Effects Of Ligand Binding Research Articles

Investigation of Protein-Lipid Interactions Using Native Mass Spectrometry.

Integral membrane proteins are embedded in biological membranes where various lipids modulate their structure and function. There exists a critical need to elucidate how these lipids participate in the physiological and pathological processes associated with the membrane protein dysfunction. Native mass spectrometry (MS), combined with ion mobility spectrometry (IM), is emerging as a powerful tool to probe membrane protein complexes and their interactions with ligands, lipids, and other small molecules. Unlike other biophysical approaches, native IM-MS can resolve individual ligand/lipid binding events. We have developed a novel method using native MS, coupled with a temperature-control apparatus, to determine the thermodynamic parameters of individual ligand or lipid binding events to proteins. This approach has been validated using several soluble protein-ligand systems wherein MS results are compared with those acquired from conventional biophysical techniques, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Using these principles, it is possible to elucidate the thermodynamics of individual lipid binding to integral membrane proteins. Herein, we use the ammonia channel (AmtB) from Escherichia coli as a model membrane protein. Remarkably, distinct thermodynamic signatures for AmtB binding to lipids with different headgroups and acyl chain configurations are observed. Additionally, using a mutant form of AmtB that abolishes a specific lipid binding site, distinct changes have been discovered in the thermodynamic signatures compared with the wild-type, implying that these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites. This chapter provides procedures and findings associated with temperature-controlled native MS as a novel approach to interrogate membrane proteins and their interactions with lipids and other molecules.

Resonance Raman studies of gas sensing heme proteins

AbstractHeme sensor proteins are present in organisms ranging from bacteria to mammals, typically consisting of a catalytic domain and a heme‐containing sensor domain, which can bind diatomic gas molecules such as O2, CO, and NO. Such ligand binding event can induce active site conformational changes that are subsequently propagated to the functional domain, ultimately activating/deactivating a series of biological events, such as aerotaxis, gene expression, changes the metabolic pathway, and so forth. Such regulatory events are crucial for the cell to fulfill the physiological functions by regulating enzyme activity in response to the external stimuli. Resonance Raman (rR) spectroscopy is an effective method to investigate the sensing mechanism of these structurally different associated sensor proteins that exhibit diverse inherent physiological functions. This review summarizes the results of rR studies on a diverse collection of these heme sensor proteins, including FixL, Escherichia coli direct oxygen sensor (EcDOS), HemAT from Bacillus subtilis (HemAT‐Bs), CooA, soluble guanylyl cyclase (sGC), heme nitric oxide and/or oxygen binding domain (H‐NOX), and nitric oxide sensing proteins (NosP).

Effects of Pooling Operations on Prediction of Ligand Rotation‐Dependent Protein–Ligand Binding in 3D Graph Convolutional Network

3D graph convolutional network (3DGCN) is a graph neural network (GNN) variant that utilizes the 3D bond information on molecules for chemical tasks. In 3DGCN, the pooling operation extracts the molecular features from the atomic features for molecule embedding and dimensionality reduction. In this work, we investigated the pooling effects on the performance of 3DGCN in the classification of protein–ligand binding events, especially when a ligand is rotated with respect to a target protein in the 3D Cartesian coordinate. 3DGCN showed the general ability for recognizing the ligand rotation, and its prediction accuracy was found to be pooling‐dependent. For example, 3DGCN with max pooling did not recognize the rotations of known active ligands for human β‐secretase 1 faithfully, compared with the other pooling operations (sum, avg, and set2set). This work would contribute to augmented architecture evolution of 3DGCN for the chemical tasks that require the 3D molecular information including chirality.

