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1.
Protein Expr Purif ; 212: 106361, 2023 12.
Article in English | MEDLINE | ID: mdl-37652393

ABSTRACT

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.


Subject(s)
Monomeric GTP-Binding Proteins , ras Proteins , Escherichia coli/genetics , Amino Acids , Biophysics
2.
J Mol Biol ; 434(9): 167527, 2022 05 15.
Article in English | MEDLINE | ID: mdl-35257782

ABSTRACT

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.


Subject(s)
Guanine Nucleotide Exchange Factors , Proto-Oncogene Proteins p21(ras) , Guanine Nucleotide Exchange Factors/chemistry , Humans , Mutation , Protein Domains , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/genetics
3.
Mol Cell ; 82(5): 950-968.e14, 2022 03 03.
Article in English | MEDLINE | ID: mdl-35202574

ABSTRACT

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.


Subject(s)
GTP Phosphohydrolases , Signal Transduction , Animals , Crystallography, X-Ray , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Guanosine Triphosphate/metabolism , Mammals/metabolism , Nucleotides , Proteins
4.
Chem Sci ; 12(38): 12827-12837, 2021 Oct 06.
Article in English | MEDLINE | ID: mdl-34703570

ABSTRACT

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.

5.
J Biomol NMR ; 74(10-11): 531-554, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32804298

ABSTRACT

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.


Subject(s)
Drug Discovery/methods , Membrane Proteins/antagonists & inhibitors , Nuclear Magnetic Resonance, Biomolecular/methods , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Signal Transduction/drug effects , Cyclic Nucleotide Phosphodiesterases, Type 6/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , Humans , Mutation , Prenylation/drug effects , Protein Binding , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism , Small Molecule Libraries/chemistry
6.
J Biol Chem ; 291(30): 15641-52, 2016 07 22.
Article in English | MEDLINE | ID: mdl-27226556

ABSTRACT

RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.


Subject(s)
Adenocarcinoma , Lung Neoplasms , Mutation, Missense , Neoplasm Proteins , Noonan Syndrome , Urologic Neoplasms , ras Proteins , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma of Lung , Amino Acid Substitution , Cell Line , Guanosine Triphosphate/chemistry , Humans , Hydrolysis , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Noonan Syndrome/genetics , Noonan Syndrome/metabolism , Protein Domains , Urologic Neoplasms/genetics , Urologic Neoplasms/metabolism , ras Proteins/chemistry , ras Proteins/genetics , ras Proteins/metabolism
7.
Proc Natl Acad Sci U S A ; 112(21): 6625-30, 2015 May 26.
Article in English | MEDLINE | ID: mdl-25941399

ABSTRACT

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.


Subject(s)
Genes, ras , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Amino Acid Sequence , Binding Sites/genetics , Humans , Lipid Bilayers , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins p21(ras)/chemistry , Sequence Homology, Amino Acid , Signal Transduction , Static Electricity , ral Guanine Nucleotide Exchange Factor/chemistry , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism
8.
J Mol Biol ; 425(21): 4034-46, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-23978697

ABSTRACT

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca(2+)) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca(2+) sensitivity, propagating sarcoplasmic reticulum luminal Ca(2+) release in the process of excitation-contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1-559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the ß-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense "columns" projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.


Subject(s)
Cardiomyopathies/genetics , Exons , Mutation , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/genetics , Amino Acid Sequence , Animals , Mice , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment/methods
9.
Methods ; 57(4): 473-85, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22750304

ABSTRACT

The Ras superfamily of small GTPases is a large family of switch-like proteins that control diverse cellular functions, and their deregulation is associated with multiple disease processes. When bound to GTP they adopt a conformation that interacts with effector proteins, whereas the GDP-bound state is generally biologically inactive. GTPase activating proteins (GAPs) promote hydrolysis of GTP, thus impeding the biological activity of GTPases, whereas guanine nucleotide exchange factors (GEFs) promote exchange of GDP for GTP and activate GTPase proteins. A number of methods have been developed to assay GTPase nucleotide hydrolysis and exchange, as well as the activity of GAPs and GEFs. The kinetics of these reactions are often studied with purified proteins and fluorescent nucleotide analogs, which have been shown to non-specifically impact hydrolysis and exchange. Most GAPs and GEFs are large multidomain proteins subject to complex regulation that is challenging to reconstitute in vitro. In cells, the activities of full-length GAPs or GEFs are typically assayed indirectly on the basis of nucleotide loading of the cognate GTPase, or by exploiting their interaction with effector proteins. Here, we describe a recently developed real-time NMR method to assay kinetics of nucleotide exchange and hydrolysis reactions by direct monitoring of nucleotide-dependent structural changes in an isotopically labeled GTPase. The unambiguous readout of this method makes it possible to precisely measure GAP and GEF activities from extracts of mammalian cells, enabling studies of their catalytic and regulatory mechanisms. We present examples of NMR-based assays of full-length GAPs and GEFs overexpressed in mammalian cells.


