Negative Cooperativity Research Articles

Negative Cooperative Binding of Thymidine, Ordered Substrate Binding, and Product Release of Human Mitochondrial Thymidine Kinase 2 Explain Its Complex Kinetic Properties and Physiological Functions.

Mitochondrial thymidine kinase 2 (TK2) catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC) and is essential for mitochondrial function in post-mitotic tissues. The phosphorylation of dT shows negative cooperativity, but the phosphorylation of dC follows classical Michaelis–Menten kinetics. The enzyme is feedback-inhibited by its end products deoxythymidine triphosphate (dTTP) and deoxycytidine triphosphate (dCTP). In order to better understand the reaction mechanism and the negative cooperative behavior, we conducted isothermal titration calorimetry (ITC) and intrinsic tryptophan fluorescence (ITF) quenching studies with purified recombinant human TK2. Cooperative binding was observed with dT but not dC by the ITC analysis in accordance with earlier enzyme kinetic studies. The phosphate donor adenosine triphosphate (ATP) did not bind to either dTTP-bound or dTTP-free enzymes but bound tightly to the dT– or dC–TK2 complexes with large differences in enthalpy and entropy changes, strongly suggesting an ordered binding of the substrates and different conformational states of the ATP and dT– and dC–TK2 ternary complexes. dTTP binding was endothermic; however, dCTP could not be shown to interact with the enzyme. ITF quenching studies also revealed tight binding of dT, dC, deoxythymidine monophosphate, deoxycytidine monophosphate, and dTTP but not adenosine 5′-diphosphate or ATP. These results strongly indicate an ordered sequential binding of the substrates and ordered release of the products as well as different conformational states of the active site of TK2. These results help to explain the different kinetics observed with dT and dC as substrates, which have important implications for TK2 regulation in vivo.

Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.

Nucleotide excision repair (NER) protects cells against diverse types of DNA damage, principally UV irradiation. In Escherichia coli, damage is recognized by 2 key enzymes: UvrA and UvrB. Despite extensive investigation, the role of UvrA’s 2 ATPase domains in NER remains elusive. Combining single-molecule fluorescence microscopy and classic biochemical methods, we have investigated the role of nucleotide binding in UvrA’s kinetic cycle. Measurement of UvrA’s steady-state ATPase activity shows it is stimulated upon binding DNA (kcat 0.71–1.07/s). Despite UvrA’s ability to discriminate damage, we find UV-damaged DNA does not alter the steady-state ATPase. To understand how damage affects UvrA, we studied its binding to DNA under various nucleotide conditions at the single molecule level. We have found that both UV damage and nucleotide cofactors affect the attached lifetime of UvrA. In the presence of ATP and UV damage, the lifetime is significantly greater compared with undamaged DNA. To reconcile these observations, we suggest that UvrA uses negative cooperativity between its ATPase sites that is gated by damage recognition. Only in the presence of damage is the second site activated, most likely in a sequential manner.—Barnett, J. T., Kad, N. M. Understanding the coupling between DNA damage detection and UvrA’s ATPase using bulk and single molecule kinetics.

A two-domain folding intermediate of RuBisCO in complex with the GroEL chaperonin

The chaperonins (GroEL and GroES in Escherichia coli) are ubiquitous molecular chaperones that assist a subset of essential substrate proteins to undergo productive folding to the native state. Using single particle cryo EM and image processing we have examined complexes of E. coli GroEL with the stringently GroE-dependent substrate enzyme RuBisCO from Rhodospirillum rubrum. Here we present snapshots of non-native RuBisCO - GroEL complexes. We observe two distinct substrate densities in the binary complex reminiscent of the two-domain structure of the RuBisCO subunit, so that this may represent a captured form of an early folding intermediate. The occupancy of the complex is consistent with the negative cooperativity of GroEL with respect to substrate binding, in accordance with earlier mass spectroscopy studies.

Characterization of methanthelinium binding and function at human M1-M5 muscarinic acetylcholine receptors.

