scholarly journals Structural Plasticity of the SARS-CoV-2 3CL Mpro Active Site Cavity Revealed by Room Temperature X-ray Crystallography

2020 ◽  
Author(s):  
Daniel W. Kneller ◽  
Gwyndalyn Phillips ◽  
Hugh M. O'Neill ◽  
Robert Jedrzejczak ◽  
Lucy Stols ◽  
...  
Keyword(s):  
Active Site ◽  
Room Temperature ◽  
Relevant Information ◽  
Docking Studies ◽  
Temperature Structure ◽  
Health Crisis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Main Protease ◽  
Active Site Cavity

Abstract The COVID-19 disease caused by the SARS-CoV-2 Coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the resting structure of the active site and the conformation of the catalytic site cavity. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

scholarly journals Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniel W. Kneller ◽  
Gwyndalyn Phillips ◽  
Hugh M. O’Neill ◽  
Robert Jedrzejczak ◽  
Lucy Stols ◽  
...  
Keyword(s):  
Active Site ◽  
Room Temperature ◽  
Structural Plasticity ◽  
X Ray ◽  
X Ray Crystallography ◽  
Active Site Cavity

scholarly journals Synthesis, Molecular Docking Analysis, and Carbonic Anhydrase Inhibitory Evaluations of Benzenesulfonamide Derivatives Containing Thiazolidinone

Molecules ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2418
Author(s):  
Zuo-Peng Zhang ◽  
Ze-Fa Yin ◽  
Jia-Yue Li ◽  
Zhi-Peng Wang ◽  
Qian-Jie Wu ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Active Site ◽  
Docking Studies ◽  
Active Site Residues ◽  
Single Crystal Diffraction ◽  
Docking Analysis ◽  
X Ray ◽  
Active Site Cavity ◽  
Molecular Docking Analysis ◽  
Positive Controls

To find novel human carbonic anhydrase (hCA) inhibitors, we synthesized thirteen compounds by combining thiazolidinone with benzenesulfonamide. The result of the X-ray single-crystal diffraction experiment confirmed the configuration of this class of compounds. The enzyme inhibition assays against hCA II and IX showed desirable potency profiles, as effective as the positive controls. The docking studies revealed that compounds (2) and (7) efficiently bound in the active site cavity of hCA IX by forming sufficient interactions with active site residues. The fragment of thiazolidinone played an important role in the binding of the molecules to the active site.

scholarly journals Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Aron Broom ◽  
Rojo V. Rakotoharisoa ◽  
Michael C. Thompson ◽  
Niayesh Zarifi ◽  
Erin Nguyen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Protein Design ◽  
Active Site ◽  
Room Temperature ◽  
De Novo ◽  
Conformational Ensemble ◽  
Enzyme Design ◽  
X Ray ◽  
Conformational Ensembles ◽  
X Ray Crystallography

Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

scholarly journals Michaelis-like complex of SARS-CoV-2 main protease visualized by room-temperature X-ray crystallography

IUCrJ ◽  
2021 ◽  
Vol 8 (6) ◽  
Author(s):  
Daniel W. Kneller ◽  
Qiu Zhang ◽  
Leighton Coates ◽  
John M. Louis ◽  
Andrey Kovalevsky
Keyword(s):  
Room Temperature ◽  
Catalytic Mechanism ◽  
Complex Structure ◽  
Catalytic Site ◽  
Strong Electron ◽  
X Ray ◽  
X Ray Crystallography ◽  
Main Protease ◽  
Inactivating Mutation ◽  
Important Drug

SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145–His41 dyad, can help in guiding drug design. Here, a 2.0 Å resolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3′ by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.

scholarly journals Identification of SARS-CoV2 Main Protease Coldspots Suitable for Drug Targeting

2020 ◽  
Author(s):  
Navaneethakrishnan Krishnamoorthy ◽  
Khalid Fakhro
Keyword(s):  
Active Site ◽  
Missense Mutations ◽  
The Novel ◽  
X Ray ◽  
X Ray Crystallography ◽  
Main Protease ◽  
New Perspective ◽  
Function Relationship ◽  
Novel Coronavirus ◽  
Relationship Of

