Binding Of Allosteric Effectors Research Articles

Toward the Design of Allosteric Effectors: Gaining Comprehensive Control of Drug Properties and Actions.

While the therapeutic potential of allosteric drugs is increasingly realized, the discovery of effectors is largely incidental. The rational design of allosteric effectors requires new state-of-the-art approaches to account for the distinct characteristics of allosteric ligands and their modes of action. We present a broadly applicable computational framework for obtaining allosteric site-effector pairs, providing targeted, highly specific, and tunable regulation to any functional site. We validated the framework using the main protease from SARS-CoV-2 and the K-RasG12D oncoprotein. High-throughput per-residue quantification of the energetics of allosteric signaling and effector binding revealed known drugs capable of inducing the required modulation upon binding. Starting from fragments of known well-characterized drugs, allosteric effectors and binding sites were designed and optimized simultaneously to achieve targeted and specific signaling to distinct functional sites, such as, for example, the switch regions of K-RasG12D. The generic framework proposed in this work will be instrumental in developing allosteric therapies aligned with a precision medicine approach.

Dissecting the Allosteric Fine-Tuning of Enzyme Catalysis.

Fully understanding the mechanism of allosteric regulation in biomolecules requires separating and examining all of the involved factors. In enzyme catalysis, allosteric effector binding shifts the structure and dynamics of the active site, leading to modified energetic (e.g., energy barrier) and dynamical (e.g., diffusion coefficient) factors underlying the catalyzed reaction rate. Such modifications can be subtle and dependent on the type of allosteric effector, representing a fine-tuning of protein function. The microscopic description of allosteric regulation at the level of function-dictating factors has prospective applications in fundamental and pharmaceutical sciences, which is, however, largely missing so far. Here, we characterize the allosteric fine-tuning of enzyme catalysis, using human Pin1 as an example, by performing more than half-millisecond all-atom molecular dynamics simulations. Changes of reaction kinetics and the dictating factors, including the free energy surface along the reaction coordinate and the diffusion coefficient of the reaction dynamics, under various enzyme and allosteric effector binding conditions are examined. Our results suggest equal importance of the energetic and dynamical factors, both of which can be modulated allosterically, and the combined effect determines the final allosteric output. We also reveal the potential dynamic basis for allosteric modulation using an advanced statistical technique to detect function-related conformational dynamics. Methods developed in this work can be applied to other allosteric systems.

Modulation of Allosteric Control and Evolution of Hemoglobin.

Allostery arises when a ligand-induced change in shape of a binding site of a protein is coupled to a tertiary/quaternary conformational change with a consequent modulation of functional properties. The two-state allosteric model of Monod, Wyman and Changeux [J. Mol. Biol. 1965; 12, 88-118] is an elegant and effective theory to account for protein regulation and control. Tetrameric hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the ideal system to test for the validity of the MWC theory. The small ligands affecting Hb's behavior (organic phosphates, protons, bicarbonate) are produced by the red blood cell during metabolism. By binding to specific sites, these messengers make Hb sensing the environment and reacting consequently. HbI and HbIV from trout and human HbA are classical cooperative models, being similar yet different. They share many fundamental features, starting with the globin fold and the quaternary assembly, and reversible cooperative O2 binding. Nevertheless, they differ in ligand affinity, binding of allosteric effectors, and stability of the quaternary assembly. Here, we recollect essential functional properties and correlate them to the tertiary and quaternary structures available in the protein databank to infer on the molecular basis of the evolution of oxygen transporters.

Emergence of allostery through reorganization of protein residue network architecture.

Despite more than a century of study, consensus on the molecular basis of allostery remains elusive. A comparison of allosteric and non-allosteric members of a protein family can shed light on this important regulatory mechanism, and the bacterial biotin protein ligases, which catalyze post-translational biotin addition, provide an ideal system for such comparison. While the Class I bacterial ligases only function as enzymes, the bifunctional Class II ligases use the same structural architecture for an additional transcription repression function. This additional function depends on allosterically activated homodimerization followed by DNA binding. In this work, we used experimental, computational network, and bioinformatics analyses to uncover distinguishing features that enable allostery in the Class II biotin protein ligases. Experimental studies of the Class II Escherichia coli protein indicate that catalytic site residues are critical for both catalysis and allostery. However, allostery also depends on amino acids that are more broadly distributed throughout the protein structure. Energy-based community network analysis of representative Class I and Class II proteins reveals distinct residue community architectures, interactions among the communities, and responses of the network to allosteric effector binding. Bioinformatics mutual information analyses of multiple sequence alignments indicate distinct networks of coevolving residues in the two protein families. The results support the role of divergent local residue community network structures both inside and outside of the conserved enzyme active site combined with distinct inter-community interactions as keys to the emergence of allostery in the Class II biotin protein ligases.

