Engineering topics

Kovalevsky, Andrii Y.

Publications and source records attributed to Kovalevsky, Andrii Y..

7 records

DOE OSTI2020

Supercomputer-Based Ensemble Docking Drug Discovery Pipeline with Application to Covid-19

In this work, we present a supercomputer-driven pipeline for in silico drug discovery using enhanced sampling molecular dynamics (MD) and ensemble docking. Ensemble docking makes use of MD results by docking compound databases into representative protein binding-site conformations, thus taking into account the dynamic properties of the binding sites. We also describe preliminary results obtained for 24 systems involving eight proteins of the proteome of SARS-CoV-2. The MD involves temperature replica exchange enhanced sampling, making use of massively parallel supercomputing to quickly sample the configurational space of protein drug targets. Using the Summit supercomputer at the Oak Ridge National Laboratory, more than 1 ms of enhanced sampling MD can be generated per day. We have ensemble docked repurposing databases to 10 configurations of each of the 24 SARS-CoV-2 systems using AutoDock Vina. Comparison to experiment demonstrates remarkably high hit rates for the top scoring tranches of compounds identified by our ensemble approach. We also demonstrate that, using Autodock-GPU on Summit, it is possible to perform exhaustive docking of one billion compounds in under 24 h. Finally, we discuss preliminary results and planned improvements to the pipeline, including the use of quantum mechanical (QM), machine learning, and artificial intelligence (AI) methods to cluster MD trajectories and rescore docking poses.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Studying the Role of a Single Mutation of a Family 11 Glycoside Hydrolase Using High-Resolution X-ray Crystallography

XynII is a family 11 glycoside hydrolase that uses the retaining mechanism for catalysis. In the active site, E177 works as the acid/base and E86 works as the nucleophile. Mutating an uncharged residue (N44) to an acidic residue (D) near E177 decreases the enzyme’s optimal pH by ~ 1.0 unit. D44 was previously suggested to be a second proton carrier for catalysis. To test this hypothesis, we abolished the activity of E177 by mutating it to be Q, and mutated N44 to be D or E. These double mutants have dramatically decreased activities. Our high-resolution crystallographic structures and the microscopic pK a calculations show that D44 has similar position and pK a value during catalysis, indicating that D44 changes electrostatics around E177, which makes it prone to rotate as the acid/base in acidic conditions, thus decreases the pH optimum. Our results could be helpful to design enzymes with different pH optimum.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Chapter Twelve - Proton transfer and drug binding details revealed in neutron diffraction studies of wild-type and drug resistant HIV-1 protease

HIV-1 protease is an essential therapeutic target for the design and development of antiviral inhibitors to treat AIDS. We used room temperature neutron crystallography to accurately determine hydrogen atom positions in several protease complexes with clinical drugs, amprenavir and darunavir. Hydrogen bonding interactions were carefully mapped to provide an unprecedented picture of drug binding to the protease target. We demonstrate that hydrogen atom positions within the enzyme catalytic site can be altered by introducing drug resistant mutations and by protonating surface residues that trigger proton transfer reactions between the catalytic Asp residues and the hydroxyl group of darunavir. When protein perdeuteration is not feasible, we validate the use of initial H/D exchange with unfolded protein and partial deuteration in pure D2O with hydrogenous glycerol to maximize deuterium incorporation into the protein, with no detrimental effects on the growth of quality crystals suitable for neutron diffraction experiments.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Rational design, synthesis, and evaluation of uncharged, “smart” bis-oxime antidotes of organophosphate-inhibited human acetylcholinesterase

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 of OPs into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are nonproductive 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, 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-human AChE conjugates. Oxime group reorientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Finally, 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.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

IMAGINE: The neutron protein crystallography beamline at the high flux isotope reactor

IMAGINE is a high intensity, quasi-Laue neutron crystallography beamline developed at the 85 MW High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory (ORNL). This state-of-the-art facility for neutron-diffraction enables neutron protein structures to be determined at or near atomic resolutions from crystals with volumes of < 1 mm 3 and unit cell edges of < 150 Å. The beamline features include elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm 2 focal spot at the sample position, and variable short and long wavelength cutoff optics that provide automated exchange between multiple wavelength configurations. The beamline is equipped with a single-axis goniometer, neutron-sensitive cylindrical image plate detector and room temperature and cryogenic sample environments. This article describes the beamline components, the diffractometer and the data collection and data analysis protocols that are used, and outlines the protein deuteration, crystallization and conventional crystallography capabilities that are available to users at ORNL's neutron facilities. We also present examples of the scientific questions being addressed at this beamline and highlight important findings in enzyme chemistry that have been made possible by IMAGINE.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Pyridoxal 5'-phosphate dependent reactions: Analyzing the mechanism of aspartate aminotransferase

Enzyme catalysis is the primary activity in energy and information metabolism and enzyme cofactors are key to the catalytic ability of most enzymes. Pyridoxal 5'-phosphate (PLP) cofactor, derived from Vitamin B6, is widely distributed in nature and has significant latitude in catalytic diversity. X-ray crystallography has revealed the structures of diverse PLP dependent enzymes from multiple families. But these structures are incomplete, lacking the positions of protons essential for understanding enzymatic mechanisms. Here, we review the diversity of PLP and discuss the use of neutron crystallography and joint X-ray/neutron refinement of Fold Type I aspartate aminotransferase to visualize the positions of protons in both the internal and external aldimine forms. Strategies used to prepare extremely large crystals required for neutron diffraction and the approach to data refinement including the PLP cofactor are discussed. The observed positions of protons, including one located in a previously unknown low-barrier hydrogen bond, have been used to create more accurate models for computational analysis. The results revealed a new mechanism for the transaminase reaction where hyperconjugation is key to reducing the energy barrier which finally provides a clear explanation of the Dunathan alignment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Protein kinase A in the neutron beam: Insights for catalysis from directly observing protons

Protein kinases transmit chemical signals by phosphorylating substrate proteins, thus regulating a multitude of cellular processes. cAMP-dependent protein kinase (PKA), a prototypical enzyme for the whole kinase family, has been the focus of research for several decades, however, the details of the chemical mechanism of phosphoryl group transfer have remained unknown. We used neutron crystallography to map key proton sites and hydrogen bonding interactions in the PKA catalytic subunit (PKAc) in a product complex containing adenosine diphosphate (ADP) and the phosphorylated high affinity protein kinase substrate (pPKS) peptide. To improve neutron diffraction, we deuterated PKAc allowing us to use very small crystals. In the product complex, the phosphoryl group of pPKS is protonated whereas the catalytic Asp166 is not. H/D exchange analysis of the main-chain amides and comparison with the NMR analysis of PKAc with inhibitor peptide complex revealed exchangeable amides that may distinguish the catalytic and inhibited states.

59 BASIC BIOLOGICAL SCIENCES↗