ABSTRACT
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
Subject(s)
Protein Kinase Inhibitors , Protein Multimerization , Proto-Oncogene Proteins B-raf , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/chemistry , Allosteric Regulation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/metabolism , Protein Multimerization/drug effects , Humans , Protein Conformation , Protein Binding , Models, MolecularABSTRACT
Conformational changes of catalytically-important structural elements are a key feature of the regulation mechanisms of protein kinases and are important for dictating inhibitor binding modes and affinities. The lack of widely applicable methods for tracking kinase conformational changes in solution has hindered our understanding of kinase regulation and our ability to design conformationally selective inhibitors. Here we provide an overview of two recently developed methods that detect conformational changes of the regulatory activation loop and αC-helix of kinases and that yield complementary information about allosteric mechanisms. An intramolecular Förster resonance energy transfer-based approach provides a scalable platform for detecting and classifying structural changes in high-throughput, as well as quantifying ligand binding cooperativity, shedding light on the energetics governing allostery. The pulsed electron paramagnetic resonance technique double electron-electron resonance provides lower throughput but higher resolution information on structural changes that allows for unambiguous assignment of conformational states and quantification of population shifts. Together, these methods are shedding new light on kinase regulation and drug interactions and providing new routes for the identification of novel kinase inhibitors and allosteric modulators.
Subject(s)
Fluorescence Resonance Energy Transfer , Protein Conformation , Protein Kinases , Electron Spin Resonance Spectroscopy/methods , Protein Kinases/chemistry , Protein Kinases/metabolism , Allosteric Regulation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Humans , Protein Binding , Models, MolecularABSTRACT
Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.
Subject(s)
Cyclin-Dependent Kinase 2 , Cyclins , Protein Kinase Inhibitors , Animals , Mice , Contraception , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclins/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
ABSTRACT
Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential. Herein, we describe the screening and optimization that led to compounds like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable, orally bioavailable, and significantly disrupts spermatogenesis, demonstrating this series' therapeutic potential. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for this series to yield an efficacious and selective allosteric CDK2 inhibitor.
Subject(s)
Contraceptive Agents, Male , Male , Humans , Animals , Mice , Cyclin-Dependent Kinase 2 , Structure-Activity Relationship , Contraceptive Agents, Male/pharmacology , Sperm Count , Semen/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistryABSTRACT
Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).
Subject(s)
Allosteric Regulation/physiology , Cyclin-Dependent Kinase 2/metabolism , Allosteric Site/genetics , Cell Cycle/physiology , Cell Cycle Proteins/metabolism , Cyclin A/metabolism , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Humans , Microtubule-Associated Proteins/metabolism , Models, Biological , Phosphorylation/physiology , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site, where many kinase inhibitors bind. We have previously discovered that 8-anilino-1-naphthalene sulfonic acid (ANS) binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Here, we detail the positive cooperativity between ANS and orthosteric Cdk2 inhibitors dinaciclib and roscovitine, which increase the affinity of ANS toward Cdk2 5-fold to 10-fold, and the relatively noncooperative effects of ATP. We observe these effects using a fluorescent binding assay and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR), where we noticed a shift from fast exchange to slow exchange upon ANS titration in the presence of roscovitine but not with an ATP mimic. The discovery of cooperative relationships between orthosteric and allosteric kinase inhibitors could further the development of selective kinase inhibitors in general.
