Allosteric Effects Research Articles

Generation of affinity maps for thiazolidinediones with human serum albumin using affinity microcolumns. I. Studies of effects by glycation on multisite drug binding

Human serum albumin (HSA) is known to undergo modifications by glucose during diabetes. This process produces glycated HSA that can have altered binding to some drugs. In this study, high-performance affinity microcolumns and competition studies were used to see how glycation affects the binding by two thiazolidinedione-class drugs (i.e., pioglitazone and rosiglitazone) at specific regions of HSA. These regions included Sudlow sites I and II, the tamoxifen and digitoxin sites, and a drug-binding site located in subdomain IB. At Sudlow site II, the association equilibrium constants (or binding constants) for pioglitazone and rosiglitazone with normal HSA were 1.7 × 105 M−1 and 2.0 × 105 M−1 at pH 7.4 and 37 °C, with values that changed by up to 5.7-fold for glycated HSA. Sudlow site I of normal HSA had binding constants for pioglitazone and rosiglitazone of 3.4 × 105 M−1 and 4.6 × 105 M−1, with these values changing by up to 1.5-fold for glycated HSA. Rosiglitazone was found to also bind a second region that had a positive allosteric effect on Sudlow site I for all the tested preparations of HSA (binding affinity, 1.1–3.2 × 105 M−1; coupling constant for Sudlow site I, 1.20–1.34). Both drugs had a strong positive allosteric effect on the tamoxifen site of HSA (coupling constants, 13.7–19.9 for pioglitazone and 3.7–11.5 for rosiglitazone). Rosiglitazone also had weak interactions at a site in subdomain IB, with a binding constant of 1.4 × 103 M−1 for normal HSA and a value that was altered by up to 6.8-fold with glycated HSA. Neither of the tested drugs had any significant binding at the digitoxin site. The results were used to produce affinity maps that described binding by these thiazolidinediones with HSA and the effects of glycation on these interactions during diabetes.

Genetic and functional modulation by agonist MRS5698 and allosteric enhancer LUF6000 at the native A3 adenosine receptor in HL-60 cells

The A3 adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A3AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1β, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1β, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A3AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000’s small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A3AR and in recombinant hA3AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A3AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA3AR.

Why classical receptor theory, which ignores allostery, can effectively measure the strength of an allosteric effect as agonist's efficacy.

The classical theory of receptor action has been used for decades as a powerful tool to estimate molecular determinants of ligand-induced receptor activation (i.e., affinity and efficacy) from experimentally observable biological responses. However, it is also a well-recognized fact that the receptor-binding and activation mechanisms, and the parameters thereof, described in the classical theory contradict with the modern view of receptor activation based on allosteric principles. We used mathematical analysis, along with some numerical simulations, to answer the key question as to what extent the classical theory is compatible-if at all-with the modern understanding of receptor activation. Here, we showed conclusively that (1) receptor activation equations based on allosteric principles contain the logic of the classical theory in disguise, and therefore, (2) estimates of "intrinsic efficacy" (ε) obtained by means of classical techniques (i.e., null methods or fitting the operational model to concentration-response data) are equivalent to the allosteric coupling factors that represent the molecular efficacy of ligands. Thus, we conclude that despite the justified criticisms it has received so far, the classical theory may continue to be useful in estimating ligand efficacy from experimental data, if used properly. Here, we also provide rigorous criteria for the proper use of the theory. These findings not only have implications for ligand classification but also resolve some long lasting discussions in the field of bias agonism in GPCR, which requires reasonable estimates of relative ligand efficacies at different signalling pathways.

Evaluation of the inhibitory potential of bioactive compounds against SARS-CoV-2 by in silico approach.