Firstly, it was determined whether methanthelinium bromide (MB) binds to human M1-M5 (hM1-hM5) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [3H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100μM of MB ranging from none in hM3 to 4.6-fold in hM2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [3H]NMS (0.2 and 2.0nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logKI (± S.E.) hM3 = 8.71 ± 0.15, hM1 = 8.68 ± 0.14, hM5 = 8.58 ± 0.07, hM2 = 8.27 ± 0.07 to hM4 = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logKB: hM1 = 9.53 ± 0.05, hM4 = 9.33 ± 0.05, hM5 = 8.80 ± 0.05, hM2 = 8,79± 0.06, to hM3 = 8.43 ± 0.04. In conclusion, MB, below 1μM, binds competitively and non-selectively (except for the difference between hM3 vs. hM4) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM1 and hM4.

Quantitation without Calibration: Response Profile as an Indicator of Target Amount.

Quantitative assessment of biomarkers is essential in numerous contexts from decision-making in clinical situations to food quality monitoring to interpretation of life-science research findings. However, appropriate quantitation techniques are not as widely addressed as detection methods. One of the major challenges in biomarker's quantitation is the need to have a calibration for correlating a measured signal to a target amount. The step complicates the methodologies and makes them less sustainable. In this work we address the issue via a new strategy: relying on position of response profile rather than on an absolute signal value for assessment of a target's amount. In order to enable the capability we develop a target-probe binding mechanism based on a negative cooperativity effect. A proof-of-concept example demonstrates that the model is suitable for quantitative analysis of nucleic acids over a wide concentration range. The general principles of the platform will be applicable toward a variety of biomarkers such as nucleic acids, proteins, peptides, and others.

Allostery and molecular machines.

Machines undergo repeated energy (fuel)-driven cycling between various functional and structural states. Such cycling involves motions of the machine parts, which take place in a highly coordinated manner in time and space. This description applies not only to man-made machines but also to the molecular machines that mediate many of the key processes in all forms of life [1]. Examples include protein synthesis by the ribosome, DNA unwinding by helicases during replication, protein degradation by the proteasome and protein folding by chaperones. The coordinated movements of most biomolecular machines are achieved by allosteric regulation of ATP binding and hydrolysis. Consequently, a molecular level understanding of how the essential machines of life work requires invoking and further developing allosteric theory. Hence, the motivation for the Royal Society Discussion Meeting entitled ‘Allostery and molecular machines' that took place in June 2017. The meeting brought together scientists interested in allostery, in general, with others who are studying specific biomolecular machines of interest. The meeting led to many interesting discussions and resulted in this special issue of the Philosophical Transactions B . In 1965, Monod, Wyman & Changeux published a seminal paper [2] that described a so-called ‘concerted model' (referred to here and generally as the MWC model) for cooperative ligand binding by oligomeric proteins. This issue contains a paper by one of the co-authors of this landmark paper in which the MWC model is explained in brief and its application to the nicotinic acetylcholine receptor is then described [3]. The MWC model remains important but its elegance has come at the price of several restrictive assumptions. One key assumption is that cooperativity is due to a shift in equilibrium between different quaternary …

Multivalency regulates activity in an intrinsically disordered transcription factor.

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation.

A Novel M1 PAM VU0486846 Exerts Efficacy in Cognition Models without Displaying Agonist Activity or Cholinergic Toxicity.

Selective activation of the M1 subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M1 ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M1, leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M1 PAM, VU0486846. VU0486846 possesses only weak agonist activity in M1-expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [3H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M1 PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M1 PAM activity (EC50 > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M1. Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.

Corrigendum: Chloroplast Glutamine Synthetase, the Key Regulator of Nitrogen Metabolism in Wheat, Performs Its Role by Fine Regulation of Enzyme Activity via Negative Cooperativity of Its Subunits

[This corrects the article on p. 191 in vol. 9, PMID: 29520285.].

A Thieno[2,3- d]pyrimidine Scaffold Is a Novel Negative Allosteric Modulator of the Dopamine D2 Receptor.