Subject(s)
Anilino Naphthalenesulfonates/chemistry , Cyclic N-Oxides/chemistry , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Indolizines/chemistry , Protein Kinase Inhibitors/chemistry , Pyridinium Compounds/chemistry , Roscovitine/chemistry , Allosteric Regulation , Anilino Naphthalenesulfonates/metabolism , Cyclic N-Oxides/metabolism , Cyclin-Dependent Kinase 2/metabolism , Drug Synergism , Humans , Indolizines/metabolism , Molecular Docking Simulation , Protein Binding , Protein Kinase Inhibitors/metabolism , Pyridinium Compounds/metabolism , Roscovitine/metabolismABSTRACT
Natriuretic peptides regulate multiple physiologic systems by activating transmembrane receptors containing intracellular guanylyl cyclase domains, such as GC-A and GC-B, also known as Npr1 and Npr2, respectively. Both enzymes contain an intracellular, phosphorylated pseudokinase domain (PKD) critical for activation of the C-terminal cGMP-synthesizing guanylyl cyclase domain. Because ATP allosterically activates GC-A and GC-B, we investigated how ATP binding to the PKD influenced guanylyl cyclase activity. Molecular modeling indicated that all the residues of the ATP-binding site of the prototypical kinase PKA, except the catalytic aspartate, are conserved in the PKDs of GC-A and GC-B. Kinase-inactivating alanine substitutions for the invariant lysine in subdomain II or the aspartate in the DYG-loop of GC-A and GC-B failed to decrease enzyme phosphate content, consistent with the PKDs lacking kinase activity. In contrast, both mutations reduced enzyme activation by blocking the ability of ATP to decrease the Michaelis constant without affecting peptide-dependent activation. The analogous lysine-to-alanine substitution in a glutamate-substituted phosphomimetic mutant form of GC-B also reduced enzyme activity, consistent with ATP stimulating guanylyl cyclase activity through an allosteric, phosphorylation-independent mechanism. Mutations designed to rigidify the conserved regulatory or catalytic spines within the PKDs increased guanylyl cyclase activity, increased sensitivity to natriuretic peptide, or reduced the Michaelis constant in the absence of ATP, consistent with ATP binding stabilizing the PKD in a conformation analogous to that of catalytically active kinases. We conclude that allosteric mechanisms evolutionarily conserved in the PKDs promote the catalytic activation of transmembrane guanylyl cyclases.
Subject(s)
Adenosine Triphosphate/metabolism , Catalytic Domain , Guanylate Cyclase/metabolism , Receptors, Atrial Natriuretic Factor/metabolism , Allosteric Regulation , Allosteric Site/genetics , Amino Acid Sequence , Animals , Binding, Competitive , COS Cells , Chlorocebus aethiops , Enzyme Activation/genetics , Guanylate Cyclase/chemistry , Guanylate Cyclase/genetics , HEK293 Cells , Humans , Models, Molecular , Mutation , Phosphorylation , Protein Binding , Receptors, Atrial Natriuretic Factor/chemistry , Receptors, Atrial Natriuretic Factor/genetics , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure-activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.
Subject(s)
Aurora Kinase A/drug effects , Aurora Kinase A/metabolism , Protein Conformation , Protein Interaction Domains and Motifs , Protein Kinase Inhibitors/pharmacology , Allosteric Regulation , Amino Acid Motifs , Binding Sites , Cell Cycle Proteins/metabolism , Cell Division , Crystallography, X-Ray , Humans , Microtubule-Associated Proteins/metabolism , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Nuclear Proteins/metabolism , Oligopeptides , Phosphorylation , Protein BindingABSTRACT
Kinases play a critical role in cellular signaling and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Therapeutics targeting kinases currently account for roughly 50% of cancer drug discovery efforts. The ability to explore human kinase biochemistry and biophysics in the laboratory is essential to designing selective inhibitors and studying drug resistance. Bacterial expression systems are superior to insect or mammalian cells in terms of simplicity and cost effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression produced high yields of Src and Abl kinase domains in bacteria, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 µg/mL of culture in an automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases and lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identifying kinase domain constructs previously expressed in bacteria and likely to express well in our protocol, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. Using a fluorescence-based thermostability assay and a fluorescent ATP-competitive inhibitor, we show that the highest-expressing kinases are folded and have well-formed ATP binding sites. We also demonstrate that these constructs can enable characterization of clinical mutations by expressing a panel of 48 Src and 46 Abl mutations. The wild-type kinase construct library is available publicly via Addgene.
Subject(s)
Bacteria/metabolism , Binding Sites , Escherichia coli/metabolism , Humans , Phosphorylation , Protein Structure, Secondary , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-abl/metabolism , src-Family Kinases/metabolismABSTRACT
Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases.