Biomolecule Exchange Dynamics on Carbon Nanotubes Via Multiplexed Fluorophore Quenching

Adsorption of biomolecules on single-walled carbon nanotubes (SWCNTs) has enabled developments in molecular sensing, in vivo imaging, and gene delivery applications.1,2 This noncovalent functionalization of SWCNTs preserves the pristine SWCNT atomic structure, thus retaining the intrinsic near-infrared fluorescence for sensing or imaging functions, and offers a reversible binding mode for cargo delivery. However, biomolecule adsorption is an inherently dynamic process, where exchange occurs between molecules in the bulk solution and molecules on the nanoparticle surface, into what is known as the corona phase. The nature, strength, and kinetics of both the biomolecule binding and unbinding processes on SWCNTs are important contributors to the success of SWCNT-based technologies. Understanding this binding process is especially important for intended uses of functionalized SWCNTs in biological environments, where native biomolecules such as proteins compete with the original biomolecule to occupy the SWCNT surface. Such biofouling of the SWCNT surface disrupts the intended functionality of the nanoparticle and can further lead to adverse biocompatibility outcomes.We present a generalizable method to study the corona exchange dynamics between solution-phase and corona-phase biomolecules on SWCNTs in real-time.3 This approach exploits the quenching property of fluorophores proximal to the SWCNT surface to monitor ligand binding and unbinding events. Real-time tracking of adsorption and desorption events on the SWCNT surface are conducted with systematic variation of the molecular entities and solution conditions, with a specific focus on DNA (as the sensing moiety) and protein (as the biofouling agent). Binding profiles are extracted from the experimental assay and used to inform a kinetic model of the system. The work presented herein develops an understanding of the fundamental corona exchange mechanism and provides insight into performance of these designed SWCNT-based systems in biologically relevant, protein-rich conditions. References Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nature Nanotechnology 1 (2019) doi:10.1038/s41565-019-0382-5.Beyene, A. G. et al. Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor. Science Advances 5, eaaw3108 (2019).Pinals, R. L., Yang, D., Lui, A., Cao, W. & Landry, M. P. Corona Exchange Dynamics on Carbon Nanotubes by Multiplexed Fluorescence Monitoring. J. Am. Chem. Soc. (2019) doi:10.1021/jacs.9b09617. Figure 1

Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor

The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor’s ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR’s extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.

Abstract 6644: A comprehensive in vitro and in vivo system to evaluate STING agonist efficacy in cancer therapeutics

Abstract A number of stimulator of interferon genes protein (STING) agonists has recently been developed to treat cancer and infectious diseases. To investigate the binding of ligand to STING and subsequent activation of the STING pathway, we used the protein thermal shift assay, a high-throughput screening method for intermolecular interaction involving a protein of interest, to identify previously unknown compounds that bind to STING. To determine the functional consequence of ligand binding, we constructed IFN-β-Luc THP1 reporter cell line, to evaluate the pathway activation together with IFNβ secretion. In addition, we incubated human peripheral blood mononuclear cells (PBMCs) with STING agonist and measured phosphorylation of STING, phosphorylation of IRF3 and the secretion of cytokines such as IFN-β by ELISA or Luminex. To determine the in vivo therapeutic effects of STING agonists, we performed efficacy studies in syngeneic mouse models of melanoma (B16F10 cell line), colorectal cancer (CT-26 cell line) or breast cancer (4T1 cell line) with intratumoral delivery of test compounds. Test Treatment with STING agonists resulted in significant tumor growth inhibition as measured by tumor volume and significantly improved survival. Tumor-reactive IFN-γ secreting CD8+ T cells were harvested from spleens, purified by immunomagnetic sorting, and immediately used in ELISPOT assays using the mouse IFN-γ ELISPOT Kit. Tumor-infiltrating lymphocytes (TILs) have also been assessed in harvested melanoma (B16F10) tumors. We found that treatment with STING agonists resulted in increased CD69 percentage in CD8+ T cells in TILs, and decreased MHCII levels in macrophages and DCs in TILs. In summary, we have established reporter-based in vitro assays and in vivo efficacy validation platforms, which can be used for STING agonists efficacy evaluation for cancer therapy and combination IO target therapy. Citation Format: Xiaohe Shi, Feifei Fan, Ruicheng Wei, Xiangyang Zuo, Xiutao Lu, Quanhong Sun. A comprehensive in vitro and in vivo system to evaluate STING agonist efficacy in cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6644.