Recently, a novel negative allosteric modulator (NAM) of the D2-like dopamine receptors 1 was identified through virtual ligand screening. This ligand comprises a thieno[2,3- d]pyrimidine scaffold that does not feature in known dopaminergic ligands. Herein, we provide pharmacological validation of an allosteric mode of action for 1, revealing that it is a NAM of dopamine efficacy and identify the structural determinants of this allostery. We find that key structural moieties are important for functional affinity and negative cooperativity, while functionalization of the thienopyrimidine at the 5- and 6-positions results in analogues with divergent cooperativity profiles. Successive compound iterations have yielded analogues exhibiting a 10-fold improvement in functional affinity, as well as enhanced negative cooperativity with dopamine affinity and efficacy. Furthermore, our study reveals a fragment-like core that maintains low μM affinity and robust negative cooperativity with markedly improved ligand efficiency.

The mechanism underlying a unique response to acetyl CoA activation in eukaryotic pyruvate carboxylase from Aspergillus nidulans

Pyruvate carboxylase (PC) is a multifunctional, allosterically regulated, biotin‐dependent enzyme that carboxylates pyruvate to oxaloacetate. The conversion of pyruvate to oxaloacetate is accomplished by coupling two half reactions that are catalyzed at spatially distinct active sites in the Biotin Carboxylase (BC) and Carboxyltransferase (CT) domains, through the movement of a carrier domain (Biotin Carboxyl Carrier Protein; BCCP). PC is a homotetramer, with two subunits contributing to each face of the tetramer, and with homodimerization mediated between the individual domains at each corner of the tetramer. Acetyl coenzyme A (acetyl CoA) is an allosteric activator of PC found in most species. However, PC from Aspergillus nidulans (AnPC) is unique in not being stimulated by acetyl CoA. Here, we provide a comprehensive structural and kinetic characterization of AnPC, and compare it with the PC from Rhizobium etli (RePC) that is highly sensitive to acetyl CoA stimulation, in order to better understand the mechanism of allosteric activation of PC by acetyl CoA.We determined the structure of AnPC by X‐ray crystallography at 3.0 Å resolution. In this structure, the BCCP domain was observed to occupy a unique position, suggesting that the carrier domain in AnPC may proceed through an alternative translocation pathway. Interestingly, despite the absence of activation by acetyl CoA in the overall reaction catalyzed by AnPC, the two half‐reactions were activated by acetyl CoA in AnPC. Kinetic studies with a series of acetyl CoA analogues suggests that AnPC binds with acetyl CoA at the conserved acetyl CoA binding site, while the acetyl group plays a less significant role in the activation of AnPC compared to RePC. Modification of the acyl group reduces the binding affinity of the analogues with AnPC but not the extent of activation.To further dissect the contributions made by the individual catalytic domains to the allosteric response in PC, we generated two chimeric enzymes (ReBC‐AnPC and AnBC‐RePC) by fusing the enzymes at the interface with the N‐terminal BC domain. The coupling efficiency and acetyl CoA sensitivity of these chimeras were determined, revealing that both of the chimeras were stimulated by acetyl CoA and that allosteric activation is largely determined by the BC domain.Most interestingly, negative cooperativity was observed for ATP cleavage in the BC domain of the acetyl CoA sensitive RePC. These observations are consistent with a ‘half‐the‐sites' reactivity mechanism and can be interpreted in light of recent cryo‐EM studies reported in Staphylococcus aureus PC1. In contrast, negative cooperativity was observed for ATP cleavage only in the presence of acetyl CoA for AnPC. The acetyl moiety of acetyl CoA is thought to bind at the BC‐domain dimer interface and is considered to be critically important for allosteric activation. Our structural and kinetic data with AnPC suggest that the BC‐domain dimer interface may differ between RePC and AnPC, contributing to the divergent responses to acetyl CoA activation..Support or Funding InformationThis work was supported by the National Institutes of Health grant GM117540.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carbamate Insecticides Bind to a Novel Allosteric Site on hMT1 Melatonin Receptors

