Valine Side Chains Research Articles

A “Hand‐In‐Hand” Thermal‐Responsive Cyclodextrin Material Designed for Liquid Smart Windows

AbstractThermochromic smart windows have emerged as a focal point in energy research. In this study, a novel approach is introduced for developing thermoresponsive materials for smart windows, utilizing microscopic agglomeration of molecules to regulate incident light. The groundbreaking material, β‐CD‐Val‐IBAm4, is constructed by grafting four isobutylated valines onto natural β‐cyclodextrins. This material exhibits a unique “hand‐in‐hand” effect, resulting from the interaction of isobutylated valine side chains, which leads to molecular aggregation and reduced light transmission at elevated temperatures. The hydration–dehydration process is related to molecular aggregation rather than crosslinking, ensuring the structural integrity and uniformity of the smart windows. Molecular dynamics simulations and 2D NOESY confirm the thermal response mechanism of β‐CD‐Val‐IBAm4. When dissolve in a glycerol‐water binary solvent system, the material forms a thermochromic liquid with outstanding optical performance (ΔTsol of 1 mm liquid can reach 69.8%) and long‐term stability. This innovative approach opens new avenues for the advancement of next‐generation smart window technologies.

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers.

The oxidation of polyunsaturated fatty acids by lipoxygenases (LOXs) is initiated by a C-H cleavage step in which the hydrogen atom is transferred quantum mechanically (i.e., via tunneling). In these reactions, protein thermal motions facilitate the conversion of ground-state enzyme-substrate complexes to tunneling-ready configurations and are thus important for transferring energy from the solvent to the active site for the activation of catalysis. In this report, we employed temperature-dependent hydrogen-deuterium exchange mass spectrometry (TDHDX-MS) to identify catalytically linked, thermally activated peptides in a representative animal LOX, human epithelial 15-LOX-2. TDHDX-MS of wild-type 15-LOX-2 was compared to two active site mutations that retain structural stability but have increased activation energies (Ea) of catalysis. The Ea value of one variant, V427L, is implicated to arise from suboptimal substrate positioning by increased active-site side chain rotamer dynamics, as determined by X-ray crystallography and ensemble refinement. The resolved thermal network from the comparative Eas of TDHDX-MS between wild-type and V426A is localized along the front face of the 15-LOX-2 catalytic domain. The network contains a clustering of isoleucine, leucine, and valine side chains within the helical peptides. This thermal network of 15-LOX-2 is different in location, area, and backbone structure compared to a model plant lipoxygenase from soybean that exhibits a low Ea value of catalysis compared to the human ortholog. The presented data provide insights into the divergence of thermally activated protein motions in plant and animal LOXs and their relationships to the enthalpic barriers for facilitating hydrogen tunneling.

Clues to the Design of Aggregation-Resistant Insulin from Proline Scanning of Highly Amyloidogenic Peptides Derived from the N-Terminal Segment of the A-Chain.

Insulin aggregation poses a significant problem in pharmacology and medicine as it occurs during prolonged storage of the hormone and in vivo at insulin injection sites. We have recently shown that dominant forces driving the self-assembly of insulin fibrils are likely to arise from intermolecular interactions involving the N-terminal segment of the A-chain (ACC1-13). Here, we study how proline substitutions within the pilot GIVEQ sequence of this fragment affect its propensity to aggregate in both neutral and acidic environments. In a reasonable agreement with in silico prediction based on the Cordax algorithm, proline substitutions at positions 3, 4, and 5 turn out to be very effective in preventing aggregation according to thioflavin T-fluorescence-based kinetic assay, infrared spectroscopy, and atomic force microscopy (AFM). Since the valine and glutamate side chains within this segment are strongly involved in the interactions with the insulin receptor, we have focused on the possible implications of the Q → P substitution for insulin's stability and interactions with the receptor. To this end, comparative molecular dynamics (MD) simulations of the Q5P mutant and wild-type insulin were carried out for both free and receptor-bound (site 1) monomers. The results point to a mild destabilization of the mutant vis à vis the wild-type monomer, as well as partial preservation of key contacts in the complex between Q5P insulin and the receptor. We discuss the implications of these findings in the context of the design of aggregation-resistant insulin analogues retaining hormonal activity.