Abstract P3-10-09: Gene deletion of IRS1 inhibited IGF-I, insulin and estradiol stimulated MCF-7L breast cancer cell growth

Abstract Targeting of the type I insulin-like growth factor receptor (IGF-IR) has not been successful in breast cancer. Data suggest the highly homologous insulin receptor (IR) may be an alternate growth stimulatory pathway used by cancer cells. Since both receptors phosphorylate the insulin receptor substrate-1 (IRS-1) protein as an immediate consequence of ligand binding, disruption of both receptors could be accomplished by suppression of IRS-1. To test this hypothesis, we created a doxycycline-inducible IRS-1 shRNA lentiviral construct to infect hormone dependent MCF-7 breast cancer cells. IRS-1 shRNA downregulation resulted in decreased responses to IGF-I, insulin, and estradiol in monolayer and anchorage independent growth assays. Decreased IRS-1 levels also suppressed estradiol stimulated gene expression and estrogen receptor binding to DNA. Xenograft growth was also inhibited by induction of IRS-1 shRNA. These data show IRS-1 is a critical regulator of endocrine responsive breast cancer. To further study the critical effects of IRS1 in ER positive breast cancer biology, CRISPR technology was used to delete IRS1 from MCF-7L cells. We analyzed 5 CRISPR IRS1 gene edited clones. Two different commercial available IRS1 antibodies were used to examine IRS1 protein expression. Clones #16, #20 did not show any IRS1 expression, while clones #22, #33 and #38 showed truncated IRS1 expression. IGFIRβ and Insulin R levels were comparable to the parent MCF-7L cells in all the clones, while IRS2 and ERα levels were significantly lower in clone #16. IRS phosphorylation, detected by PY20 antibody, was significantly lower in clone #16. The downstream signaling measured by pAKT and pErk1,2 did not change in all clones compared to those in MCF-7L. In all the clones, ERα ser118 phosphorylation by estradiol stimulation (10 nM) and ERα downregulation by fulvestrant treatment (100 nM) were not affected by IRS1 knockout. IGF-I (5 nM), insulin (10 nM) and estradiol (10 nM) stimulated monolayer cell growth in clone #16 was completely inhibited, while other clones were partially inhibited. In contrast, growth in clone #22 was not inhibited due to extremely high IRS2 expression and compensation. In conclusion, when the IRS1 gene was deleted, multiple growth stimulatory ligands (IGF-I, insulin and estradiol) were inhibited in breast cancer cells. However, IRS-2 compensated for IRS-1 gene deletion in one of the clones we studied. Thus, targeting the IRS proteins may inhibit multiple ligands that cause growth stimulation in ER positive breast cancer therapy. Citation Format: Xihong Zhang, Douglas Yee. Gene deletion of IRS1 inhibited IGF-I, insulin and estradiol stimulated MCF-7L breast cancer cell growth [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-09.

Origin of cooperativity in the activation of dimeric transcription factors

Cooperative behavior in the binding of ligands to a protein is often viewed as a complex phenomenon where conformational changes induced by the binding of the first ligand leads to tighter binding of subsequent ligands. We revisit the ligand-dependent activation of dimeric transcription factors and show that this process may appear cooperative even when it results from independent ligand binding events. This effect is further accentuated through binding of the activated transcription factor to its cognate operator site on the DNA, where we demonstrate that cooperative activation is a stable fixed point. Our analysis nicely accounts for the apparent co-operativity inherent in the biological activity of many dimeric transcription factors.

