Molecular basis for the allosteric activation mechanism of the heterodimeric imidazole glycerol phosphate synthase complex.
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ABSTRACT:
SUBMITTER: Wurm JP
PROVIDER: S-EPMC8115485 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protein allosteric pathways are investigated in the imidazole glycerol phosphate synthase heterodimer in an effort to elucidate how the effector (PRFAR, N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) activates glutaminase catalysis at a distance of 25 ? from the glutamine-binding site. We apply solution NMR techniques and community analysis of dynamical networks, based on mutual information of correlated protein motions in the active and inactive enzymes. We find evidence that the allosteric pathways in the PRFAR bound enzyme involve conserved residues that correlate motion of the PRFAR binding loop to motion at the protein-protein interface, and ultimately at the glutaminase active site. The imidazole glycerol phosphate synthase bienzyme is an important branch point for the histidine and nucleotide biosynthetic pathways and represents a potential therapeutic target against microbes. The proposed allosteric mechanism and the underlying allosteric pathways provide fundamental insights for the design of new allosteric drugs and/or alternative herbicides.
| S-EPMC3365145 | biostudies-literature
Project description:The enzyme imidazole glycerol phosphate synthase (IGPS) is a model for studies of long-range allosteric regulation in enzymes. Binding of the allosteric effector ligand N'-[5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) stimulates millisecond (ms) timescale motions in IGPS that enhance its catalytic function. We studied the effect of temperature on these critical conformational motions and the catalytic mechanism of IGPS from the hyperthermophile Thermatoga maritima in an effort to understand temperature-dependent allostery. Enzyme kinetic and NMR dynamics measurements show that apo and PRFAR-activated IGPS respond differently to changes in temperature. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments performed at 303, 323, and 343 K (30, 50, and 70°C) reveal that millisecond flexibility is enhanced to a higher degree in apo IGPS than in the PRFAR-bound enzyme as the sample temperature is raised. We find that the flexibility of the apo enzyme is nearly identical to that of its PRFAR activated state at 343 K, whereas conformational motions are considerably different between these two forms of the enzyme at room temperature. Arrhenius analyses of these flexible sites show a varied range of activation energies that loosely correlate to allosteric communities identified by computational methods and reflect local changes in dynamics that may facilitate conformational sampling of the active conformation. In addition, kinetic assays indicate that allosteric activation by PRFAR decreases to 65-fold at 343 K, compared to 4,200-fold at 303 K, which mirrors the decreased effect of PRFAR on ms motions relative to the unactivated enzyme. These studies indicate that at the growth temperature of T. maritima, PFRAR is a weaker allosteric activator than it is at room temperature and illustrate that the allosteric mechanism of IGPS is temperature dependent.
| S-EPMC5808140 | biostudies-literature
Project description:Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond time scale interconversion between these functional states hamper their experimental and computational characterization. Here, we combine extensive molecular dynamics simulations, enhanced sampling techniques, and dynamical networks to describe the allosteric activation of imidazole glycerol phosphate synthase (IGPS) from the substrate-free form to the active ternary complex. IGPS is a heterodimeric bienzyme complex whose HisH subunit is responsible for hydrolyzing glutamine and delivering ammonia for the cyclase activity in HisF. Despite significant advances in understanding the underlying allosteric mechanism, essential molecular details of the long-range millisecond allosteric activation of IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound in HisF, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that the combined effector and substrate binding dramatically decreases the conformational barrier associated with oxyanion hole formation, in line with the experimentally observed 4500-fold activity increase in glutamine hydrolysis. The allosteric activation is controlled by correlated time-evolving dynamic networks connecting the effector and substrate binding sites. This computational strategy tailored to describe millisecond events can be used to rationalize the effect of mutations on the allosteric regulation and guide IGPS engineering efforts.
| S-EPMC9052757 | biostudies-literature
Project description:Imidazole glycerol phosphate synthase (IGPS) is a class-I glutamine amidotransferase (GAT) that hydrolyzes glutamine. Ammonia is produced and transferred to a second active site, where it reacts with N1-(5'-phosphoribosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) to form precursors to purine and histidine biosynthesis. Binding of PrFAR over 25 Å away from the active site increases glutaminase efficiency by ∼4500-fold, primarily altering the glutamine turnover number. IGPS has been the focus of many studies on allosteric communication; however, atomic details for how the glutamine hydrolysis rate increases in the presence of PrFAR are lacking. We present a density functional theory study on 237-atom active site cluster models of IGPS based on crystallized structures representing the inactive and allosterically active conformations and investigate the multistep reaction leading to thioester formation and ammonia production. The proposed mechanism is supported by similar, well-studied enzyme mechanisms, and the corresponding energy profile is consistent with steady-state kinetic studies of PrFAR + IGPS. Additional active site models are constructed to examine the relationship between active site structural change and transition-state stabilization via energy decomposition schemes. The results reveal that the inactive IGPS conformation does not provide an adequately formed oxyanion hole structure and that repositioning of the oxyanion strand relative to the substrate is vital for a catalysis-competent oxyanion hole, with or without the hVal51 dihedral flip. These findings are valuable for future endeavors in modeling the IGPS allosteric mechanism by providing insight into the atomistic changes required for rate enhancement that can inform suitable reaction coordinates for subsequent investigations.