Backbone and ILV side-chain NMR resonance assignments of the catalytic domain of human deubiquitinating enzyme USP7.

Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme, which removes ubiquitin tag from numerous protein substrates involved in diverse cellular processes such as cell cycle regulation, apoptosis and DNA damage response. USP7 affects stability, interaction network and cellular localization of its cellular and viral substrates by controlling their ubiquitination status. The large 41kDa catalytic domain of USP7 harbors the active site of the enzyme. Here we present a nearly complete (93%) NMR resonance assignment of isoleucine, leucine and valine (ILV) side-chains of the USP7 catalytic domain along with a refined nearly complete (93%) assignment of its backbone resonances. The reported ILV methyl group assignment will facilitate further NMR investigations of structure, interactions and conformational dynamics of the USP7 enzyme.

Deuterium solid-state NMR quadrupolar order rotating frame relaxation with applications to amyloid-β fibrils.

We describe a new method for measuring molecular dynamics based on the deuterium solid-state nuclear magnetic resonance (NMR) quadrupolar order rotating frame relaxation rateR1ρ,Q under static conditions. The observed quadrupolar order coherence is created using the broad-band Jeener-Broekaert excitation and is locked with a weak radio frequency (RF) field. We describe the experimental and theoretical approaches and show applications to a selectively deuterated valine side chain of the phosphorylated amyloid-β (1-40) fibrils phosphorylated at the serine-8 position. The R1ρ,Q rate is sensitive to the rotameric exchange mode. For biological samples, the low spin-lock field in the 5- to 10-kHz range has the advantage of avoiding sample heating and dehydration. Thus, it provides an alternative to approaches based on single-quantum coherence, which require larger spin-lock fields.

Cycloalkyl Groups as Subunits of Artificial Carbohydrate Receptors: Effect of Ring Size of the Cycloalkyl Unit on the Receptor Efficiency

1,3,5‐Trisubstituted 2,4,6‐triethylbenzenes consisting of isopropyl groups as recognition units have previously been shown to have interesting binding properties towards selected carbohydrates. The design of such artificial carbohydrate receptors was inspired by the mode of action of carbohydrate‐binding proteins, namely by the participation of the isopropyl side chain of valine in the formation of van der Waals interactions with the carbohydrate substrate. This study aimed to investigate how the replacement of the isopropyl groups by other structural motifs, such as cycloalkyl groups of varying sizes (three to seven‐membered rings), influences the binding properties of the acyclic compounds towards carbohydrates. The cyclopropyl moiety represents a cyclic analog of the isopropyl group, whereas the other cycloalkyl units can be regarded as structural elements, which differ from the structure of the isopropyl group by a –(CH2)n‐bridge (n = 1–4) between the two methyl groups. Furthermore, according to the natural interactions, the cyclopentyl group should be able to participate in the formation of van der Waals contacts in a similar way as that observed for the pyrrolidine ring of proline in some protein‐carbohydrate complexes. Such systematic binding studies allow the identification of interesting structure‐activity relationships, which are very useful for the development of artificial carbohydrate receptors with predictable binding properties.

Statistically Optimal Continuous Free Energy Surfaces from Biased Simulations and Multistate Reweighting

Free energies as a function of a selected set of collective variables are commonly computed in molecular simulation and of significant value in understanding and engineering molecular behavior. These free energy surfaces are most commonly estimated using variants of histogramming techniques, but such approaches obscure two important facets of these functions. First, the empirical observations along the collective variable are defined by an ensemble of discrete observations, and the coarsening of these observations into a histogram bin incurs unnecessary loss of information. Second, the free energy surface is itself almost always a continuous function, and its representation by a histogram introduces inherent approximations due to the discretization. In this study, we relate the observed discrete observations from biased simulations to the inferred underlying continuous probability distribution over the collective variables and derive histogram-free techniques for estimating this free energy surface. We reformulate free energy surface estimation as minimization of a Kullback-Leibler divergence between a continuous trial function and the discrete empirical distribution and show that this is equivalent to likelihood maximization of a trial function given a set of sampled data. We then present a fully Bayesian treatment of this formalism, which enables the incorporation of powerful Bayesian tools such as the inclusion of regularizing priors, uncertainty quantification, and model selection techniques. We demonstrate this new formalism in the analysis of umbrella sampling simulations for the χ torsion of a valine side chain in the L99A mutant of T4 lysozyme with benzene bound in the cavity.

