Kovalevsky A


Full name : Kovalevsky Andrey

First name : Andrey

Mail : Oak Ridge National Laboratory, Oak Ridge, Tennessee

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Country : USA

Email : kovalevskyay@ornl.gov

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References (9)

Title : Structural and dynamic effects of paraoxon binding to human acetylcholinesterase by X-ray crystallography and inelastic neutron scattering - Gerlits_2022_Structure_30_1538
Author(s) : Gerlits O , Fajer M , Cheng X , Blumenthal DK , Radic Z , Kovalevsky A
Ref : Structure , 30 :1538 , 2022
Abstract : Organophosphorus (OP) compounds, including nerve agents and some pesticides, covalently bind to the catalytic serine of human acetylcholinesterase (hAChE), thereby inhibiting acetylcholine hydrolysis necessary for efficient neurotransmission. Oxime antidotes can reactivate the OP-conjugated hAChE, but reactivation efficiency can be low for pesticides, such as paraoxon (POX). Understanding structural and dynamic determinants of OP inhibition and reactivation can provide insights to design improved reactivators. Here, X-ray structures of hAChE with unaged POX, with POX and oximes MMB4 and RS170B, and with MMB4 are reported. A significant conformational distortion of the acyl loop was observed upon POX binding, being partially restored to the native conformation by oximes. Neutron vibrational spectroscopy combined with molecular dynamics simulations showed that picosecond vibrational dynamics of the acyl loop soften in the -20-50 cm(-1) frequency range. The acyl loop structural perturbations may be correlated with its picosecond vibrational dynamics to yield more comprehensive template for structure-based reactivator design.
ESTHER : Gerlits_2022_Structure_30_1538
PubMedSearch : Gerlits_2022_Structure_30_1538
PubMedID: 36265484
Gene_locus related to this paper: human-ACHE

Title : Room temperature crystallography of human acetylcholinesterase bound to a substrate analogue 4K-TMA: Towards a neutron structure - Gerlits_2021_Curr.Res.Struct.Biol_3_206
Author(s) : Gerlits O , Blakeley MP , Keen DA , Radic Z , Kovalevsky A
Ref : Current Research in Structural Biology , 3 :206 , 2021
Abstract : Acetylcholinesterase (AChE) catalyzes hydrolysis of acetylcholine thereby terminating cholinergic nerve impulses for efficient neurotransmission. Human AChE (hAChE) is a target of nerve agent and pesticide organophosphorus compounds that covalently attach to the catalytic Ser203 residue. Reactivation of inhibited hAChE can be achieved with nucleophilic antidotes, such as oximes. Understanding structural and electrostatic (i.e. protonation states) determinants of the catalytic and reactivation processes is crucial to improve design of oxime reactivators. Here we report X-ray structures of hAChE conjugated with a reversible covalent inhibitor 4K-TMA (4K-TMA:hAChE) at 2.8 A resolution and of 4K-TMA:hAChE conjugate with oxime reactivator methoxime, MMB4 (4K-TMA:hAChE:MMB4) at 2.6 A resolution, both at physiologically relevant room temperature, as well as cryo-crystallographic structure of 4K-TMA:hAChE at 2.4 A resolution. 4K-TMA acts as a substrate analogue reacting with the hydroxyl of Ser203 and generating a reversible tetrahedral hemiketal intermediate that closely resembles the first tetrahedral intermediate state during hAChE-catalyzed acetylcholine hydrolysis. Structural comparisons of room temperature with cryo-crystallographic structures of 4K-TMA:hAChE and published mAChE complexes with 4K-TMA, as well as the effect of MMB4 binding to the peripheral anionic site (PAS) of the 4K-TMA:hAChE complex, revealed only discrete, minor differences. The active center geometry of AChE, already highly evolved for the efficient catalysis, was thus indicative of only minor conformational adjustments to accommodate the tetrahedral intermediate in the hydrolysis of the neurotransmitter acetylcholine (ACh). To map protonation states in the hAChE active site gorge we collected 3.5 A neutron diffraction data paving the way for obtaining higher resolution datasets that will be needed to determine locations of individual hydrogen atoms.
ESTHER : Gerlits_2021_Curr.Res.Struct.Biol_3_206
PubMedSearch : Gerlits_2021_Curr.Res.Struct.Biol_3_206
PubMedID: 34541552
Gene_locus related to this paper: human-ACHE