| S-EPMC10729027 | biostudies-literature
Project description:Allosteric drugs have the potential to revolutionize biomedicine due to their enhanced selectivity and protection against overdosage. However, we need to better understand allosteric mechanisms in order to fully harness their potential in drug discovery. In this study, molecular dynamics simulations and nuclear magnetic resonance spectroscopy are used to investigate how increases in temperature affect allostery in imidazole glycerol phosphate synthase. Results demonstrate that temperature increase triggers a cascade of local amino acid-to-amino acid dynamics that remarkably resembles the allosteric activation that takes place upon effector binding. The differences in the allosteric response elicited by temperature increase as opposed to effector binding are conditional to the alterations of collective motions induced by either mode of activation. This work provides an atomistic picture of temperature-dependent allostery, which could be harnessed to more precisely control enzyme function.
| S-EPMC10115891 | biostudies-literature
Project description:Understanding the relationship between protein structures and their function is still an open question that becomes very challenging when allostery plays an important functional role. Allosteric proteins, in fact, exploit different ranges of motions (from sidechain local fluctuations to long-range collective motions) to effectively couple distant binding sites, and of particular interest is whether allosteric proteins of the same families with similar functions and structures also necessarily share the same allosteric mechanisms. Here, we compared the early dynamics initiating the allosteric communication of a prototypical allosteric enzyme from two different organisms, i.e., the imidazole glycerol phosphate synthase (IGPS) enzymes from the thermophilic bacteria and the yeast, working at high and room temperatures, respectively. By combining molecular dynamics simulations and network models derived from graph theory, we found rather distinct early allosteric dynamics in the IGPS from the two organisms, involving significatively different allosteric pathways in terms of both local and collective motions. Given the successful prediction of key allosteric residues in the bacterial IGPS, whose mutation disrupts its allosteric communication, the outcome of this study paves the way for future experimental studies on the yeast IGPS that could foster therapeutic applications by exploiting the control of IGPS enzyme allostery.
| S-EPMC8758406 | biostudies-literature
Project description:IGPS is a 51 kDa heterodimeric enzyme comprised of two proteins, HisH and HisF, that catalyze the hydrolysis of glutamine to produce NH(3) in the HisH active site and the cyclization of ammonia with N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in HisF to produce imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR). Binding of PRFAR and IGP stimulates glutaminase activity in the HisH enzyme over 5,000 and 100-fold, respectively, despite the active sites being >25 A apart. The details of this long-range protein communication process were investigated by solution NMR spectroscopy and CPMG relaxation dispersion experiments. Formation of the heterodimer enzyme results in a reduction in millisecond motions in HisF that extend throughout the protein. Binding of lGP results in an increase in protein-wide millisecond dynamics evidenced as severe NMR line broadening and elevated R (ex) values. Together, these data demonstrate a grouping of flexible residues that link the HisF active site with the protein interface to which HisH binds and provide a model for the path of communication between the IGPS active sites.
| S-EPMC2918893 | biostudies-literature
Project description:Allosteric enzymes regulate a wide range of catalytic transformations, including biosynthetic mechanisms of important human pathogens, upon binding of substrate molecules to an orthosteric (or active) site and effector ligands at distant (allosteric) sites. We find that enzymatic activity can be impaired by small molecules that bind along the allosteric pathway connecting the orthosteric and allosteric sites, without competing with endogenous ligands. Noncompetitive allosteric inhibitors disrupted allostery in the imidazole glycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima as evidenced by nuclear magnetic resonance, microsecond time-scale molecular dynamics simulations, isothermal titration calorimetry, and kinetic assays. The findings are particularly relevant for the development of allosteric antibiotics, herbicides, and antifungal compounds because IGPS is absent in mammals but provides an entry point to fundamental biosynthetic pathways in plants, fungi, and bacteria.
| S-EPMC5283573 | biostudies-literature
Project description:Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.
| S-EPMC2955087 | biostudies-literature
Project description:The bi-enzyme HisF-HisH heterodimer is part of the pathway that produces histidine and purines in bacteria and lower eukaryotes, but it is absent in mammals. This heterodimer has been largely studied probing the basis of the allosteric effects and the structural stability in proteins. It is also a potential target for antibacterial drugs. In this work, we developed a simple method to evaluate changes in the affinity between HisF and HisH in the heterodimer of the bacteria Thermotoga maritima. HisH contains a single tryptophan residue, which is exposed in the free protein, but buried in the heterodimer interface. Hence, the intrinsic fluorescence maximum of this residue changes to shorter wavelengths upon dimerization. Thus, we used the fluorescence intensity at this shorter wavelength to monitor heterodimer accumulation when HisH was combined with sub-stoichiometric HisF. Under conditions where the HisF-HisH heterodimer is in equilibrium with the free states of these enzymes, when [HisH] > [HisF], we deduced a linear function connecting [HisF-HisH] to [HisF], in which the slope depends on the heterodimer dissociation constant (Kd). Based on this equation, taking fluorescence intensities as proxies of the heterodimer and HisF concentrations, we experimentally determined the Kd at four different temperatures. These Kd values were compared to those evaluated using ITC. Both methods revealed an increase in the HisF and HisH binding affinity as the temperature increases. In spite of differences in their absolute values, the Kd determined using these methods presented an evident linear correlation. To demonstrate the effectiveness of the fluorescence method we determined the effect on the Kd caused by 12 single mutations in HisF. Coherently, this test singled out the only mutation in the binding interface. In brief, the method described here effectively probes qualitative effects on the Kd, can be carried out using common laboratory equipment and is scalable.
| S-EPMC9032424 | biostudies-literature