Biased Signaling of the G-Protein-Coupled Receptor β2AR Is Governed by Conformational Exchange Kinetics

G-protein-coupled receptors (GPCRs) mediate a wide range of human physiological functions by transducing extracellular ligand binding events into intracellular responses. GPCRs can activate parallel, independent signaling pathways mediated by G proteins or β-arrestins. Whereas "balanced" agonists activate both pathways equally, "biased" agonists dominantly activate one pathway, which is of interest for designing GPCR-targeting drugs because it may mitigate undesirable side effects. Previous studies demonstrated that β-arrestin activation is associated with transmembrane helix VII (TM VII) of GPCRs. Here, single-molecule fluorescence spectroscopy with the β2-adrenergic receptor (β2AR) in the ligand-free state showed that TM VII spontaneously fluctuates between one inactive and one active-like conformation. The presence of the β-arrestin-biased agonist isoetharine prolongs the dwell time of TM VII in the active-like conformation compared with the balanced agonist formoterol, suggesting that ligands can induce signaling bias by modulating the kinetics of receptor conformational exchange.

A multi-state model of the CaMKII dodecamer suggests a role for calmodulin in maintenance of autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzyme’s subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of a twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous experimental measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca2+/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca2+/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca2+/CaM and protein phosphatase do or do not structurally exclude each other’s binding to CaMKII. Our results suggest a functional mechanism for the so-called “CaM trapping” phenomenon, wherein Ca2+/CaM may structurally exclude phosphatase binding and thereby prolong CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca2+/CaM may be an important mechanism for regulation of synaptic plasticity.

Structural Features of Tight-Junction Proteins.

Tight junctions are complex supramolecular entities composed of integral membrane proteins, membrane-associated and soluble cytoplasmic proteins engaging in an intricate and dynamic system of protein–protein interactions. Three-dimensional structures of several tight-junction proteins or their isolated domains have been determined by X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. These structures provide direct insight into molecular interactions that contribute to the formation, integrity, or function of tight junctions. In addition, the known experimental structures have allowed the modeling of ligand-binding events involving tight-junction proteins. Here, we review the published structures of tight-junction proteins. We show that these proteins are composed of a limited set of structural motifs and highlight common types of interactions between tight-junction proteins and their ligands involving these motifs.

Development of a Cell-Based Ligand-Screening System for Identifying Hsp90 Inhibitors.

Because of its critical roles in regulating cellular signal transduction, the molecular chaperone heat-shock protein 90 (Hsp90) has become a novel therapeutic target for various diseases, including cancer, inflammation, and neurological diseases. However, the lack of methods that allow us to directly evaluate the binding of small molecule ligands to intracellular Hsp90 makes the inhibitor development more difficult. Here, we report a simple cell-based assay system for the Hsp90 inhibitor in live-cell environments. In this strategy, the binding activity of ligands of interest is evaluated by competitive inhibition of ligand-directed N-acyl-N-alkyl sulfonamide (LDNASA) chemistry-mediated Hsp90 labeling. Using several known Hsp90 inhibitors, we demonstrated that our method could easily detect the ligand-binding event of Hsp90 in live cells. Our system is applicable to high-throughput ligand screening, and we discovered a new small molecule candidate that binds to the N-terminal ATP binding domain of Hsp90. These results demonstrate the use of the competitive LDNASA-based approach to directly evaluate ligand activity in live cells and identify potent drug candidates from chemical libraries.

Spectroscopic X-ray and Mössbauer Characterization of M6 and M5 Iron(Molybdenum)-Carbonyl Carbide Clusters: High Carbide-Iron Covalency Enhances Local Iron Site Electron Density Despite Cluster Oxidation