Coevolution-based prediction of key allosteric residues for protein function regulation.

Allostery is fundamental to many biological processes. Due to the distant regulation nature, how allosteric mutations, modifications, and effector binding impact protein function is difficult to forecast. In protein engineering, remote mutations cannot be rationally designed without large-scale experimental screening. Allosteric drugs have raised much attention due to their high specificity and possibility of overcoming existing drug-resistant mutations. However, optimization of allosteric compounds remains challenging. Here, we developed a novel computational method KeyAlloSite to predict allosteric site and to identify key allosteric residues (allo-residues) based on the evolutionary coupling model. We found that protein allosteric sites are strongly coupled to orthosteric site compared to non-functional sites. We further inferred key allo-residues by pairwise comparing the difference of evolutionary coupling scores of each residue in the allosteric pocket with the functional site. Our predicted key allo-residues are in accordance with previous experimental studies for typical allosteric proteins like BCR-ABL1, Tar, and PDZ3, as well as key cancer mutations. We also showed that KeyAlloSite can be used to predict key allosteric residues distant from the catalytic site that are important for enzyme catalysis. Our study demonstrates that weak coevolutionary couplings contain important information of protein allosteric regulation function. KeyAlloSite can be applied in studying the evolution of protein allosteric regulation, designing and optimizing allosteric drugs, and performing functional protein design and enzyme engineering.

Engineering a Conformationally Switchable Artificial Metalloprotein.

Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3-, H3C-, and iPr-). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.

Computational interpretation of the effect of omecamtiv mecarbil on the recovery stroke of human cardiac beta myosin, using metadynamics

The small molecule omecamtiv mecarbil (OM) is an allosteric effector binding to the motor domain of the cardiac myosin II, observed to enhance contraction of cardiac muscle. The drug affects the major steps of the myosin mechanochemical cycle central to the contractile function, the recovery stroke being one of them. The recovery stroke primes the lever arm of the myosin leading to transition from the post-rigor state to the pre-powerstroke state, preparing the active residues for catalyzing ATP hydrolysis.

Deciphering the Molecular Terms of Arp2/3 Allosteric Regulation from All-Atom Simulations and Dynamical Network Theory.

The Arp2/3 molecular machine stimulates the generation of branched actin networks at the cytosolic surface of cellular membranes. Arp2/3 is thus pivotal for cell motility and migration, and its aberrant function is implicated in cancer invasion and metastasis. Here, all-atom multi μs-long molecular dynamics simulations and dynamical NetWork Analysis (NWA) unprecedentedly disclose the molecular terms of Arp2/3 regulation (activation/inhibition) by positive/negative allosteric modulators. After identifying the crucial structural elements underlying Arp2/3's conformational transition toward its active actin-polymerization-competent state, we decrypt the activating signaling paths heading from the allosteric effector (ATP) binding sites to these pivotal regions, also elucidating how small-molecule inhibitors scramble this signal-exchange. As a result, while ATP-induced signaling triggers a harmonious conformational transition toward active Arp2/3, the inhibitors disturb these information channels, desynchronizing Arp2/3 functional movements, thus hindering its activation. Our outcomes supply a conceptual basis for devising small-molecule inhibitors to block infiltrative cancer migration.

Different Solvent and Conformational Entropy Contributions to the Allosteric Activation and Inhibition Mechanisms of Yeast Chorismate Mutase.

Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. Saccharomyces cerevisiae chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector. We utilized variable temperature isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments to better understand the entropic contributions to allosteric effector binding, including changes to solvent entropy and protein conformational entropy. Upon binding either Tyr or Trp, ScCM experiences a quenching of motions on the picosecond-to-nanosecond time scale, which we could relate to a loss of protein conformational entropy. Further ITC and NMR studies were consistent with the Tyr-bound form of ScCM being associated with more water molecules compared to the Trp-bound form and Tyr binding being associated with a less positive solvent entropy change. These studies provide insight into the role of structural dynamics in ScCM function and highlight the importance of solvent entropy changes in allosteric regulation, a historically underappreciated concept.