The COVID-19 (coronavirus disease 19) pandemic brought on by the SARS-CoV-2 outbreak (severe acute respiratory syndrome coronavirus 2) has stimulated the exploration of various available chemical compounds that could be used to treat the infection. This has driven numerous researchers to investigate the antiviral potential of several bioactive compounds from medicinal plants due to their reduced adverse effects compared to chemicals. Some of the bioactive compounds used in folklore treatment strategies are reported as effective inhibitors against the proliferative and infective cycles of SARS-CoV-2. The secondary metabolites from plants are generally used to treat various diseases due to their intact medicinal properties. The present study analyzes the inhibitory potential of phytochemicals from medicinal plants like Sphaeranthus indicus, Lantana camara, and Nelumbo nucifera against SARS-CoV-2 by molecular docking. Ten druggable protein targets from SARS-CoV-2 are docked against the phytochemicals from the selected medicinal plants. The phytocompounds astragalin, isoquercetin, and 5-hydroxy-7-methoxy-6-c-glycosy flavone were found to have lower binding energy depicting their inhibitive potential compared with the reported inhibitors that are used in the treatment of SARS-CoV-2 infection. The phytocompounds found to have the least binding energy were selected for further analyses. To assess the compounds' potential as drugs, their ADMET characteristics were also examined. Sphaeranthus indicus, Lantana camara, and Nelumbo nucifera six possible compounds were separately screened for ADME and toxicity characteristics; then, the results were analyzed. To assess the impact of the phytocompound binding on the dynamics of SARS-CoV-2 ribonuclease protein NSP15, microsecond-level all atomistic molecular dynamics simulations were performed, and their dynamics were analyzed. Microsecond-level molecular dynamics simulations of both the ligands complexed with NSP15 revealed that the ligand induces allosteric effects on NSP15, which could lead to destabilization of NSP15 hexameric interface and loss of RNA binding. The low binding energy exhibited by the phytochemicals from Lantana camera, Sphaeranthus indicus, and Nelumbo nucifera against the protein targets of SARS-CoV-2 showed inhibitory potential by the selected molecules. Their predicted interference of the enzymes involved in the molecular mechanisms aiding the proliferation of SARS-CoV-2 indicated the inhibitive ability of the phytochemicals.

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.

Membrane-Mediated Allosteric Action of Serotonin on a Noncognate G-Protein-Coupled Receptor.

The structure and dynamics of the lipid membrane can affect the activity of membrane proteins. Therefore, small lipophilic molecules that alter membrane properties (such as the neurotransmitter serotonin) can potentially modulate receptor activity without binding to the receptor. Here, we investigated how the activity of neuropeptide Y type 4 receptor (Y4R, reconstituted in lipid bicelles) is modulated by serotonin, which has no known interaction with Y4R. We found a serotonin-concentration-dependent decrease (down to 0.1 mM of serotonin) in the ligand affinity of Y4R. This effect correlates with a serotonin-induced reduction of the resistance of the bilayer to indentation (measured by atomic force microscopy) and bilayer thickness (measured by solid state NMR) in two different types of zwitterionic lipid bicelles. Our findings indicate a "membrane-mediated allosteric effect" of serotonin on the activation of Y4R and suggest the potential for developing pharmacophores, which can modulate cellular signaling without directly interacting with any receptor.

Pathogenic COPA mutations decrease binding of dilysine motifs through allosteric effects

Pathogenic COPA mutations decrease binding of dilysine motifs through allosteric effects

PopShift: A Thermodynamically Sound Approach to Estimate Binding Free Energies by Accounting for Ligand-Induced Population Shifts from a Ligand-Free Markov State Model.

Obtaining accurate binding free energies from in silico screens has been a long-standing goal for the computational chemistry community. However, accuracy and computational cost are at odds with one another, limiting the utility of methods that perform this type of calculation. Many methods achieve massive scale by explicitly or implicitly assuming that the target protein adopts a single structure, or undergoes limited fluctuations around that structure, to minimize computational cost. Others simulate each protein-ligand complex of interest, accepting lower throughput in exchange for better predictions of binding affinities. Here, we present the PopShift framework for accounting for the ensemble of structures a protein adopts and their relative probabilities. Protein degrees of freedom are enumerated once, and then arbitrarily many molecules can be screened against this ensemble. Specifically, we use Markov state models (MSMs) as a compressed representation of a protein's thermodynamic ensemble. We start with a ligand-free MSM and then calculate how addition of a ligand shifts the populations of each protein conformational state based on the strength of the interaction between that protein conformation and the ligand. In this work we use docking to estimate the affinity between a given protein structure and ligand, but any estimator of binding affinities could be used in the PopShift framework. We test PopShift on the classic benchmark pocket T4 Lysozyme L99A. We find that PopShift is more accurate than common strategies, such as docking to a single structure and traditional ensemble docking─producing results that compare favorably with alchemical binding free energy calculations in terms of RMSE but not correlation─and may have a more favorable computational cost profile in some applications. In addition to predicting binding free energies and ligand poses, PopShift also provides insight into how the probability of different protein structures is shifted upon addition of various concentrations of ligand, providing a platform for predicting affinities and allosteric effects of ligand binding. Therefore, we expect PopShift will be valuable for hit finding and for providing insight into phenomena like allostery.

Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S.

During progression of rheumatoid arthritis (RA), angiogenesis provides oxygen and nutrients for the cells' increased metabolic demands and number. To turn on angiogenesis, pro-angiogenic factors must outweigh anti-angiogenic factors. We have previously shown that CD147/extracellular matrix metalloproteinase inducer (EMMPRIN) can induce the expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) in a co-culture of the human HT1080 fibrosarcoma and U937 monocytic-like cell lines. However, whether CD147 influences anti-angiogenic factors was not known. We now show that relative to single cultures, the co-culture of these cells not only enhanced pro-angiogenic factors but also decreased the anti-angiogenic factors endostatin and thrombospondin-1 (Tsp-1), generally increasing the angiogenic potential as measured by a wound assay. Using anti-CD147 antibody, CD147 small interfering RNA (siRNA), and recombinant CD147, we demonstrate that CD147 hormetically regulates the generation of endostatin but has no effect on Tsp-1. Since endostatin is cleaved from collagen XVIII (Col18A), we applied different protease inhibitors and established that MMP-9 and proteasome 20S, but not cathepsins, are responsible for endostatin generation. MMP-9 and proteasome 20S collaborate to synergistically enhance endostatin generation, and in a non-cellular system, CD147 enhanced MMP-9 activity and hormetically regulated proteasome 20S activity. Serum samples obtained from RA patients and healthy controls mostly corroborated these findings, indicating clinical relevance. Cumulatively, these findings suggest that secreted CD147 mediates a possibly allosteric effect on MMP-9 and proteasome 20S activities and can serve as a switch that turns angiogenesis on or off, depending on its ambient concentrations in the microenvironment.

Single-molecule FRET probes allosteric effects on protein-translocating pore loops of a AAA+ machine

AAA+ proteins (ATPases associated with various cellular activities) comprise a family of powerful ring-shaped ATP-dependent translocases that carry out numerous vital substrate-remodeling functions. ClpB is a AAA+ protein disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops protruding into its center and binding to substrate proteins. It remains unknown whether these pore loops contribute only passively to substrate-protein threading or have a more active role. Recently, we have applied single-molecule FRET spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the microsecond timescale that are likely to be mechanistically important for disaggregation. Here, using single-molecule FRET, we study the allosteric coupling between the pore loops and the two nucleotide-binding domains of ClpB (NBD1–2). By mutating the conserved Walker B motifs within the NBDs to abolish ATP hydrolysis, we demonstrate how the nucleotide state of each NBD tunes pore-loop dynamics. This effect is surprisingly long-ranged; in particular, PL2 and PL3 respond differentially to a Walker B mutation in either NBD1 or NBD2, as well as to mutations in both. We characterize the conformational dynamics of pore loops and the allosteric paths connecting NBDs to pore loops by molecular dynamics simulations and find that both principal motions and allosteric paths can be altered by changing the ATPase state of ClpB. Remarkably, PL3, which is highly conserved in AAA+ machines, is found to favor an upward conformation when only NBD1 undergoes ATP hydrolysis but a downward conformation when NBD2 is active. These results explicitly demonstrate a significant long-range allosteric effect of ATP hydrolysis sites on pore-loop dynamics. Pore loops are therefore established as active participants that undergo ATP-dependent conformational changes to translocate substrate proteins through the central pores of AAA+ machines.

Molecular recognition of ITIM/ITSM domains with SHP2 and their allosteric effect.

Src homology 2-domain-containing tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase that is widely expressed in a variety of cells and regulates the immune response of T cells through the PD-1 pathway. However, the activation mechanism and allosteric effects of SHP2 remain unclear, hindering the development of small molecule inhibitors. For the first time, in this study, the complex structure formed by the intact PD-1 tail and SHP2 was modeled. The molecular recognition and conformational changes of inactive/active SHP2 versus ITIM/ITSM were compared based on prolonged MD simulations. The relative flexibility of the two SH2 domains during MD simulations contributes to the recruitment of ITIM/ITSM and supports the subsequent conformational change of SHP2. The binding free energy calculation shows that inactive SHP2 has a higher affinity for ITIM/ITSM than active SHP2, mainly because the former's N-SH2 refers to the α-state. In addition, a significant decrease in the contribution to the binding energy of certain residues (e.g., R32, S34, K35, T42, and K55) of conformationally transformed SHP2 contributes to the above result. These detailed changes during conformational transition will provide theoretical guidance for the molecular design of subsequent novel anticancer drugs.