Abstract Most attempts to target the novel coronavirus SARS-CoV2 are focusing on the main protease (Mpro) 1,2. We already have access to high resolution 3D-structures of the SARS-CoV2 Mpro, which were developed with inhibitors as co-crystals using X-ray crystallography 3-9. However, >19,000 missense mutations in the Mpro have already been reported 10. The mutations encompassing 282 amino acid positions and these “hotspots” might change the Mpro structure and activity, potentially rendering novel antivirals and vaccines ineffective. Here we identified 24 mutational “coldspots” that have resisted mutation since the virus was first detected. We compared the structure-function relationship of these coldspots with several SARS-CoV2 Mpro X-ray crystal structures. We found that three coldspot residues (Leu141, Phe185 and Gln192) help to form the active site, while six (Gly2, Arg4, Tyr126, Lys137, Leu141 and Leu286) contribute to dimer formation that is required for Mpro activity. Importantly, seven coldpots are conserved among other coronaviruses and available on the surface of the active site and at the dimer interface for targeting. The identification and short list of these coldspots offers a new perspective to target the SARS-CoV2 Mpro while avoiding mutation-based drug resistance.

scholarly journals Evolution of an enzyme conformational ensemble guides design of an efficient biocatalyst

2020 ◽  
Author(s):  
Aron Broom ◽  
Rojo V. Rakotoharisoa ◽  
Michael C. Thompson ◽  
Niayesh Zarifi ◽  
Erin Nguyen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Protein Design ◽  
Active Site ◽  
Room Temperature ◽  
De Novo ◽  
Conformational Ensemble ◽  
Enzyme Design ◽  
X Ray ◽  
Conformational Ensembles ◽  
X Ray Crystallography

AbstractThe creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we used room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 160 M−1s−1). We observed that catalytic residues were increasingly rigidified, the active site became better pre-organized, and its entrance was widened. Based on these observations, we engineered HG4, an efficient biocatalyst (kcat/KM 120,000 M−1s−1) containing active-site mutations found during evolution but not distal ones. HG4 structures revealed that its active site was pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

Synthesis, structural characterization and catalytic activity of chlororuthenium(II) complexes with substituted Schiff base/phosphine ancillary ligands

2020 ◽  
Vol 75 (9-10) ◽  
pp. 851-857
Author(s):  
Chong Chen ◽  
Fule Wu ◽  
Jiao Ji ◽  
Ai-Quan Jia ◽  
Qian-Feng Zhang
Keyword(s):  
Schiff Base ◽  
Catalytic Activity ◽  
Structural Characterization ◽  
Room Temperature ◽  
Molecular Structures ◽  
Cationic Complex ◽  
Neutral Complex ◽  
Ancillary Ligands ◽  
X Ray ◽  
X Ray Crystallography

AbstractTreatment of [(η6-p-cymene)RuCl2]2 with one equivalent of chlorodiphenylphosphine in tetrahydrofuran at reflux afforded a neutral complex [(η6-p-cymene)RuCl2(κ1-P-PPh2OH)] (1). Similarly, the reaction of [Ru(bpy)2Cl2·2H2O] (bpy = 2,2′-bipyridine) and chlorodiphenylphosphine in methanol gave a cationic complex [Ru(bpy)2Cl(κ1-P-PPh2OCH3)](PF6) (2), while treatment of [RuCl2(PPh3)3] with [2-(C5H4N)CH=N(CH2)2N(CH3)2] (L1) in tetrahydrofuran at room temperature afforded a ruthenium(II) complex [Ru(PPh3)Cl2(κ3-N,N,N-L1)] (3). Interaction of the chloro-bridged complex [Ru(CO)2Cl2]n with one equivalent of [Ph2P(o-C6H4)CH=N(CH2)2N(CH3)2] (L2) led to the isolation of [Ru(CO)Cl2(κ3-P,N,N-L2)] (4). The molecular structures of the ruthenium(II) complexes 1–4 have been determined by single-crystal X-ray crystallography. The properties of the ruthenium(II) complex 4 as a hydrogenation catalyst for acetophenone were also tested.

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