The present study employs a suite of spectroscopic techniques to evaluate the electronic and bonding characteristics of the interstitial carbide in a set of iron-carbonyl-carbide clusters, one of which is substituted with a molybdenum atom. The M6C and M5C clusters are the dianions (Et4N)2[Fe6(μ6-C)(μ2-CO)2(CO)14] (1), [K(benzo-18-crown-6)]2[Fe5(μ5-C)(μ2-CO)1(CO)13] (2), and [K(benzo-18-crown-6)]2[Fe5Mo(μ6-C)(μ2-CO)2(CO)15] (3). Because 1 and 2 have the same overall cluster charge (2−) but different numbers of iron sites (1: 6 sites → 2: 5 sites), the metal atoms of 2 are formally oxidized compared to those in 1. Despite this, Mössbauer studies indicate that the iron sites in 2 possess significantly greater electron density (lower spectroscopic oxidation state) compared with those in 1. Iron K-edge X-ray absorption and valence-to-core X-ray emission spectroscopy measurements, paired with density functional theory spectral calculations, revealed the presence of significant metal-to-metal and carbide 2p-based character in the filled valence and low-lying unfilled electronic manifolds. In all of the above experiments, the presence of the molybdenum atom in 3 (Fe5Mo) results in somewhat unremarkable spectroscopic properties that are essentially a “hybrid” of 1 (Fe6) and 2 (Fe5). The overall electronic portrait that emerges illustrates that the central inorganic carbide ligand is essential for distributing charge and maximizing electronic communication throughout the cluster. It is evident that the carbide coordination environment is quite flexible and adaptive: it can drastically modify the covalency of individual Fe–C bonds based on local structural changes and redox manipulation of the clusters. In light of these findings, our data and calculations suggest a potential role for the central carbon atom in FeMoco, which likely performs a similar function in order to maintain cluster integrity through multiple redox and ligand binding events.

Understanding Corona Exchange Dynamics on Single-Walled Carbon Nanotubes with Multiplexed Fluorescence Monitoring

Adsorption of polymers on single-walled carbon nanotubes (SWCNTs) has enabled developments in molecular sensing, in vivo imaging, gene and drug delivery, and chirality sorting applications. Noncovalent functionalization offers a modification route that preserves the pristine atomic structure, thus retaining the intrinsic near-infrared fluorescence of the SWCNTs for sensing or imaging functions, and offers a reversible binding mode for cargo delivery or chirality separation processes. However, noncovalent adsorption is an inherently dynamic process, where exchange occurs between molecules in the bulk solution and molecules on the surface, into what is known as the ‘corona phase’. In the case of polymers on SWCNTs, the nature, strength, and kinetics of both the polymer binding and unbinding processes are important contributors to the success of polymer-SWCNT based technologies. Understanding this binding process is especially important for intended uses of functionalized SWCNTs in biological environments, where native biomolecules compete with the original polymer to occupy the SWCNT surface. Binding of proteins and other biomolecules to the SWCNT not only disrupts the intended functionality of the nanoparticle, but further leads to potentially adverse biocompatibility outcomes. We present an assay to study the process of corona exchange dynamics between solution-phase and corona-phase polymers on SWCNTs. Real-time tracking of polymer and protein adsorption and desorption events are conducted with systematic variation of the molecular entities and solution conditions. This assay exploits the quenching property of fluorophores in close proximity to the SWCNT surface to monitor ligand binding and unbinding events. Binding profiles are thus extracted from the experimental assay and used to inform a kinetic model of the system. This study encompasses exchange of SWCNT-adsorbed ssDNA and either ssDNA, surfactant, or protein added to the bulk solution with multiplexed fluorescence tracking of all entities. Moreover, the kinetics of these binding profiles are validated against an orthogonal and more ubiquitously-used platform monitoring solvatochromic shifting of the near-infrared SWCNT spectrum as a proxy for SWCNT corona perturbations (Beyene et al., Nano Letters, 2018; Heller et al., PNAS, 2011; Jena et al., ACS Appl. Mater. Interfaces, 2017). The work presented herein develops an understanding of the fundamental corona exchange mechanism, contextualizes the nature of the ligand exchange process versus SWCNT solvatochromic shifting, and provides insight into performance of these designed SWCNT-based systems in biologically relevant, protein-rich conditions.

Communication between the Ligand-Binding Pocket and the Activation Function-2 Domain of Androgen Receptor Revealed by Molecular Dynamics Simulations.