Assessing the role of residue Phe108 of cytochrome P450 3A4 in allosteric effects of midazolam metabolism.

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more drugs in clinical use than any other xenobiotic-metabolizing enzyme. CYP3A4-mediated drug metabolism is usually allosterically modulated by substrate concentration (homotropic allostery) and other drugs (heterotropic allostery), exhibiting unusual kinetic profiles and regiospecific metabolism. Recent studies suggest that residue Phe108 (F108) of CYP3A4 may have an important role in drug metabolism. In this work, residue mutations were coupled with well-tempered metadynamics simulations to assess the importance of F108 in the allosteric effects of midazolam metabolism. Comparing the simulation results of the wild-type and mutation systems, we identify that the π-π interaction and steric effect between the F108 side chain and midazolam is favorable for the stable binding of substrate in the active site. F108 also plays an important role in the transition of substrate binding mode, which mainly induces the transition of substrate binding mode by forming π-π interactions with multiple aromatic rings of the substrate. Moreover, the side chain of F108 is closely related to the radius and depth of the 2a and 2f channels, and F108 may further regulate drug metabolism by affecting the pathway, orientation, or time of substrate entry into the CYP3A4 active site or product egress from the active site. Altogether, we suggest that F108 affects drug metabolism and regulatory mechanisms by affecting substrate binding stability, binding mode transition, and channel characteristics of CYP3A4. Our findings could promote the understanding of complicated allosteric mechanisms in CYP3A4-mediated drug metabolism, and the knowledge could be used for drug development and disease treatment.

Comprehensive structural and functional analysis of hVEGFR1: Insights into phosphorylation, molecular interactions, and potential inhibitors through docking and dynamics simulations

Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), is an enzyme with tyrosine kinase activity that plays a pivotal role in angiogenesis, the process of new blood vessel formation. This receptor is of significant clinical importance as it is implicated in various cancers, particularly non-small cell lung cancer (NSCLC), where its dysregulation leads to uncontrolled cell growth through ligand-induced phosphorylation. While commercially available drugs target VEGFR1, their prolonged use often leads to drug resistance and the emergence of mutations in cancer patients. To address these challenges, researchers have identified the human tyrosine kinase (hTK) domain of VEGFR1 as a potential therapeutic marker for lung malignancies. The 3D crystal structure of the hTK domain, obtained from Protein Data Bank (PDB ID: 3HNG), has provided vital structural insights of hVEGFR1. This study has revealed variations within the hVEGFR1 tyrosine kinase domain, distinguishing between regions associated with phosphorylase kinase and transferase activities. We identified numerous potential phosphorylation sites within the TK domain, shedding light on the protein's regulation and signaling possible. Detailed molecular interaction analyses have elucidated the binding forces between lead molecules and hVEGFR1, including hydrogen bonds, electrostatic, hydrophobic, and π-sigma interactions. The stability observed during molecular dynamics simulations further underscores the biological relevance of these interactions. Furthermore, docked complexes has highlighted localized structural fluctuations, offering insight into potential allosteric effects and dynamic conformational changes induced by lead molecules. These findings not only provide a comprehensive characterization of hVEGFR1 but also pave the way for the development of targeted therapies. Eventually, this study has the potential in identifying drug to combat diseases associated with hVEGFR1 dysregulation, including cancer and angiogenesis-related disorders, contributing to effective treatment strategies.

Lithium chloride selective ion-pair recognition by heteroditopic [2]rotaxanes.