The water molecule arrangement over the side chain of some aliphatic amino acids: A quantum chemical and bottom‐up investigation

AbstractThe geometry of surrounding water molecules on the side chain of glycine, alanine, α‐aminoisobutyric acid, α‐aminobutyric acid, valine, and related hydrocarbons has been analyzed combining bottom‐up and quantum chemical methodologies. To minimize the cavity size and to prevent water‐water hydrogen bonding loss, the water molecules adopt a shape, resembling the one found in crystal structure of gas clathrate hydrates, with water molecules tangentially oriented to the surface of hydrophobic side chain. The cage is directly hydrogen bonded to the backbone's polar groups, thus hydration shells around hydrophobic and hydrophilic groups are folded together in amphiphilic molecules. The hydrophobe enclathration implies a substantial freedom degree reduction which makes it entropically disfavored. This disadvantageous entropic contribution is partially compensated by the favorable van der Waals interactions with guest in stabilizing clathrate hydrate formation. The water shell around the side chain relates intimately with the side‐chain rotational isomerism. Present data are correlated with the experimental determined populations of the three rotamers, yielding promising results for both α‐aminobutyric acid and valine.

Enhancing Side Chain Rotamer Sampling Using Nonequilibrium Candidate Monte Carlo.

Molecular simulations are a valuable tool for studying biomolecular motions and thermodynamics. However, such motions can be slow compared to simulation time scales, yet critical. Specifically, adequate sampling of side chain motions in protein binding pockets is crucial for obtaining accurate estimates of ligand binding free energies from molecular simulations. The time scale of side chain rotamer flips can range from a few ps to several hundred ns or longer, particularly in crowded environments like the interior of proteins. Here, we apply a mixed nonequilibrium candidate Monte Carlo (NCMC)/molecular dynamics (MD) method to enhance sampling of side chain rotamers. The NCMC portion of our method applies a switching protocol wherein the steric and electrostatic interactions between target side chain atoms and the surrounding environment are cycled off and then back on during the course of a move proposal. Between NCMC move proposals, simulation of the system continues via traditional molecular dynamics. Here, we first validate this approach on a simple, solvated valine-alanine dipeptide system and then apply it to a well-studied model ligand binding site in T4 lysozyme L99A. We compute the rate of rotamer transitions for a valine side chain using our approach and compare it to that of traditional molecular dynamics simulations. Here, we show that our NCMC/MD method substantially enhances side chain sampling, especially in systems where the torsional barrier to rotation is high (≥10 kcal/mol). These barriers can be intrinsic torsional barriers or steric barriers imposed by the environment. Overall, this may provide a promising strategy to selectively improve side chain sampling in molecular simulations.

Enzymatic Preparation of Modified DNA: Study of the Kinetics by Real-Time PCR

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

Binding, folding and insertion of a β-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKVdPlPTKVKVKVK), an engineered AMP and anti-cancer β-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a “flip and dip” mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers.

Molecular, Local, and Network-Level Basis for the Enhanced Stiffness of Hydrogel Networks Formed from Coassembled Racemic Peptides: Predictions from Pauling and Corey.

Hydrogels prepared from self-assembling peptides are promising materials for medical applications, and using both l- and d-peptide isomers in a gel’s formulation provides an intuitive way to control the proteolytic degradation of an implanted material. In the course of developing gels for delivery applications, we discovered that a racemic mixture of the mirror-image β-hairpin peptides, named MAX1 and DMAX1, provides a fibrillar hydrogel that is four times more rigid than gels formed by either peptide alone—a puzzling observation. Herein, we use transmission electron microscopy, small angle neutron scattering, solid state NMR, diffusing wave, infrared, and fluorescence spectroscopies, and modeling to determine the molecular basis for the increased mechanical rigidity of the racemic gel. We find that enantiomeric peptides coassemble in an alternating fashion along the fibril long axis, forming an extended heterochiral pleat-like β-sheet, a structure predicted by Pauling and Corey in 1953. Hydrogen bonding between enantiomers within the sheet dictates the placement of hydrophobic valine side chains in the fibrils’ dry interior in a manner that allows the formation of nested hydrophobic interactions between enantiomers, interactions not accessible within enantiomerically pure fibrils. Importantly, this unique molecular arrangement of valine side chains maximizes inter-residue contacts within the core of the fibrils resulting in their local stiffening, which in turn, gives rise to the significant increase in bulk mechanical rigidity observed for the racemic hydrogel.