Subject(s)
Cell Extracts/chemistry , GTP Phosphohydrolases/chemistry , GTPase-Activating Proteins/chemistry , Guanine Nucleotide Exchange Factors/chemistry , Animals , Dyneins/chemistry , Enzyme Assays , Fluorescent Dyes/chemistry , Guanosine Triphosphate/chemistry , HEK293 Cells , Humans , Hydrolysis , Magnetic Resonance Spectroscopy , Monomeric GTP-Binding Proteins/chemistry , Neuropeptides/chemistry , Ras Homolog Enriched in Brain Protein , Recombinant Proteins/chemistry , Signal Transduction , Transfection , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/chemistry
10.
Proc Natl Acad Sci U S A ; 109(16): 6078-83, 2012 Apr 17.
Article in English | MEDLINE | ID: mdl-22474372

ABSTRACT

Forkhead box class O 3a (FOXO3a) is a transcription factor and tumor suppressor linked to longevity that determines cell fate through activating transcription of cell differentiation, survival, and apoptotic genes. Recruitment of the coactivator CBP/p300 is a crucial step in transcription, and we revealed that in addition to conserved region 3 (CR3) of FOXO3a, the C-terminal segment of CR2 (CR2C) binds CBP/p300 and contributes to transcriptional activity. CR2C and CR3 of FOXO3a interact with the KIX domain of CBP/p300 at both "MLL" and "c-Myb" binding sites simultaneously. A FOXO3a CR2C-CR3 peptide in complex with KIX exists in equilibrium between two equally populated conformational states, one of which has CR2C bound to the MLL site and CR3 bound to the c-Myb site, whereas in the other, CR2C and CR3 bind the c-Myb and MLL sites, respectively. This promiscuous interaction between FOXO3a and CBP/p300 is further supported by additional binding sites on CBP/p300, namely, the TAZ1 and TAZ2 domains. In functional studies, our structure-guided mutagenesis showed that both CR2C and CR3 are involved in the activation of certain endogenous FOXO3a target genes. Further, phosphorylation of S626, a known AMP-dependent protein kinase target in CR3, increased affinity for CBP/p300 and the phosphomimetic mutation enhanced transactivation of luciferase. These findings underscore the significance of promiscuous multivalent interactions and posttranslational modification in the recruitment of transcriptional coactivators, which may allow transcription factors to adapt to various gene-specific genomic and chromatin structures and respond to cell signals.


Subject(s)
Forkhead Transcription Factors/chemistry , Protein Structure, Tertiary , Transcriptional Activation , p300-CBP Transcription Factors/chemistry , Amino Acid Sequence , Animals , Binding Sites/genetics , Calorimetry , Cells, Cultured , Circular Dichroism , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , HCT116 Cells , HEK293 Cells , Humans , Kinetics , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Myeloid-Lymphoid Leukemia Protein/chemistry , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-myb/chemistry , Proto-Oncogene Proteins c-myb/genetics , Proto-Oncogene Proteins c-myb/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , p300-CBP Transcription Factors/genetics , p300-CBP Transcription Factors/metabolism
11.
J Mol Biol ; 403(3): 468-79, 2010 Oct 29.
Article in English | MEDLINE | ID: mdl-20826161

ABSTRACT

Immunoglobulin (Ig)-like domains are found frequently on the surface of tailed double-stranded DNA bacteriophages, yet their functional role remains obscure. Here, we have investigated the structure and function of the C-terminal Ig-like domain of gpV (gpV(C)), the tail tube protein of phage λ. This domain has been predicted through sequence similarity to be a member of the bacterial Ig-like domain 2 (Big_2) family, which is composed of more than 1300 phage and bacterial sequences. Using trypsin proteolysis, we have delineated the boundaries of gpV(C) and have shown that its removal reduces the biological activity of gpV by 100-fold; thus providing a definitive demonstration of a functional role for this domain. Determination of the solution structure of gpV(C) by NMR spectroscopy showed that it adopts a canonical Ig-like fold of the I-set class. This represents the first structure of a phage-encoded Ig-like domain and only the second structure of a Big_2 domain. Structural and sequence comparisons indicate that the gpV(C) structure is more representative of both the phage-encoded Big_2 domains and Big_2 domains in general than the other available Big_2 structure. Bioinformatics analyses have identified two conserved clusters of residues on the surface of gpV(C) that may be important in mediating the function of this domain.


Subject(s)
Bacteriophage lambda/chemistry , Immunoglobulins/chemistry , Viral Tail Proteins/chemistry , Bacteriophage lambda/metabolism , Crystallography, X-Ray , Immunoglobulins/metabolism , Kinetics , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Conformation , Thermodynamics , Viral Tail Proteins/metabolism
12.
J Biol Chem ; 285(8): 5132-6, 2010 Feb 19.
Article in English | MEDLINE | ID: mdl-20018863

ABSTRACT

The Ras family of small GTPases control diverse signaling pathways through a conserved "switch" mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2'(3')-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is approximately 10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay.


Subject(s)
Guanosine Triphosphate/analogs & derivatives , Monomeric GTP-Binding Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , ortho-Aminobenzoates/chemistry , Animals , Guanosine Triphosphate/chemistry , Humans , Hydrolysis , Kinetics , Mice , Recombinant Proteins/chemistry
13.
J Biol Chem ; 285(8): 5137-45, 2010 Feb 19.
Article in English | MEDLINE | ID: mdl-20018869

ABSTRACT

Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PH(PRG)). Using the non-hydrolyzable analogue guanosine-5'-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 x 10(-4) min(-1). This reaction is markedly accelerated to 1179 x 10(-4) min(-1) in the presence of DH-PH(PRG) at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PH(PRG), together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PH(PRG). Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles.


Subject(s)
Guanine Nucleotide Exchange Factors/chemistry , Guanosine Diphosphate/chemistry , Guanosine Triphosphate/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , rhoA GTP-Binding Protein/chemistry , Amino Acid Substitution , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Mutagenesis , Mutation, Missense , Protein Structure, Tertiary , Rho Guanine Nucleotide Exchange Factors , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism
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