Single point mutant inverts stereoselectivity of a carbonyl reductase toward β‐ketoesters with enhanced activity

Abstract The interaction between the receptor-binding domain of the SARS-CoV-2 spike glycoprotein and the ACE2 enzyme is believed to be the entry point of the virus into various cells in the body, including the lungs, heart, liver, and kidneys. The current focus of several therapeutic design efforts explore attempts at affecting the binding interaction between the two proteins to limit the activity of the virus and disease progression. In this work, we analyze the stability of the spike protein under all possible single-point mutations in the receptor-binding domain and computationally explore mutations that can affect the binding with the ACE2 enzyme. We unravel the mutation landscape of the receptor region and assess the toxicity potential of single and multi-point mutations, generating insights for future vaccine efforts on potential mutations that might further stabilize the spike protein and increase its infectivity. We developed a tool, called SpikeMutator, to construct full atomic protein structures of the mutant spike proteins and shared a database of 3,800 single-point mutant structures. We analyzed the recent 65,000 reported spike sequences across the globe and observed the emergence of stable multi-point mutant structures. Using the landscape, we searched through 7.5 million possible 2-point mutation combinations and report that the (R355D K424E) mutation produces one of the strongest spike proteins that therapeutic efforts should investigate for the sake of developing an effective vaccine.

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K+ Conduction and Mg2+ Blockade in a Shaker Kv-Channel Single Point Mutant with an Unusually High Conductance

Biophysical Journal ◽

10.1016/j.bpj.2012.08.015 ◽

2012 ◽

Vol 103 (6) ◽

pp. 1198-1207 ◽

Cited By ~ 6

Author(s):

Cristian Moscoso ◽

Ariela Vergara-Jaque ◽

Valeria Márquez-Miranda ◽

Romina V. Sepúlveda ◽

Ignacio Valencia ◽

...

Keyword(s):

Single Point ◽

Point Mutant ◽

Kv Channel ◽

High Conductance

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Expression, purification and preliminary crystallographic studies of a single-point mutant of Mos1 mariner transposase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444904003798 ◽

2004 ◽

Vol 60 (5) ◽

pp. 962-964 ◽

Cited By ~ 14

Author(s):

Julia M. Richardson ◽

Lei Zhang ◽

Severine Marcos ◽

David J. Finnegan ◽

Marjorie M. Harding ◽

...

Keyword(s):

Single Point ◽

Point Mutant

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An MHV macrodomain mutant predicted to lack ADP-ribose binding activity is severely attenuated, indicating multiple roles for the macrodomain in coronavirus replication

10.1101/2021.03.30.437796 ◽

2021 ◽

Author(s):

Lynden S Voth ◽

Joseph J O'Connor ◽

Catherine M Kerr ◽

Ethan Doerger ◽

Nancy Schwarting ◽

...

Keyword(s):

Hepatitis Virus ◽

Single Point ◽

Binding Activity ◽

Multiple Roles ◽

Structural Protein ◽

Transcript Accumulation ◽

Murine Hepatitis Virus ◽

Point Mutant

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes ADP-ribose covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutant of a highly conserved asparagine-to-alanine mutation, which is known to largely eliminate Mac1 ADP-ribosylhydrolase activity. To determine if Mac1 ADP-ribose binding separately contributes to CoV replication, we compared the replication of a murine hepatitis virus (MHV) Mac1 mutant predicted to dramatically reduce ADP-ribose binding, D1329A, to the previously mentioned asparagine mutant, N1347A. D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. However, D1329A was significantly more attenuated than N1347A in all cell lines tested that were susceptible to MHV infection. In addition, D1329A retained some ability to block IFN-β transcript accumulation compared to N1347A, indicating that these two mutants impacted distinct Mac1 functions. Mac1 mutants predicted to eliminate both binding and hydrolysis activities were unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple roles in promoting the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics.

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Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity

Biochemical Journal ◽

10.1042/bj20040735 ◽

2004 ◽

Vol 383 (3) ◽

pp. 429-437 ◽

Cited By ~ 47

Author(s):

David E. LANCASTER ◽

Luke A. McNEILL ◽

Michael A. McDONOUGH ◽

Robin T. APLIN ◽

Kirsty S. HEWITSON ◽

...

Keyword(s):

Single Point ◽

Hypoxia Inducible Factor ◽

Transcriptional Response ◽

Structural Data ◽

Dimer Interface ◽

Point Mutant ◽

Catalytically Active ◽

Substrate Phosphorylation ◽

Asparagine Residue ◽

Inhibit Factor

HIF (hypoxia-inducible factor) is an αβ transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-α are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-α (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340→Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344→Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.

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Structure of the His44 → Ala Single Point Mutant of the Distal Finger Motif of HIV-1 Nucleocapsid Protein: A Combined NMR, Molecular Dynamics Simulation, and Fluorescence Study†

Biochemistry ◽

10.1021/bi036137u ◽

2004 ◽

Vol 43 (24) ◽

pp. 7687-7697 ◽

Cited By ~ 16

Author(s):

Roland H. Stote ◽

Esther Kellenberger ◽

Hervé Muller ◽

Elisa Bombarda ◽

Bernard P. Roques ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Nucleocapsid Protein ◽

Single Point ◽

Protein A ◽

Dynamics Simulation ◽

Fluorescence Study ◽

Point Mutant ◽

Hiv 1

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Thermodynamics and Kinetics of Non-native Interactions in Protein Folding: A Single Point Mutant Significantly Stabilizes the N-terminal Domain of L9 by Modulating Non-native Interactions in the Denatured State

Journal of Molecular Biology ◽

10.1016/j.jmb.2004.02.073 ◽

2004 ◽

Vol 338 (4) ◽

pp. 827-837 ◽

Cited By ~ 86

Author(s):

Jae-Hyun Cho ◽

Satoshi Sato ◽

Daniel P. Raleigh

Keyword(s):

Protein Folding ◽

Single Point ◽

Denatured State ◽

Point Mutant ◽

Thermodynamics And Kinetics ◽

Terminal Domain ◽

Kinetics Of

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Unraveling the stability landscape of mutations in the SARS-CoV-2 receptor-binding domain

Scientific Reports ◽

10.1038/s41598-021-88696-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mohamed Raef Smaoui ◽

Hamdi Yahyaoui

Keyword(s):

Receptor Binding ◽

Single Point ◽

Protein Structures ◽

Point Mutations ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Point Mutant ◽

The Stability ◽

Spike Proteins

AbstractThe interaction between the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and the ACE2 enzyme is believed to be the entry point of the virus into various cells in the body, including the lungs, heart, liver, and kidneys. The current focus of several therapeutic design efforts explores attempts at affecting the binding potential between the two proteins to limit the activity of the virus and disease progression. In this work, we analyze the stability of the spike protein under all possible single-point mutations in the RBD and computationally explore mutations that can affect the binding with the ACE2 enzyme. We unravel the mutation landscape of the receptor region and assess the toxicity potential of single and multi-point mutations, generating insights for future vaccine efforts on mutations that might further stabilize the spike protein and increase its infectivity. We developed a tool, called SpikeMutator, to construct full atomic protein structures of the mutant spike proteins and shared a database of 3800 single-point mutant structures. We analyzed the recent 65,000 reported spike sequences across the globe and observed the emergence of stable multi-point mutant structures. Using the landscape, we searched through 7.5 million possible 2-point mutation combinations and report that the (R355D K424E) mutation produces one of the strongest spike proteins that therapeutic efforts should investigate for the sake of developing effective vaccines.

Download Full-text