Probing Protein Allostery as a Residue-Specific Concept via Residue Response Maps

Allosteric regulation is a well-established phenomenon defined as a distal conformational or dynamical change of the protein upon allosteric effector binding. Here, we developed a novel approach to delineate allosteric effects in proteins. In this approach, we applied robust machine learning methods, including deep neural network and random forest, on extensive molecular dynamics (MD) simulations to distinguish otherwise similar allosteric states of proteins. Using the PDZ3 domain of PDS-95 as a model protein, we demonstrated that the allosteric effects could be represented as residue-specific properties through two-dimensional property-residue maps, which we refer to as "residue response maps". These maps were constructed through two machine learning methods and could accurately describe how different properties of various residues are affected upon allosteric perturbation on protein. Based on the "residue response maps", we propose allostery as a residue-specific concept, suggesting that all residues could be considered as allosteric residues because each residue "senses" the allosteric events through changing its single or multiple attributes in a quantitatively unique way. The "residue response maps" could be used to fingerprint a protein based on the unique patterns of residue responses upon binding events, providing a novel way to systematically describe the protein allosteric effects of each residue upon perturbation.

ACR11 modulates levels of reactive oxygen species and salicylic acid-associated defense response in Arabidopsis

The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis.

Dissimilar flexibility of α and β subunits of human adult hemoglobin influences the protein dynamics and its alteration induced by allosteric effectors.

The general question by what mechanism an “effector” molecule and the hemes of hemoglobin interact over widely separated intramolecular distances to change the oxygen affinity has been extensively investigated, and still has remained of central interest. In the present work we were interested in clarifying the general role of the protein matrix and its dynamics in the regulation of human adult hemoglobin (HbA). We used a spectroscopy approach that yields the compressibility (κ) of the protein matrix around the hemes of the subunits in HbA and studied how the binding of heterotropic allosteric effectors modify this parameter. κ is directly related to the variance of volume fluctuation, therefore it characterizes the molecular dynamics of the protein structure. For the experiments the heme groups either in the α or in the β subunits of HbA were replaced by fluorescent Zn-protoporphyrinIX, and series of fluorescence line narrowed spectra were measured at varied pressures. The evaluation of the spectra yielded the compressibility that showed significant dynamic asymmetry between the subunits: κ of the α subunit was 0.17±0.05/GPa, while for the β subunit it was much higher, 0.36±0.07/GPa. The heterotropic effectors, chloride ions, inositol hexaphosphate and bezafibrate did not cause significant changes in κ of the α subunits, while in the β subunits the effectors lead to a significant reduction down to 0.15±0.04/GPa. We relate our results to structural data, to results of recent functional studies and to those of molecular dynamics simulations, and find good agreements. The observed asymmetry in the flexibility suggests a distinct role of the subunits in the regulation of Hb that results in the observed changes of the oxygen binding capability.

Structural basis of potassium activation in plant asparaginases.

l-asparaginases (EC 3.5.1.1) play an important role in nitrogen mobilization in plants. Here, we investigated the biochemical and biophysical properties of potassium-dependent (PvAspG1) and potassium-independent (PvAspG-T2) l-asparaginases from Phaseolus vulgaris. Our previous studies revealed that PvAspG1 requires potassium for catalytic activation and its crystal structure suggested that Ser-118 in the activation loop plays a critical role in coordinating the metal cation. This amino acid residue is replaced by isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, allosteric effector binding, secondary structure conformation, and pH profile were studied. Introduction of the serine residue conferred potassium activation in PvAspG-T2. Conversely, the PvAspG1-S118I mutant could no longer be activated by potassium. PvAspG1 and the PvAspG-T2-I117S mutant had a similar half-maximal effective concentration (EC50 ) value for potassium activation, between 0.1 and 0.3 mm. Potassium binding elicited a similar conformational change in PvAspG1 and PvAspG-T2-I117S, as studied by circular dichroism. However, no change in conformation was observed for PvAspG-T2 and PvAspG1-S118I. Analysis of kinetic parameters in function of pH indicated that potassium activation mediated by Ser-118 influences the ionization of specific functional groups in the enzyme-substrate complex. Together, the results indicate that Ser-118 of PvAspG1 is essential and sufficient for potassium activation in plant l-asparaginases. ENZYME: l-Asparaginase (EC 3.5.1.1).

Quaternary Structure and Activity Modulation in Human Ribonucleotide Reductase

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to 2´‐deoxynucleotides, providing the monomeric precursors necessary for DNA replication and repair. Precise regulation of RNR activity is essential for fidelity of DNA replication, as RNR is responsible for maintaining both the correct absolute cellular concentration of deoxynucleotides, as well as the appropriate relative concentration ratios. The class Ia RNRs found in many aerobic bacteria and all eukaryotes consist of 2 protein subunits. The larger subunit α houses the active site where nucleoside diphosphate substrates (CDP, ADP, GDP, UDP) are reduced, and two additional sites for allosteric effector (ATP, dGTP, TTP, and dATP) binding. The specificity site dictates which substrate is reduced, while the activity site binds ATP/dATP and regulates the overall reduction rate. Nucleotide reduction depends on precise interaction of α with a dimeric smaller subunit β2 that contains a diferric‐tyrosyl radical cofactor essential for catalysis. We are using single particle electron microscopy and advanced image analysis methods to understand the way in which nucleotide‐induced structural rearrangements and shifts in complex subunit equilibria combine to modulate the activity and specificity of human RNR.