Mechanism Underlying Conformational Effects of a Disease-Associated Hydrophobic-to-Hydrophobic Substitution on an Intrinsically Disordered Region

Disease-associated Single Nucleotide Polymorphisms (SNPs) are common in the disordered regions of proteins (21.7 % of SNPs are found in the disordered region). Most mutational studies of IDP's consider loss or gain of a charged residue. In this study, we explore the local and global effect of a charge-neutral mutation between two hydrophobic residues, with known effects on function: the Val66Met SNP in the 100-residue disordered prodomain of Brain Derived Neurotropic Factor (BDNF). Val66Met is the most frequently found SNP in the BDNF disordered domain, and is associated with various neurological and psychiatric disorders such as bipolar disorder and Alzheimer's disease. Previously, NMR studies demonstrated that the prodomain is disordered with different secondary structure preferences for Val and Met at 275K (Anastasia et al 2013). We attempt to elucidate this phenomenon at the microscopic level using large-scale, fully atomistic temperature replica exchange calculations of both the Val and Met forms of the BDNF prodomain. MD simulations indicate high propensity of secondary structure in regions similar to those identified in NMR studies. Interestingly, we observe reversed temperature dependence of the secondary structure around the SNP between Val and Met from 300K to 420K. With increasing temperature, Val66 is less likely to assume helical secondary structure, while Met66 is more likely, which is consistent with previous observations that entropy of the valine side-chain is significantly reduced upon helix formation. This cost likely offsets the entropic gain of nearby water molecules upon helix formation, associated with the hydrophobic effect and common to Val66 and Met66 helices. At room temperature, we also observe an increase in the radius of gyration of the Met66 prodomain relative to the Val66 prodomain, which can be reliably attributed to differential hydrogen bonding preferences of residues near in sequence to the SNP, affecting their likelihood of forming beta-bridges with distant residues. This is consistent with an experimentally observed higher hydrodynamic radius for the Met66 proregion than for Val66 proregion. These results indicate the seemingly subtle substitution may exert its effects by critically adjusting entropic cost of local secondary-structure elements, which, in turn, affects the conformational ensemble via differential long-range beta bridging patterns. Furthermore, although the SNP is neutral, it alters the exposure of charged residues around the SNP, which can potentially affect the binding characteristics of the prodomain to its highly charged receptor, SORCS2.

Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe279. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe279. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe279, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe229.

Aromatic Amide Polymers that Form Two Helical Conformations with Twist Sense Bias in Water

AbstractTwo benzene/2,2′‐bipyridine‐alternately incorporated amide polymers have been prepared, which are driven by hydrophobicity to form two different helical conformations. Both helices exhibit twist sense bias in water induced by chiral valine side chains and the coordination of the 2,2′‐bipyridine unit to the Ni2+ ions.

Exploring the Insertion Mechanism of SVS-1 β-Hairpin Peptide into an Anionic Lipid Bilayer

Antimicrobial peptides (AMPs) are ubiquitous in many biological systems and act as host defenses against microbial pathogens. Existing theories on how AMPs insert and cause pore formation still lack mechanistic detail. In this study, we investigate the insertion mechanism of SVS-1 (KVKVKVKVDPPTKVKVKVK), an anti-cancer beta-hairpin peptide, into an anionic lipid bilayer using atomistic molecular dynamics (MD) simulations.There is an energy barrier needed to overcome the strong electrostatic interactions of positive peptide sidechains and negatively charged lipid head groups. By comparing the folding of SVS-1 in charged and uncharged lipid bilayers, we found that folding occurs at different rates and lead to different secondary structures. The introduction of one valine sidechain site into the bilayer promotes folding at the surface. We see correct folding and misfolding in the forms of a beta bulge and partial α-helical formation in our simulations. We infer these single molecule structures as intermediates and suggest a cooperative peptide effect will induce correct folding and insertion. Further insight gained from our studies can potentially aid in design of novel AMPs as therapeutic or cancer targets and classify the mechanisms of insertion and membrane disruption.

