Decreased Interfacial Dynamics Caused by the N501Y Mutation in the SARS-CoV-2 S1 Spike:ACE2 Complex

Ahmed, Wesam S.; Philip, Angelin M.; Biswas, Kabir H.

doi:10.3389/fmolb.2022.846996

Cited by 10 publications

(10 citation statements)

References 84 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We then performed dynamic cross-correlation (DCC) analysis of residues using the MD-TASK suite of MD simulation analysis software 43 to elucidate any alterations in the correlated motions in the WT and E50A mutant picALuc proteins. 44 The WT picALuc showed both positively and negatively correlated motions in the residues with prominent correlations observed for residues close to the residue E50 (Fig. 7C).…”

Section: Resultsmentioning

confidence: 90%

“…We utilized the available nuclear magnetic resonance (NMR)-derived structure of GLuc (PDB: 7D2O) 1 to generate a homology-based structural model of the picALuc using the SWISS-MODEL webserver 51 . 41, 44, 52 The two proteins, picALuc and GLuc show considerable sequence similarity (92.5%; and 85% identity) with key differences in the N- and C-terminal (Supplementary Figure 1A) allowing high confidence structural modelling of picALuc. Notably, picALuc lacks N-terminal a-helices 1 and 2 and some residues in the C-terminal region of ALuc (Supplementary Figure 1B).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

Biswas

2023

Preprint

View full text Add to dashboard Cite

Recently, a miniaturized variant of an artificial luciferase (ALuc), named picALuc, with a molecular weight of 13 kDa and thus, the smallest luciferase, was reported. While picALuc was found to be as active as the ALuc, questions remained on the structural organization and residue-residue interactions in the protein. Here, combining structural modeling, molecular dynamics (MD) simulations and mutational analysis, we show that the loss of a salt bridge interaction formed by Glu50 (E50) residue results in an increased enzymatic activity of picALuc. Specifically, we generated a model of picALuc using the available structure of the Gaussia luciferase (GLuc) and performed a 1 μs long Gaussian accelerated molecular dynamics (GaMD) simulation which revealed a general compaction of the protein structure as well as residue level interactions in the protein. Given that picALuc contains a number of charged residues, we focused our attention to salt bridge interactions and decided to mutate E10, E50 and D94 that were found to form a fluctuating, stable or a new salt bridge interaction, respectively. Live cell assays showed an enhanced bioluminescence in cells expressing the E50A mutant picALuc while in vitro assays revealed an increased Vmax of the E50A mutant without affecting its thermal stability. Dynamic cross-correlation and principal component analyses of the GaMD simulation trajectories revealed altered collective dynamics in the protein, in which residue E50 contributed substantially. Finally, we developed a protein fragment complementation assay using picALuc that allows monitoring protein-protein interaction in live cells. We envisage that the brighter variant of picALuc and the protein fragment complementation assay reported here will find a general applicability in developing bioluminescence-based assays and the strategy developed here will pave the way for further engineering of brighter variants of picALuc.

show abstract

Section: Resultsmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

Biswas

2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Following energy minimization, the system was slowly heated from 60 to 310 at 1 K interval to reach the 310 K equilibrium temperature using a temperature ramp that runs 500 steps after each temperature increment. Following thermalization, temperature was maintained at 310 K using Langevin temperature control and at 1.0 atm using Nose–Hoover Langevin piston pressure control 93 , 94 . The system was then equilibrated with 500,000 steps (1 ns) using Periodic Boundary Conditions.…”

Section: Methodsmentioning

confidence: 99%

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

et al. 2022

Self Cite

View full text Add to dashboard Cite

The main protease, Mpro, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting Mpro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with Mpro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in Mpro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

show abstract

“…Molecular mechanics/generalized Born surface area (MM/GBSA) free-energy calculations were conducted using VMD to evaluate the enthalpic contribution to binding free energy. − This follows examples set by several recent works in which MM/GBSA proved useful in estimating protein–protein binding free energy. − In particular, MM/GBSA and related methods have been used to study the effects of mutations on the complex between the SARS-Cov-2 S RDB and the ACE2 ectodomain. − We consider here the same PDB structure (6M0J) as a basis to use the same method and to estimate the enthalpic contribution to binding free energy between SARS-CoV-2 S RBD and ACE2.…”

Section: Methodsmentioning

confidence: 99%

Entropic Overcompensation of the N501Y Mutation on SARS-CoV-2 S Binding to ACE2

Vergara

Gatchel

Abrams

2022

J. Chem. Inf. Model.

View full text Add to dashboard Cite

Recent experimental work has shown that the N501Y mutation in the SARS-CoV-2 S glycoprotein’s receptor binding domain (RBD) increases binding affinity to the angiotensin-converting enzyme 2 (ACE2), primarily by overcompensating for a less favorable enthalpy of binding by greatly reducing the entropic penalty for complex formation, but the basis for this entropic overcompensation is not clear [J. Biol. Chem.2021297101151]. We use all-atom molecular dynamics simulations and free-energy calculations to qualitatively assess the impact of the N501Y mutation on the enthalpy and entropy of binding of RBD to ACE2. Our calculations correctly predict that N501Y causes a less favorable enthalpy of binding to ACE2 relative to the original strain. Furthermore, we show that this is overcompensated for by a more entropically favorable increase in large-scale quaternary flexibility and intraprotein root mean square fluctuations of residue positions upon binding in both RBD and ACE2. The enhanced quaternary flexibility stems from N501Y’s ability to remodel the inter-residue interactions between the two proteins away from interactions central to the epitope and toward more peripheral interactions. These findings suggest that an important factor in determining protein–protein binding affinity is the degree to which fluctuations are distributed throughout the complex and that residue mutations that may seem to result in weaker interactions than their wild-type counterparts may yet result in increased binding affinity thanks to their ability to suppress unfavorable entropy changes upon binding.

show abstract

Decreased Interfacial Dynamics Caused by the N501Y Mutation in the SARS-CoV-2 S1 Spike:ACE2 Complex

Cited by 10 publications

References 84 publications

A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

Entropic Overcompensation of the N501Y Mutation on SARS-CoV-2 S Binding to ACE2

Contact Info

Product

Resources

About