Allosteric Coupling via Distant Disorder-to-Order Transitions

Intrinsic disorder provides a means of maximizing allosteric coupling in proteins. However, the mechanisms by which the disorder functions in allostery remain to be elucidated. Small ligand, bio-5′-AMP, binding and dimerization of the Escherichia coli biotin repressor are allosterically coupled. Folding of a disordered loop in the allosteric effector binding site is required to realize the full coupling free energy of −4.0±0.3kcal/mol observed in the wild-type protein. Alanine substitution of a glycine residue on the dimerization surface that does not directly contribute to the dimerization interface completely abolishes this coupling. In this work, the structure of this variant, solved by X-ray crystallography, reveals a monomeric corepressor-bound protein. In the structure loops, neither of which contains the alanine substitution, on both the dimerization and effector binding surfaces that are folded in the corepressor-bound wild-type protein are disordered. The structural data combined with functional measurements indicate that allosteric coupling between ligand binding and dimerization in BirA (E. coli biotin repressor/biotin protein ligase) is achieved via reciprocal communication of disorder-to-order transitions on two distant functional surfaces.

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

The epithelial Na(+) channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na(+), Cl(-), protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na(+) concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na(+) binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na(+). Mutations at selected sites altered the cation inhibitory preference to favor Li(+) or K(+) rather than Na(+). Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na(+). Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family.

The first ribonucleotide reductase without an activating radical cysteine

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several different mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, for over 30 years the paradigm has been that this initial radical is always channeled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction. Such a cysteine residue has been present in the structure of each of the many RNRs determined to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), both alone and in complex with allosteric effector dATP and substrate CTP. Remarkably, tmNrdD lacks a cysteine for radical transfer to the substrate, and is the first structurally or biochemically characterized RNR to do so. However in many other respects tmNrdD appears to be a normal anaerobic RNR, including gene structure, expression levels, metal cofactor and binding of allosteric effectors and substrates in the expected conformations. Furthermore, it is possible to generate a glycyl radical as expected. We present evidence that the structure of tmNrdD is representative for the new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria, all lacking the canonical cysteine residue. The wide distribution provides further evidence that the subclass IIIh is a functional RNR. Taken together, the results imply that an alternative initiation route for the RNR reduction reaction must exist that do not require channeling through a cysteine side chain.

Diversity of Allosteric Regulation in Proteases

Allostery is a fundamental regulatory mechanism that is based on a functional modulation of a site by a distant site. Allosteric regulation can be triggered by binding of diverse allosteric effectors, ranging from small molecules to macromolecules, and is therefore offering promising opportunities for functional modulation in a wide range of applications including the development of chemical probes or drug discovery. Here, we provide an overview of key classes of allosteric protease effectors, their corresponding molecular mechanisms, and their practical implications.

The importance of post-translational modifications in regulating Saccharomyces cerevisiae metabolism

Regulation of the flow of mass and energy through cellular metabolic networks is fundamental to the operation of all living organisms. Such metabolic fluxes are determined by the concentration of limiting substrates and by the amount and kinetic properties of the enzymes. Regulation of the amount of enzyme can be exerted, on a long-term scale, at the level of gene and protein expression. Enzyme regulation by post-translational modifications (PTMs) and noncovalent binding of allosteric effectors are shorter-term mechanisms that modulate enzyme activity. PTMs, in particular protein phosphorylation, are increasingly being recognized as key regulators in many cellular processes, including metabolism. For example, about half of the enzymes in the Saccharomyces cerevisiae metabolic network have been detected as phosphoproteins, although functional relevance has been demonstrated only in a few cases. Direct regulation of enzymes by PTMs provides one of the fastest ways for cells to adjust to environmental cues and internal stimulus. This review charts the so far identified metabolic enzymes undergoing reversible PTMs in the model eukaryote S. cerevisiae and reviews their underlying mechanistic principles - both at the individual enzyme level and in the context of the entire metabolic network operation.

Ligand‐induced formation of transient dimers of mammalian 12/15‐lipoxygenase: A key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.