Title : Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects - Blumenthal_2021_J.Biol.Chem__
Author(s) : Blumenthal DK , Cheng X , Fajer M , Ho KY , Rohrer J , Gerlits O , Taylor P , Juneja P , Kovalevsky A , Radic Z
Ref : Journal of Biological Chemistry , :101007 , 2021
Abstract : Acetylcholinesterase (EC; AChE), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission, be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human AChE (hAChE) in solution occurs through a C-terminal 4-helix bundle (4HB) at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the R(P) enantiomer of sarin promotes a ten-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6 or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of a S(P)-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS (TR-SAXS) occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wild-type hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket towards the 4HB dimerization interface 25 A away.
ESTHER : Blumenthal_2021_J.Biol.Chem__
PubMedSearch : Blumenthal_2021_J.Biol.Chem__
PubMedID: 34324828
Gene_locus related to this paper: human-ACHE

Title : Characterization and structural analysis of a thermophilic GH11 xylanase from compost metatranscriptome - Yi_2021_Appl.Microbiol.Biotechnol_105_7757
Author(s) : Yi Y , Xu S , Kovalevsky A , Zhang X , Liu D , Wan Q
Ref : Applied Microbiology & Biotechnology , 105 :7757 , 2021
Abstract : Xylanase is efficient for xylan degradation and widely applied in industries. We found a GH11 family xylanase (Xyn11A) with high thermostability and catalytic activity from compost metatranscriptome. This xylanase has the optimal reaction temperature at 80 degreesC with the activity of 2907.3 U/mg. The X-ray crystallographic structure shows a typical "right hand" architecture, which is the characteristics of the GH11 family enzymes. Comparing it with the mesophilic XYN II, a well-studied GH11 xylanase from Trichoderma reesei, Xyn11A is more compact with more H-bonds. Our mutagenic results show that the electrostatic interactions in the thumb and palm region of Xyn11A could result in its high thermostability and activity. Introducing a disulfide bond at the N-terminus further increased its optimal reaction temperature to 90 degreesC with augmented activity. KEY POINTS: A hyperthermophilic xylanase with high activity was discovered using the metatranscriptomic method. The mechanisms of thermophilicity and high activity were revealed using X-ray crystallography, mutagenesis, and molecular dynamics simulations. The thermostability and activity were further improved by introducing a disulfide bond.
ESTHER : Yi_2021_Appl.Microbiol.Biotechnol_105_7757
PubMedSearch : Yi_2021_Appl.Microbiol.Biotechnol_105_7757
PubMedID: 34553251

Title : Rational design, synthesis and evaluation of uncharged, smart bis-oxime antidotes of organophosphate-inhibited human acetylcholinesterase - Gorecki_2020_J.Biol.Chem_295_4079
Author(s) : Gorecki L , Gerlits O , Kong X , Cheng X , Blumenthal DK , Taylor P , Ballatore C , Kovalevsky A , Radic Z
Ref : Journal of Biological Chemistry , 295 :4079 , 2020
Abstract : Corrected : Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime-based therapies whose success rates are currently limited. The pyridinium cation hampers uptake into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are non-productive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)-inhibited human AChE (hAChE), here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved in vitro reactivation of diverse OP-hAChE conjugates. Oxime group re-orientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Our findings provide detailed structure-activity properties of several CNS-directed, uncharged aliphatic bis-oximes holding promise for use as protonation-dependent, conformationally adaptive, "smart" accelerated antidotes against OP toxicity.
ESTHER : Gorecki_2020_J.Biol.Chem_295_4079
PubMedSearch : Gorecki_2020_J.Biol.Chem_295_4079
PubMedID: 32019865
Gene_locus related to this paper: human-ACHE

Title : A new crystal form of human acetylcholinesterase for exploratory room-temperature crystallography studies - Gerlits_2019_Chem.Biol.Interact_13ChEPon_
Author(s) : Gerlits O , Ho KY , Cheng X , Blumenthal D , Taylor P , Kovalevsky A , Radic Z
Ref : Chemico-Biological Interactions , : , 2019
Abstract : Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 A resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under closer-to-physiological temperature conditions.
ESTHER : Gerlits_2019_Chem.Biol.Interact_13ChEPon_
PubMedSearch : Gerlits_2019_Chem.Biol.Interact_13ChEPon_
PubMedID: 31176713
Gene_locus related to this paper: human-ACHE