The first heteroditopic [2]rotaxane host systems capable of strong and selective binding of lithium chloride ion-pair species are described. Importantly, a cooperative 'switch on' mechanism was found to operate, in which complexation of a lithium metal cation enhances the halide anion affinity of the rotaxanes via a combination of favourable proximal electrostatic and preorganised allosteric effects. The mechanically bonded rotaxane host design features a macrocycle component possessing a 2,6-dialkoxy pyridyl cation binding motif and an isophthalamide anion binding group, as well as an axle component functionalised with either a halogen bonding (XB) iodotriazole or hydrogen bonding (HB) prototriazole moiety. Extensive quantitative 1H NMR titration studies in CD3CN/CDCl3 solvent mixtures determined enhanced ion-pair binding affinities for lithium halides over the corresponding sodium or potassium halide salts, with the axle prototriazole-containing HB rotaxane in particular demonstrating a marked selectivity for lithium chloride. Solid-state X-ray crystallographic studies and computational DFT investigations provide evidence for a [2]rotaxane host axle-separated ion-pair binding mode, in which complementary cation and anion binding motifs from both the macrocycle and axle components act convergently to recognise each of the charged guest species.

Synergy and anti-cooperativity in allostery: Molecular dynamics study of WT and oncogenic KRAS-RGL1.

This study focuses on investigating the effects of an oncogenic mutation (G12V) on the stability and interactions within the KRAS-RGL1 protein complex. The KRAS-RGL1 complex is of particular interest due to its relevance to KRAS-associated cancers and the potential for developing targeted drugs against the KRAS system. The stability of the complex and the allosteric effects of specific residues are examined to understand their roles as modulators of complex stability and function. Using molecular dynamics simulations, we calculate the mutual information, MI, between two neighboring residues at the interface of the KRAS-RGL1 complex, and employ the concept of interaction information, II, to measure the contribution of a third residue to the interaction between interface residue pairs. Negative II indicates synergy, where the presence of the third residue strengthens the interaction, while positive II suggests anti-cooperativity. Our findings reveal that MI serves as a dominant factor in determining the results, with the G12V mutation increasing the MI between interface residues, indicating enhanced correlations due to the formation of a more compact structure in the complex. Interestingly, although II plays a role in understanding three-body interactions and the impact of distant residues, it is not significant enough to outweigh the influence of MI in determining the overall stability of the complex. Nevertheless, II may nonetheless be a relevant factor to consider in future drug design efforts. This study provides valuable insights into the mechanisms of complex stability and function, highlighting the significance of three-body interactions and the impact of distant residues on the binding stability of the complex. Additionally, our findings demonstrate that constraining the fluctuations of a third residue consistently increases the stability of the G12V variant, making it challenging to weaken complex formation of the mutated species through allosteric manipulation. The novel perspective offered by this approach on protein dynamics, function, and allostery has potential implications for understanding and targeting other protein complexes involved in vital cellular processes. The results contribute to our understanding of the effects of oncogenic mutations on protein-protein interactions and provide a foundation for future therapeutic interventions in the context of KRAS-associated cancers and beyond.

Multi-aptamer-mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of S. pneumoniae and S. aureus.

A novel nucleic acid aptamer nanoprobes-mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of Streptococcus pneumoniae and Staphylococcus aureus is presented.In this fluorescence assay system, utilizing the hairpin allosteric effect caused by the aptamer binding to the target bacteria, the detection of S. pneumoniae is first achieved through changes in fluorescence due to FRET. Subsequently, aCas12a protein mixture is added to detect S. aureus. The amplified output signal is triggered by two methods to ensure the sensitivity of the method: the synergistic FRET effect is achieved by the assembly of multi-aptamer through the conjugation of streptavidin-biotin, and the trans-cleavage function of CRISPR/Cas 12a. Under the optimized conditions, the proposed hairpin allosteric aptasensor could achieve high sensitivity (a detection limit of 135cfu/mL) and broad-concentration quantification (dynamic range of 103-107cfu/mL) of S. pneumoniae. The aptamer-assisted CRISPR system for S. aureus detection showed good linearity (R2 = 0.996) in the concentration range 102-108cfu/mL, with a detection limit of 39cfu/mL. No cross-reactivity with other foodborne pathogenic bacteria was observed in both systems. Taking only 55min, this method of multiple pathogen detection proved to be promising.