In silico screening paired with 2‐[125I]‐iodomelatonin competition binding has implicated carbamate insecticides as potential endocrine disruptors due to their affinity for human (h) MT1 and MT2 melatonin receptors (Popovska‐Gorevski et al. 2017). hMT1 and hMT2 receptor modelling predicts that these compounds may be binding to the orthosteric site and/or to previously uncharacterized hydrophobic pockets proximal to the melatonin‐binding pocket. The location of the specific binding site for carbamate insecticides is currently unknown. The goal of this study was to assess the mechanism(s) by which they inhibit orthosteric ligand binding. Melatonin (1 μM), luzindole (a prototypical non‐selective MT1/MT2 competitive melatonin receptor antagonist, 100 μM), and four carbamate insecticides (carbaryl, fenobucarb, bendiocarb, and carbofuran, 1 mM) were screened for binding to allosteric sites by estimating the Maximal Fractional Inhibition (MFI) of a saturating 2‐[125I]‐iodomelatonin concentration (1200 pM) binding to CHO‐hMT1 and CHO‐hMT2 cell membranes. All six compounds at hMT2 and melatonin at hMT1 completely inhibited the saturating concentration of radioligand (MFI≥60%) suggesting an orthosteric binding mechanism, while luzindole and the carbamate insecticides only partially inhibited binding at hMT1 (MFI<60%), alluding to binding through an allosteric mechanism. To confirm these results, full competition binding curves in CHO‐hMT1 and CHO‐hMT2 cells were performed using five different concentrations of 2‐[125I]‐iodomelatonin (50–1200 pM) to assess binding to the receptor orthosteric site (α=0) and to determine the degree of cooperativity (α>0.01) between orthosteric and allosteric ligand binding. For both hMT1 and hMT2 receptors, melatonin (MT1 KB=0.131 nM, MT2 KB=0.154 nM) completely inhibited binding of saturating concentrations of radioligand confirming that both melatonin and 2‐[125I]‐iodomelatonin bind to the same site. Luzindole (MT1 KB=354 nM, MT2 KB=11.3 nM), and the carbamate insecticide with the highest MT1 and MT2 melatonin receptor affinities, carbaryl (MT1 KB=3790 nM, MT2 KB=389 nM), also completely inhibited 1200 pM 2‐[125I]‐iodomelatonin binding at hMT2 confirming that they bind to the orthosteric site. Interestingly, for luzindole and carbaryl at hMT1, the levels of maximal fractional inhibition of binding decreased with increasing concentrations of radioligand. These patterns show that luzindole and carbaryl bind to an allosteric site on hMT1 with strong negative cooperativity (αluzindole=0.037±0.013, n=3; αcarbaryl=0.017±0.001, n=3). Interactions of carbamate insecticides with a melatonin receptor allosteric site may provide novel mechanisms by which environmental melatonin receptor modulators disrupt signaling of the endogenous ligand.Support or Funding InformationSupported by ES 023684 to MLD and RVR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence for a Stereoselective Mechanism of Action for Non‐competitive Antagonism of the D3 Dopamine Receptor by Extended‐Length Bitopic Ligands