Subject(s)
Allosteric Regulation , Aurora Kinase A/chemistry , Aurora Kinase A/metabolism , Enzyme Activation , Protein Processing, Post-Translational , Electron Spin Resonance Spectroscopy , Molecular Dynamics Simulation , Phosphorylation , Spectrum AnalysisABSTRACT
The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift toward the active DFG-in state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium. Tpx2 further activates AurA by stabilizing a water-mediated allosteric network that links the C-helix to the active site through an unusual polar residue in the regulatory spine. The polar spine residue and water network of AurA are essential for phosphorylation-driven activation, but an alternative form of the water network found in related kinases can support Tpx2-driven activation, suggesting that variations in the water-mediated hydrogen bond network mediate regulatory diversification in protein kinases.
Subject(s)
Aurora Kinase A/metabolism , Water/metabolism , Allosteric Regulation , Enzyme Activation , Humans , Models, Molecular , Water/chemistryABSTRACT
Kinase inhibitors are important cancer drugs, but they tend to display limited target specificity, and their target profiles are often challenging to rationalize in terms of molecular mechanism. Here we report that the clinical kinase inhibitor bosutinib recognizes its kinase targets by engaging a pair of conserved structured water molecules in the active site and that many other kinase inhibitors share a similar recognition mechanism. Using the nitrile group of bosutinib as an infrared probe, we show that the gatekeeper residue and one other position in the ATP-binding site control access of the drug to the structured water molecules and that the amino acids found at these positions account for the kinome-wide target spectrum of the drug. Our work highlights the importance of structured water molecules for inhibitor recognition, reveals a new role for the kinase gatekeeper and showcases an effective approach for elucidating the molecular origins of selectivity patterns.
Subject(s)
Aniline Compounds/chemistry , Models, Biological , Nitriles/chemistry , Quinolines/chemistry , Water/chemistry , src-Family Kinases/chemistry , Amino Acid Substitution , Conserved Sequence , Humans , Hydrogen Bonding , Substrate Specificity , src-Family Kinases/metabolismABSTRACT
Hydrogen bonds are ubiquitous in chemistry and biology. The physical forces that govern hydrogen-bonding interactions have been heavily debated, with much of the discussion focused on the relative contributions of electrostatic vs quantum mechanical effects. In principle, the vibrational Stark effect, the response of a vibrational mode to electric field, can provide an experimental method for parsing such interactions into their electrostatic and nonelectrostatic components. In a previous study we showed that, in the case of relatively weak O-H···π hydrogen bonds, the O-H bond displays a linear response to an electric field, and we exploited this response to demonstrate that the interactions are dominated by electrostatics (Saggu, M.; Levinson, N. M.; Boxer, S. G. J. Am. Chem. Soc.2011, 133, 17414-17419). Here we extend this work to other X-H···π interactions. We find that the response of the X-H vibrational probe to electric field appears to become increasingly nonlinear in the order O-H < N-H < S-H. The observed effects are consistent with differences in atomic polarizabilities of the X-H groups. Nonetheless, we find that the X-H stretching vibrations of the model compounds indole and thiophenol report quantitatively on the electric fields they experience when complexed with aromatic hydrogen-bond acceptors. These measurements can be used to estimate the electrostatic binding energies of the interactions, which are found to agree closely with the results of energy calculations. Taken together, these results highlight that with careful calibration vibrational probes can provide direct measurements of the electrostatic components of hydrogen bonds.
Subject(s)
Hydrogen Bonding , Static Electricity , Models, Molecular , Spectroscopy, Fourier Transform InfraredABSTRACT
Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib, nilotinib and dasatinib are currently used to treat CML, but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML, but its binding mode is unknown. We present the 2.4 Å structure of bosutinib bound to the kinase domain of Abl, which explains the inhibitor's activity against several imatinib-resistant mutants, and reveals that similar inhibitors that lack a nitrile moiety could be effective against the common T315I mutant. We also report that two distinct chemical compounds are currently being sold under the name "bosutinib", and report spectroscopic and structural characterizations of both. We show that the fluorescence properties of these compounds allow inhibitor binding to be measured quantitatively, and that the infrared absorption of the nitrile group reveals a different electrostatic environment in the conserved ATP-binding sites of Abl and Src kinases. Exploiting such differences could lead to inhibitors with improved selectivity.