C-terminal domain dimerization in yeast Hsp90 is moderately modulated by the other domains

Heat shock protein 90 (Hsp90) serves as a crucial regulator of cellular proteostasis by stabilizing and regulating the activity of numerous substrates, many of which are oncogenic proteins. Therefore, Hsp90 is a drug target for cancer therapy. Hsp90 comprises three structural domains, a highly conserved amino-terminal domain (NTD), a middle domain (MD), and a carboxyl-terminal domain (CTD). The CTD is responsible for protein dimerization, is crucial for Hsp90’s activity, and has therefore been targeted for inhibiting Hsp90. Here we addressed the question of whether the CTD dimerization in Hsp90, in the absence of bound nucleotides, is modulated by allosteric effects from the other domains. We studied full length (FL) and isolated CTD (isoC) yeast Hsp90 spin-labeled with a Gd(III) tag by double electron-electron resonance measurements to track structural differences and to determine the apparent dissociation constant (Kd). We found the distance distributions for both the FL and isoC to be similar, indicating that the removal of the NTD and MD does not significantly affect the structure of the CTD dimer. The low-temperature double electron-electron resonance-derived Kd values, as well as those obtained at room temperature using microscale thermophoresis and native mass spectrometry, collectively suggested the presence of some allosteric effects from the NTDs and MDs on the CTD dimerization stability in the apo state. This was evidenced by a moderate increase in the Kd for the isoC compared with the FL mutants. Our results reveal a fine regulation of the CTD dimerization by allosteric modulation, which may have implications for drug targeting strategies in cancer therapy.

Studying early structural changes in SOS1 mediated KRAS activation mechanism

KRAS activation is known to be modulated by a guanine nucleotide exchange factor (GEF), namely, Son of Sevenless1 (SOS1). SOS1 facilitates the exchange of GDP to GTP thereby leading to activation of KRAS. The binding of GDP/GTP to KRAS at the REM/allosteric site of SOS1 regulates the activation of KRAS at CDC25/catalytic site by facilitating its exchange. Different aspects of the allosteric activation of KRAS through SOS1 are still being explored. To understand the SOS1 mediated activation of KRAS, molecular dynamics simulations for a total of nine SOS1 complexes (KRAS-SOS1-KRAS) were performed. These nine systems comprised different combinations of KRAS-bound nucleotides (GTP/GDP) at REM and CDC25 sites of SOS1. Various conformational and thermodynamic parameters were analyzed for these simulation systems. MMPBSA free energy analysis revealed that binding at CDC25 site of SOS1 was significantly low for GDP-bound KRAS as compared to that of GTP-bound KRAS. It was observed that presence of either GDP/GTP bound KRAS at the REM site of SOS1 affected the activation related changes in the KRAS present at CDC25 site. The conformational changes at the catalytic site of SOS1 resulting from GDP/GTP-bound KRAS at the allosteric changes may hint at KRAS activation through different pathways (slow/fast/rare). The allosteric effect on activation of KRAS at CDC25 site may be due to conformations adopted by switch-I, switch-II, beta2 regions of KRAS at REM site. The effect of structural rearrangements occurring at allosteric KRAS may have led to increased interactions between SOS1 and KRAS at both the sites. The SOS1 residues involved in these important interactions with KRAS at the REM site were R694, S732 and K735. Whereas the ones interacting with KRAS at CDC25 site were S807, W809 and K814. This may suggest the crucial role of these residues in guiding the allosteric activation of KRAS at CDC25 site. The conformational shifts observed in the switch-I, switch-II and alpha3 regions of KRAS at CDC25 site may be attributed to be a part of allosteric activation. The binding affinities, interacting residues and conformational dynamics may provide an insight into development of inhibitors targeting the SOS1 mediated KRAS activation.

Mechanisms of DNA-Mediated Allostery.

Proteins often regulate their activities via allostery-or action at a distance-in which the binding of a ligand at one binding site influences the affinity for another ligand at a distal site. Although less studied than in proteins, allosteric effects have been observed in experiments with DNA as well. In these experiments two or more proteins bind at distinct DNA sites and interact indirectly with each other, via a mechanism mediated by the linker DNA molecule. We develop a mechanical model of DNA/protein interactions which predicts three distinct mechanisms of allostery. Two of these involve an enthalpy-mediated allostery, while a third mechanism is entropy driven. We analyze experiments of DNA allostery and highlight the distinctive signatures allowing one to identify which of the proposed mechanisms best fits the data.

Unveiling the RNA-mediated allosteric activation discloses functional hotspots in CRISPR-Cas13a.

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377,N378, andR973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.