Dopamine receptors (DRs) are comprised of two subfamilies based on structure and pharmacology: D1‐like (D1R and D5R) and D2‐like (D2R, D3R, and D4R). The D3R expression is more restricted to the limbic system, as compared to the D2R, suggesting that modulation of the D3R may be useful for treating substance abuse or psychosis‐related disorders without the motor side effects associated with D2R blockade. Extensive structure‐activity relationship investigations indicate that D3R‐selective antagonists with extended‐length structures engage two distinct sites on the receptor. The extended‐length ligand's primary pharmacophore (PP) binds to the orthosteric site whereas the ligand's secondary pharmacophore (SP) interacts with a unique secondary binding pocket (SBP). Bivalent compounds that concurrently engage both the orthosteric site and a SBP that has allosteric properties, have been termed “bitopic”. We previously synthesized and characterized several extended‐length compounds with a conformationally restricted trans‐cyclopropyl linking chain based on the structure of SB269,652, a potent D3R‐preferring bitopic antagonist that acts in a noncompetitive/allosteric manner at both the D3R and the D2R. We found that relatively subtle changes in both the linker and the SP can affect the competitive/noncompetitive nature of the compounds, and identified two novel ligands that behave as noncompetitive antagonists at the D3R. We resolved the optical isomers of one of these compounds (VK04‐87) to gain a better understanding of the molecular basis of their allosteric activity. We found that the (+)‐isomer has higher affinity and is more D3R>D2R selective than the (−)‐isomer. Strikingly, using beta‐arrestin recruitment assays as a functional output, we found that (+)‐VK04‐87 inhibits the D3R in a non‐competitive manner, while (−)‐VK04‐87 behaves as a competitive antagonist. These data suggest that the bitopic interactions of VK04‐87 with the D3R are stereoselective in nature with the allosteric properties residing with the (+)‐isomer. To further investigate, we made a mutation (E90A) in the secondary binding pocket of the D3R that was previously found to decrease the affinity and negative cooperativity of SB269,652 for the D2R. We found that this mutation decreased the affinity of extended‐length ligands, including SB269,652, for the D3R, but had no effect on the affinities of smaller, competitive antagonists consisting of PPs. Further analyses of the interactions of the extended‐length ligands with the D3R E90A mutant are ongoing. These studies could help to determine how ligand interactions with the SBP can be utilized to engender allosterism, potentially producing compounds with unique pharmacological effects.Support or Funding InformationNINDS IRPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of the potassium/lysine cationic center in catalysis and functional asymmetry in membrane-bound pyrophosphatases.

Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H+ and/or Na+ ions, are divided into K+,Na+-independent, Na+-regulated, and K+-dependent families. The first two families include H+-transporting mPPases (H+-PPases), whereas the last family comprises one Na+-transporting, two Na+- and H+-transporting subfamilies (Na+-PPases and Na+,H+-PPases, respectively), and three H+-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K+ binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K+-independent mPPases. Here, we performed a systematic analysis of the K+/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K+-dependent mPPases abolished the K+ dependence of hydrolysis and transport activities and decreased these activities close to the level (4-7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K+,Na+-independent mPPases conferred partial K+ dependence on the enzyme by unmasking an otherwise conserved K+-binding site. Na+ could partially replace K+ as an activator of K+-dependent mPPases and the Lys → Ala variants of K+,Na+-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K+/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.

Protein plasticity driven by disorder and collapse governs the heterogeneous binding of CytR to DNA.

The amplitude of thermodynamic fluctuations in biological macromolecules determines their conformational behavior, dimensions, nature of phase transitions and effectively their specificity and affinity, thus contributing to fine-tuned molecular recognition. Unique among large-scale conformational changes in proteins are temperature-induced collapse transitions in intrinsically disordered proteins (IDPs). Here, we show that CytR DNA-binding domain, an IDP that folds on binding DNA, undergoes a coil-to-globule transition with temperature in the absence of DNA while exhibiting energetically decoupled local and global structural rearrangements, and maximal thermodynamic fluctuations at the optimal bacterial growth temperature. The collapse is shown to be a continuous transition through a combination of statistical-mechanical modeling and all-atom implicit solvent simulations. Surprisingly, CytR binds single-site cognate DNA with negative cooperativity, described by Hill coefficients less than one, resulting in a graded binding response. We show that heterogeneity arising from varying binding-competent CytR conformations or orientations at the single-molecular level contributes to negative binding cooperativity at the level of bulk measurements due to the conflicting requirements of collapse transition, large fluctuations and folding-upon-binding. Our work reports strong evidence for functionally driven thermodynamic fluctuations in determining the extent of collapse and disorder with implications in protein search efficiency of target DNA sites and regulation.