Title : Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase - Gerlits_2019_J.Biol.Chem_294_10607
Author(s) : Gerlits O , Kong X , Cheng X , Wymore T , Blumenthal DK , Taylor P , Radic Z , Kovalevsky A
Ref : Journal of Biological Chemistry , 294 :10607 , 2019
Abstract : Exposure to organophosphorus compounds (OPs) may be fatal if untreated, and a clear and present danger posed by nerve agent OPs has become palpable in recent years. OPs inactivate acetylcholinesterase (AChE) by covalently modifying its catalytic serine. Inhibited AChE cannot hydrolyze the neurotransmitter acetylcholine leading to its build-up at the cholinergic synapses and creating an acute cholinergic crisis. Current antidotes, including oxime reactivators that attack the OP-AChE conjugate to free the active enzyme, are inefficient. Better reactivators are sought, but their design is hampered by a conformationally rigid portrait of AChE extracted exclusively from 100K X-ray crystallography and scarcity of structural knowledge on human AChE (hAChE). Here, we present room temperature X-ray structures of native and VX-phosphonylated hAChE with an imidazole-based oxime reactivator, RS-170B. We discovered that inhibition with VX triggers substantial conformational changes in bound RS-170B from a "nonproductive" pose (the reactive aldoxime group points away from the VX-bound serine) in the reactivator-only complex to a "semi-productive" orientation in the VX-modified complex. This observation, supported by concurrent molecular simulations, suggested that the narrow active-site gorge of hAChE may be significantly more dynamic than previously thought, allowing RS-170B to reorient inside the gorge. Furthermore, we found that small molecules can bind in the choline-binding site hindering approach to the phosphorous of VX-bound serine. Our results provide structural and mechanistic perspectives on the reactivation of OP-inhibited hAChE and demonstrate that structural studies at physiologically relevant temperatures can deliver previously overlooked insights applicable for designing next-generation antidotes.
ESTHER : Gerlits_2019_J.Biol.Chem_294_10607
PubMedSearch : Gerlits_2019_J.Biol.Chem_294_10607
PubMedID: 31138650
Gene_locus related to this paper: human-ACHE

Title : IMAGINE: neutrons reveal enzyme chemistry - Schroder_2018_Acta.Crystallogr.D.Struct.Biol_74_778
Author(s) : Schroder GC , O'Dell WB , Myles DAA , Kovalevsky A , Meilleur F
Ref : Acta Crystallographica D Struct Biol , 74 :778 , 2018
Abstract : Neutron diffraction is exquisitely sensitive to the positions of H atoms in protein crystal structures. IMAGINE is a high-intensity, quasi-Laue neutron crystallography beamline developed at the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory. This state-of-the-art facility for neutron diffraction has enabled detailed structural analysis of macromolecules. IMAGINE is especially suited to resolve individual H atoms in protein structures, enabling neutron protein structures to be determined at or near atomic resolutions from crystals with volumes of less than 1mm(3) and unit-cell edges of less than 150A. Beamline features include elliptical focusing mirrors that deliver neutrons into a 2.0 x 3.2mm focal spot at the sample position, and variable short- and long-wavelength cutoff optics that provide automated exchange between multiple wavelength configurations. This review gives an overview of the IMAGINE beamline at the HFIR, presents examples of the scientific questions being addressed at this beamline, and highlights important findings in enzyme chemistry that have been made using the neutron diffraction capabilities offered by IMAGINE.
ESTHER : Schroder_2018_Acta.Crystallogr.D.Struct.Biol_74_778
PubMedSearch : Schroder_2018_Acta.Crystallogr.D.Struct.Biol_74_778
PubMedID: 30082513

Title : Limitations in current acetylcholinesterase structure-based design of oxime antidotes for organophosphate poisoning - Kovalevsky_2016_Ann.N.Y.Acad.Sci_1378_41
Author(s) : Kovalevsky A , Blumenthal DK , Cheng X , Taylor P , Radic Z
Ref : Annals of the New York Academy of Sciences , 1378 :41 , 2016
Abstract : Acetylcholinesterase (AChE; EC, an essential enzyme of cholinergic neurotransmission in vertebrates, is a primary target in acute nerve agent and organophosphate (OP) pesticide intoxication. Catalytically inactive OP-AChE conjugates formed between the active-center serine and phosphorus of OPs can, in principle, be reactivated by nucleophilic oxime antidotes. Antidote efficacy is limited by the structural diversity of OP-AChE conjugates resulting from differences in the structure of the conjugated OP, the different active-center volumes they occupy when conjugated to the active-center serine of AChE, and the distinct chemical characteristics of both OPs and oximes documented in numerous X-ray structures of OP-conjugated AChEs. Efforts to improve oxime reactivation efficacy by AChE structure-based enhancement of oxime structure have yielded only limited success. We outline here the potential limitations of available AChE X-ray structures that preclude an accurate prediction of oxime structures, which are necessary for association in the OP-AChE gorge and nucleophilic attack of the OP-conjugated phosphorus.
ESTHER : Kovalevsky_2016_Ann.N.Y.Acad.Sci_1378_41
PubMedSearch : Kovalevsky_2016_Ann.N.Y.Acad.Sci_1378_41
PubMedID: 27371941