Androgen receptor (AR), as a member of the nuclear receptor (NR) superfamily, regulates the gene transcription in response to the sequential binding of diverse agonists and coactivators. Great progress has been made in studies on the pharmacology and structure of AR, but the atomic level mechanism of the bidirectional communications between the ligand-binding pocket (LBP) and the activation function-2 (AF2) region of AR remains poorly understood. Therefore, in this study, molecular dynamics (MD) simulations and free energy calculations were carried out to explore the interactions among water, agonist (DHT) or antagonist (HFT), AR, and coactivator (SRC3). Upon the binding of an agonist (DHT) or antagonist (HFT), the LBP structure would transform to the agonistic or antagonistic state, and the conformational changes of the LBP would regulate the structure of the AF2 surface. As a result, the binding of the androgen DHT could promote the recruitment of the coactivator SRC3 to the AF2, and on the contrary, the binding of the antagonist HFT would induce a perturbation to the shape of the AF2 and then weaken its accommodating capability of the coactivators with the LXXLL motif. The simulation results illustrated that the DHT-AR binding affinity was enhanced by the association of the coactivator SRC3, which would reduce the conformational fluctuation of the AR-LBD and expand the size of the AR LBP. On the other hand, the coactivator-to-HFT allosteric pathway, which involves the SRC3, helix 3 (H3), helix 4 (H4), the loop (L1-3) between helix 1 (H1) and H3, and HFT, was characterized. The HFT's skewness and different interactions between HFT and the LBP were observed in the SRC3-present AR. The mutual communications between the AF2 surface and LBP, together with the processes involving the interplay of the ligand binding and coactivator recruitment events, would help in understanding the association of coactivators and rationally develop potent drugs to inhibit the activity of AR.

An Ion-Permeable State of the Glycine Receptor Captured by Molecular Dynamics

Glycine receptors (GlyR) mediate fast inhibitory neurotransmission by switching between discrete states in response to ligand-binding events. Recent high-resolution structures from cryoelectron microscopy (cryo-EM) and X-ray crystallography have provided atomistic models for the open and closed states. Notably, the cryo-EM structure in complex with glycine illuminated a previously unreported wide-open state, whose physiological significance is debated. Here, we present the structure of an ion-conducting state of GlyR α1 captured by molecular dynamics and validate its physiological relevance with computational electrophysiology and polyatomic anion permeation simulations. Our analysis suggests that none of the experimental structures is a true representation of the physiologically active state, although previously characterized open channels in GLIC at pH 4, or GluCl/GlyR with ivermectin bound, provide reasonable models. These results open the door to an original functional annotation and support the conclusion that pore closing by desensitization versus deactivation involves the reorientation of the pore-lining helices in opposite directions.

Network-level allosteric effects are elucidated by detailing how ligand-binding events modulate utilization of catalytic potentials.

Allosteric regulation has traditionally been described by mathematically-complex allosteric rate laws in the form of ratios of polynomials derived from the application of simplifying kinetic assumptions. Alternatively, an approach that explicitly describes all known ligand-binding events requires no simplifying assumptions while allowing for the computation of enzymatic states. Here, we employ such a modeling approach to examine the “catalytic potential” of an enzyme—an enzyme’s capacity to catalyze a biochemical reaction. The catalytic potential is the fundamental result of multiple ligand-binding events that represents a “tug of war” among the various regulators and substrates within the network. This formalism allows for the assessment of interacting allosteric enzymes and development of a network-level understanding of regulation. We first define the catalytic potential and use it to characterize the response of three key kinases (hexokinase, phosphofructokinase, and pyruvate kinase) in human red blood cell glycolysis to perturbations in ATP utilization. Next, we examine the sensitivity of the catalytic potential by using existing personalized models, finding that the catalytic potential allows for the identification of subtle but important differences in how individuals respond to such perturbations. Finally, we explore how the catalytic potential can help to elucidate how enzymes work in tandem to maintain a homeostatic state. Taken together, this work provides an interpretation and visualization of the dynamic interactions and network-level effects of interacting allosteric enzymes.