Chloroplast Glutamine Synthetase, the Key Regulator of Nitrogen Metabolism in Wheat, Performs Its Role by Fine Regulation of Enzyme Activity via Negative Cooperativity of Its Subunits.

Glutamine synthetase (GS) is of central interest as the main route of ammonia assimilation in plants, and as a connection point between the organic and inorganic worlds. Even though GS activity is critical for producing high yields of crop plants, the autoregulation of substrate consumption of wheat GS remained unknown until now. Here we show kinetic evidence, that the chloroplast localized GS isoform (GS2) of wheat (Triticum aestivum L. cv. Jubilejnaja-50) takes place at the carbon-nitrogen metabolic branch point, where it is a mediator, and its enzymatic activity is regulated in a negatively cooperative allosteric manner. We have discovered that GS2 activity is described by a tetraphasic kinetic curve in response to increasing levels of glutamate supply. We constructed a model that explains the kinetic properties of glutamate consumption and this unique allosteric behavior. We also studied the subunit composition of both wheat leaf GS isoenzymes by a combination of two dimensional gel electrophoresis and protein blotting. Both leaf isozymes have homogeneous subunit composition. Glutamate is both a substrate, and an allosteric regulator of the biosynthetic reaction. We have concluded on the basis of our results and previous reports, that wheat GS2 is probably a homooctamer, and that it processes its substrate in a well-regulated, concentration dependent way, as a result of its negatively cooperative, allosteric activity. Thus, GS2 has a central role as a regulator between the nitrogen and the carbon cycles via maintaining glutamine-glutamate pool in the chloroplast on the level of substrates, in addition to its function in ammonia assimilation.

Activation of Preformed EGFR Dimers by Binding of Single EGF Molecules: Negative Cooperativity

Epidermal growth factor receptor (EGFR) plays roles in cell proliferation, inhibition of apoptosis and angiogenesis among others. It has long been thought that binding of the ligand induces the dimer formation of monomeric EGFR (the “ligand-induced dimerization model”). Upon ligand binding, tyrosine residues in the C-terminal tail of EGFR are phosphorylated, and recruit effector proteins such as Shc1 and Grb2, which trigger activation of downstream signaling cascades. However, a pletora of recent studies indicate that EGFR exists in dimeric form (a preformed dimer), which is incompatible with the ligand-induced dimerization model. To understand the molecular mechanism of activation of EGFR, we directly observed interactions among EGFR, EGF labeled with two different fluorescent compounds, and GFP-tagged Shc1 by multi-color TIRF microscopy. Within 30 seconds of application of the dye-conjugated EGF to cultured cells expressing EGFR, three different fluorescent spots of EGF and Shc1 appeared on the cell surface. EGFR mutant that have no kinase activity could bind EGF but not recruit Shc1. Two different fluorescent colors derived from EGF molecules do not co-localize, and binding of a single EGF molecule induces Shc1 recruitment to EGFR. These results suggest that binding of a single EGF molecule can activate preformed EGFR dimer, consistently with negative cooperativity in EGF binding to EGFR.

Allostery in NMDA Receptors

Ionotropic glutamate receptors, of which the N-methyl-D-aspartate receptor (NMDAR) is one subtype, play a crucial role in excitatory neurotransmission within the mammalian central nervous system. In order to fully understand the role the NMDAR plays in neurotransmission, the conformational states and transitions that the receptor undergoes must be understood. Although the structures of the various conformational states of the NMDAR are known, the conformational changes by which the NMDAR transitions from state to state are not well understood. One question concerning NMDAR dynamics that remains to be answered is that of the nature of allosteric communication between the glycine binding sites and glutamate binding sites on the various subunits of the heterotetrameric receptor. In order to understand this allosteric communication, we utilized both electrophysiology and single molecule Förster Resonance Energy Transfer (smFRET) to examine the changes in the ligand binding domain (LBD) brought about by the binding of glycine and/or glutamate. The electrophysiological studies showed that there is negative cooperativity between the glycine and glutamate binding sites. The smFRET studies allowed the observation of the various states that a single receptor adopts under various ligand conditions. These single molecule experiments demonstrated that the conformational dynamics of the NMDAR LBD change when the receptor is in the presence or absence of various ligands; the NMDAR exhibits altered conformational dynamics when it is bound to glycine, glutamate, both, or neither. This data illustrates a potential mechanism for the negative allosteric effects between the glycine and glutamate binding sites that was observed in the electrophysiological studies.