Subject(s)
Aniline Compounds , Fusion Proteins, bcr-abl/antagonists & inhibitors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Nitriles , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinolines , Recombinant Proteins/antagonists & inhibitors , Aniline Compounds/chemistry , Aniline Compounds/pharmacology , Benzamides , Binding Sites , Dasatinib , Drug Resistance, Neoplasm , Escherichia coli , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , Isomerism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Models, Molecular , Mutation , Nitriles/chemistry , Nitriles/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/metabolism , Pyrimidines/chemistry , Pyrimidines/pharmacology , Quinolines/chemistry , Quinolines/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrophotometry, Infrared , Static Electricity , Thiazoles/chemistry , Thiazoles/pharmacology , X-Ray DiffractionABSTRACT
The physical properties of solvents strongly affect the spectra of dissolved solutes, and this phenomenon can be exploited to gain insight into the solvent-solute interaction. The large solvatochromic shifts observed for many dye molecules in polar solvents are due to variations in the solvent reaction field, and these shifts are widely used to estimate the change in the dye's dipole moment upon photoexcitation, which is typically on the order of â¼1-10 D. In contrast, the change in dipole moment for vibrational transitions is approximately 2 orders of magnitude smaller. Nonetheless, vibrational chromophores display significant solvatochromism, and the relative contributions of specific chemical interactions and electrostatic interactions are debated, complicating the interpretation of vibrational frequency shifts in complex systems such as proteins. Here we present a series of substituted benzonitriles that display widely varying degrees of vibrational solvatochromism. In most cases, this variation can be quantitatively described by the experimentally determined Stark tuning rate, coupled with a simple Onsager-like model of solvation, reinforcing the view that vibrational frequency shifts are largely caused by electrostatic interactions. In addition, we discuss specific cases where continuum solvation models fail to predict solvatochromic shifts, revealing the necessity for more advanced theoretical models that capture local aspects of solute-solvent interactions.
Subject(s)
Nitriles/chemistry , Solvents/chemistry , Spectroscopy, Fourier Transform Infrared , Static Electricity , VibrationABSTRACT
Hydrogen bonds and aromatic interactions are of widespread importance in chemistry, biology, and materials science. Electrostatics play a fundamental role in these interactions, but the magnitude of the electric fields that support them has not been quantified experimentally. Phenol forms a weak hydrogen bond complex with the π-cloud of benzene, and we used this as a model system to study the role of electric fields in weak OH···π hydrogen bonds. The effects of complex formation on the vibrational frequency of the phenol OH or OD stretches were measured in a series of benzene-based aromatic solvents. Large shifts are observed and these can be converted into electric fields via the measured vibrational Stark effect. A comparison of the measured fields with quantum chemical calculations demonstrates that calculations performed in the gas phase are surprisingly effective at capturing the electrostatics observed in solution. The results provide quantitative measurements of the magnitude of electric fields and electrostatic binding energies in these interactions and suggest that electrostatics dominate them. The combination of vibrational Stark effect (VSE) measurements of electric fields and high-level quantum chemistry calculations is a general strategy for quantifying and characterizing the origins of intermolecular interactions.
Subject(s)
Hydroxides/chemistry , Phenols/chemistry , Electromagnetic Fields , Hydrogen Bonding , Quantum Theory , Solvents/chemistryABSTRACT
The role of electric fields in important biological processes such as binding and catalysis has been studied almost exclusively by computational methods. Experimental measurements of the local electric field in macromolecules are possible using suitably calibrated vibrational probes. Here we demonstrate that the vibrational transitions of phosphate groups are highly sensitive to an electric field and show how that sensitivity can be quantified, allowing electric field measurements to be made in phosphate-containing biological systems without chemical modification.
Subject(s)
Phosphates/chemistry , Electricity , Models, Molecular , Phospholipids/chemistry , Spectrophotometry, Infrared , Water/chemistryABSTRACT
The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.
Subject(s)
Protein-Tyrosine Kinases/chemistry , Binding Sites , CSK Tyrosine-Protein Kinase , Chromatography, Gel , Crystallization , Dimerization , Humans , Kinetics , Ligands , Molecular Conformation , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Tyrosine/chemistry , src Homology Domains , src-Family KinasesABSTRACT
The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.9 A resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site used by most tyrosine kinases to recognize substrates is destabilized in Csk by a deletion in the activation loop.