Clusters of isoleucine, leucine, and valine side chains define cores of stability in high‐energy states of globular proteins: Sequence determinants of structure and stability

Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates.

Probing the Secondary Structure of Membrane Protein using Bacterial Expression System and Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

Membrane proteins possess a variety of functions essential to the survival of organisms. However, due to the inherent hydrophobic property, it is extremely difficult to gain the structural information. Previously, we probed the structure of an α-helical component of a small model membrane peptide, AchR M2δ, by detecting 2H modulation from a 2H-labeled Val side chain that was 3 or 4 residues away from a nitroxide spin label using ESEEM spectroscopy. In this study, for the first time, ESEEM technique was applied to determine the local secondary structure of a biological membrane protein system using bacterial expression method for incorporating the 2H-labeled Val side chain both in micelle and lipid bilayer environment. An integral membrane protein, KCNE1 with known 3D structure, was utilized as a model membrane protein to test the feasibility and validity of this ESSEM technique. The side chain of valine at position 21 (outside domain), 50 (transmembrane domain), and 95 (cytoplasmic domain) was 2H labeled (denoted as ith position). An MTSL nitroxide spin label was positioned at 2, 3, and 4 residues away (denoted i+2 to i+4, respectively) from 2H-labeled Val residue of each domain. Our results indicated that i+3 and i+4 positions have a significant 2H modulation while 2H modulation is absent at i+2 position, which is consistent with the 3.6 residue/turn periodicity of the α-helix. These results are also consistent with the micelle structure of KCNE1. This ESEEM technique was further validated using the protein incorporated into lipid bilayer of DMPC/DPC bicelle and the transmembrane domain was probed to be α-helical as well. This ESEEM technique is very powerful, efficient, and quick to examine the local secondary structure of any protein system without size limitation.

Computational screening and selection of cyclic peptide hairpin mimetics by molecular simulation and kinetic network models.

Designing peptidomimetic compounds to have a preorganized structure in solution is highly nontrivial. To show how simulation-based approaches can help speed this process, we performed an extensive simulation study of designed cyclic peptide mimics of a β-hairpin from bacterial protein LapD involved in a protein-protein interaction (PPI) pertinent to bacterial biofilm formation. We used replica exchange molecular dynamics (REMD) simulation to screen 20 covalently cross-linked designs with varying stereochemistry and selected the most favorable of these for massively parallel simulation on Folding@home in explicit solvent. Markov state models (MSMs) built from the trajectory data reveal how subtle chemical modifications can have a significant effect on conformational populations, leading to the overall stabilization of the target structure. In particular, we identify a key steric interaction between a methyl substituent and a valine side chain that acts to allosterically shift population between native and near-native states, which could be exploited in future designs. Visualization of this mechanism is aided considerably by the tICA method, which identifies degrees of freedom most important in slow conformational transitions. The combination of quantitative detail and human comprehension provided by MSMs suggests such approaches will be increasingly useful for design.

Structure-guided Mutation of the Conserved G3-box Glycine in Rheb Generates a Constitutively Activated Regulator of Mammalian Target of Rapamycin (mTOR)

Constitutively activated variants of small GTPases, which provide valuable functional probes of their role in cellular signaling pathways, can often be generated by mutating the canonical catalytic residue (e.g. Ras Q61L) to impair GTP hydrolysis. However, this general approach is ineffective for a substantial fraction of the small GTPase family in which this residue is not conserved (e.g. Rap) or not catalytic (e.g. Rheb). Using a novel engineering approach, we have manipulated nucleotide binding through structure-guided substitutions of an ultraconserved glycine residue in the G3-box motif (DXXG). Substitution of Rheb Gly-63 with alanine impaired both intrinsic and TSC2 GTPase-activating protein (GAP)-mediated GTP hydrolysis by displacing the hydrolytic water molecule, whereas introduction of a bulkier valine side chain selectively blocked GTP binding by steric occlusion of the γ-phosphate. Rheb G63A stimulated phosphorylation of the mTORC1 substrate p70S6 kinase more strongly than wild-type, thus offering a new tool for mammalian target of rapamycin (mTOR) signaling.