An allelic series at the KARRIKIN INSENSITIVE2 locus of Arabidopsis thaliana decouples ligand hydrolysis and receptor degradation from downstream signalling.

Karrikins are butenolide compounds present in post-fire environments that can stimulate seed germination in many species, including Arabidopsis thaliana. Plants also produce endogenous butenolide compounds that serve as hormones, namely strigolactones (SLs). The receptor for karrikins (KARRIKIN INSENSITIVE2; KAI2) and the receptor for SLs (DWARF14; D14) are homologous proteins that share many similarities. The mode of action of D14 as a dual enzyme receptor protein is well established, but the nature of KAI2-dependent signalling and its function as a receptor are not fully understood. To expand our knowledge of how KAI2 operates, we screened ethyl methanesulphonate (EMS)-mutagenized populations of A.thaliana for mutants with kai2-like phenotypes and isolated 13 new kai2 alleles. Among these alleles, kai2-10 encoded a D184N protein variant that was stable inplanta. Differential scanning fluorimetry assays indicated that the KAI2 D184N protein could interact normally with bioactive ligands. We developed a KAI2-active version of the fluorescent strigolactone analogue Yoshimulactone Green to show that KAI2 D184N exhibits normal rates of ligand hydrolysis. KAI2 D184N degraded in response to treatment with exogenous ligands, suggesting that receptor degradation is a consequence of ligand binding and hydrolysis, but is insufficient for signalling activity. Remarkably, KAI2 D184N degradation was hypersensitive to karrikins, but showed a normal response to strigolactone analogues, implying that these butenolides may interact differently with KAI2. These results demonstrate that the enzymatic and signalling functions of KAI2 can be decoupled, and provide important insights into the mechanistic events that underpin butenolide signalling in plants.

Mechano-redox control of integrin de-adhesion.

How proteins harness mechanical force to control function is a significant biological question. Here we describe a human cell surface receptor that couples ligand binding and force to trigger a chemical event which controls the adhesive properties of the receptor. Our studies of the secreted platelet oxidoreductase, ERp5, have revealed that it mediates release of fibrinogen from activated platelet αIIbβ3 integrin. Protein chemical studies show that ligand binding to extended αIIbβ3 integrin renders the βI-domain Cys177-Cys184 disulfide bond cleavable by ERp5. Fluid shear and force spectroscopy assays indicate that disulfide cleavage is enhanced by mechanical force. Cell adhesion assays and molecular dynamics simulations demonstrate that cleavage of the disulfide induces long-range allosteric effects within the βI-domain, mainly affecting the metal-binding sites, that results in release of fibrinogen. This coupling of ligand binding, force and redox events to control cell adhesion may be employed to regulate other protein-protein interactions.

Steered Molecular Dynamics Simulations of Coumarin2 5Z/5E Pulling Reveal Different Interaction Profiles for Four Human Cytosolic Carbonic Anhydrases

Carbonic anhydrase (CA) is an important enzyme, which has multiple isoforms each performing different functions in humans. Characterizing the interactions of carbonic anhydrase isoforms with ligands is a difficult but important task. In recent years, steered molecular dynamics (SMD) has been used successfully to characterize ligand binding and unbinding events. In this study, constant velocity SMD simulations were performed on four cytosolic human carbonic anhydrases (carbonic anhydrase I, II, III and VII) using a ligand called Coumarin2 5Z/5E to understand better the nature of interactions between this ligand and the various enzyme isoforms. The influence of force constant and pulling velocity on SMD simulations were investigated. When the force constant and the pulling velocity were changed, no significant effect on interaction profiles was observed. Finally, hydrogen bond interactions, Histidine 64 positions and the differences between force-distance profiles for all isozymes were analyzed. These results demonstrate that human cytosolic carbonic anhydrases I, III, and VII show similar Coumarin2 5Z/5E unbinding patterns with different rupture forces, while carbonic anhydrase II has a distinct interaction profile.