. NMR-derived insight into targeting the RAS-membrane interface has revealed a new strategy to enhance the potency of small molecules, while another approach has been development of peptidyl inhibitors that bind through large interfaces rather than deep pockets. Remarkable progress has been made with mutation-specific covalent inhibitors that target the thiol of a G12C mutant, and these are now in clinical trials. Here we review the history of RAS inhibitor development and highlight the utility of NMR and integrated biophysical approaches in RAS drug discovery.


Assuntos
Descoberta de Drogas/métodos , Proteínas de Membrana/antagonistas & inibidores , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Mutação , Prenilação/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/química
16.
Cell Rep ; 32(8): 108074, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32846131

RESUMO

The small guanosine triphosphatase (GTPase) RAS serves as a molecular switch in signal transduction, and its mutation and aberrant activation are implicated in tumorigenesis. Here, we perform real-time, in-cell nuclear magnetic resonance (NMR) analyses of non-farnesylated RAS to measure time courses of the fraction of the active GTP-bound form (fGTP) within cytosol of live mammalian cells. The observed intracellular fGTP is significantly lower than that measured in vitro for wild-type RAS as well as oncogenic mutants, due to both decrease of the guanosine diphosphate (GDP)-GTP exchange rate (kex) and increase of GTP hydrolysis rate (khy). In vitro reconstitution experiments show that highly viscous environments promote a reduction of kex, whereas the increase of khy is stimulated by unidentified cytosolic proteins. This study demonstrates the power of in-cell NMR to directly detect the GTP-bound levels of RAS in mammalian cells, thereby revealing that the khy and kex of RAS are modulated by various intracellular factors.


Assuntos
Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Humanos
17.
Proc Natl Acad Sci U S A ; 117(22): 12101-12108, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414921

RESUMO

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD ß7-8), as well as an alternative interface comprising ß6 and the C terminus of CRD and ß2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD ß7-8 and KRAS α4-α5 while state B involved the C terminus of CRD, ß3-5 of RBD, and part of KRAS α5. Notably, α4-α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.


Assuntos
Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Cisteína/química , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética
18.
Int J Mol Sci ; 21(8)2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32326637

RESUMO

Calmodulin (CaM) is a Ca2+-sensor that regulates a wide variety of target proteins, many of which interact through short basic helical motifs bearing two hydrophobic 'anchor' residues. CaM comprises two globular lobes, each containing a pair of EF-hand Ca2+-binding motifs that form a Ca2+-induced hydrophobic pocket that binds an anchor residue. A central flexible linker allows CaM to accommodate diverse targets. Several reported CaM interactors lack these anchors but contain Lys/Arg-rich polybasic sequences adjacent to a lipidated N- or C-terminus. Ca2+-CaM binds the myristoylated N-terminus of CAP23/NAP22 with intimate interactions between the lipid and a surface comprised of the hydrophobic pockets of both lobes, while the basic residues make electrostatic interactions with the negatively charged surface of CaM. Ca2+-CaM binds farnesylcysteine, derived from the farnesylated polybasic C-terminus of KRAS4b, with the lipid inserted into the C-terminal lobe hydrophobic pocket. CaM sequestration of the KRAS4b farnesyl moiety disrupts KRAS4b membrane association and downstream signaling. Phosphorylation of basic regions of N-/C-terminal lipidated CaM targets can reduce affinity for both CaM and the membrane. Since both N-terminal myristoylated and C-terminal prenylated proteins use a Singly Lipidated Polybasic Terminus (SLIPT) for CaM binding, we propose these polybasic lipopeptide elements comprise a non-canonical CaM-binding motif.


Assuntos
Sinalização do Cálcio/genética , Calmodulina/química , Calmodulina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Motivos de Aminoácidos , Cálcio/metabolismo , Calmodulina/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosforilação , Plantas/química , Plantas/genética , Plantas/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Conformação Proteica , Isoformas de Proteínas , Prenilação de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Eletricidade Estática
19.
Angew Chem Int Ed Engl ; 59(27): 11037-11045, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32227412

RESUMO

KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4-α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, "cross"-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.


Assuntos
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Dimerização , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas p21(ras)/química
20.
Sci Signal ; 13(625)2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32234958

RESUMO

KRAS4b is a small guanosine triphosphatase (GTPase) protein that regulates several signal transduction pathways that underlie cell proliferation, differentiation, and survival. KRAS4b function requires prenylation of its C terminus and recruitment to the plasma membrane, where KRAS4b activates effector proteins including the RAF family of kinases. The Ca2+-sensing protein calmodulin (CaM) has been suggested to regulate the localization of KRAS4b through direct, Ca2+-dependent interaction, but how CaM and KRAS4b functionally interact is controversial. Here, we determined a crystal structure, which was supported by solution nuclear magnetic resonance (NMR), that revealed the sequestration of the prenyl moiety of KRAS4b in the hydrophobic pocket of the C-terminal lobe of Ca2+-bound CaM. Our engineered fluorescence resonance energy transfer (FRET)-based biosensor probes (CaMeRAS) showed that, upon stimulation of Ca2+ influx by extracellular ligands, KRAS4b reversibly translocated in a Ca2+-CaM-dependent manner from the plasma membrane to the cytoplasm in live HeLa and HEK293 cells. These results reveal a mechanism underlying the inhibition of KRAS4b activity by Ca2+ signaling pathways.


Assuntos
Calmodulina , Membrana Celular , Lipídeos de Membrana , Proteínas Proto-Oncogênicas p21(ras) , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Células HeLa , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
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