Negative Cooperativity in the Interaction of Prostaglandin H Synthase-1 with the Competitive Inhibitor Naproxen Can Be Described as the Interaction of a Non-competitive Inhibitor with Heterogeneous Enzyme Preparation.

The kinetic mechanism of the interaction of nonsteroidal anti-inflammatory drugs (NSAIDs) with their main pharmacological target, prostaglandin H synthase (PGHS), has not yet been established. We showed that inhibition of PGHS-1 from sheep vesicular glands by naproxen (a representative of NSAIDs) demonstrates a non-competitive character with respect to arachidonic acid and cannot be described within a framework of the commonly used kinetic schemes. However, it can be described by taking into account the negative cooperativity of naproxen binding to the cyclooxygenase active sites of the PGHS-1 homodimer (the first naproxen molecule forms a more stable complex (K1 = 0.1 µM) with the enzyme than the second naproxen molecule (K2 = 9.2 µM)). An apparent non-competitive interaction of PGHS-1 with naproxen is due to slow dissociation of the enzyme-inhibitor complexes. The same experimental data could also be described using commonly accepted kinetic schemes, assuming that naproxen interacts was a mixture of two enzyme species with the inhibition constants Kα = 0.05 µM and Kβ = 18.3 µM. Theoretical analysis and numerical calculations show that the phenomenon of kinetic convergence of these two models has a general nature: when K2 >> K1, the kinetic patterns (for transient kinetics and equilibrium state) generated by the cooperative model could be described by a scheme assuming the presence of two enzyme forms with the inhibition constants Kα = K1/2, Kβ = 2·K2. When K2 << K1, the cooperative model can be presented as a scheme with two inhibitor molecules simultaneously binding to the enzyme with the observed inhibition constant K (K = K1·K2). The assumption on the heterogeneity of the enzyme preparation in relation to its affinity to the inhibitor can be used instead of the assumption on the negative cooperativity of the enzyme-inhibitor interactions for convenient and easy practical description of such phenomena in enzymology, biotechnology, pharmacology, and other fields of science.

Use multiscale simulation to explore the effects of the homodimerizations between different conformation states on the activation and allosteric pathway for the μ-opioid receptor.

Recently, oligomers of G-protein coupled receptors (GPCRs) have been an important topic in the GPCR fields. However, knowledge about their structures and activation mechanisms is very limited due to the absence of crystal structures reported. In this work, we used multiscale simulations to study the effects of homodimerization between different conformation states on their activation, dynamic behaviors, and allosteric communication pathways for μ-OR. The results indicated that the dimerization of one inactive monomer with either one inactive monomer or one active one could enhance its constitutive activation. However, the conformation state of the other protomer (e.g., active or inactive) can influence the activated extent. The dimerization between the two inactive protomers leads to a negative cooperativity for their activation, which should contribute to the asymmetric activation of GPCR dimers observed in some experiments. On the other hand, for the active monomer, its dimerization with one inactive receptor could alleviate its deactivation, whereby negative and positive cooperativities can be observed between the two subunits of the dimer, depending on the different regions. Observations from protein structure network (PSN) analysis indicated that the dimerization of one inactive monomer with one active one would cause a significant drop in the number of main pathways from the ligand binding pocket to the G-protein coupled region for the inactive protomer, while the impact is minor for the active protomer. But, for the active monomer or the inactive one, its dimerization with one inactive monomer would significantly change the types of residues participating in the pathway with the highest frequency.