Crystallographic models of SARS-CoV-2 3CL^pro: in-depth assessment of structure quality and validation

Abstract

The appearance at the end of 2019 of the new SARS-CoV-2 coronavirus led to an unprecedented response by the structural biology community, resulting in the rapid determination of many hundreds of structures of proteins encoded by the virus. As part of an effort to analyze and, if necessary, remediate these structures as deposited in the Protein Data Bank (PDB), this work presents a detailed analysis of 81 crystal structures of the main protease 3CLpro, an important target for the design of drugs against COVID-19. The structures of the unliganded enzyme and its complexes with a number of inhibitors were determined by multiple research groups using different experimental approaches and conditions; the resulting structures span 13 different polymorphs representing seven space groups. The structures of the enzyme itself, all determined by molecular replacement, are highly similar, with the exception of one polymorph with a different inter-domain orientation. However, a number of complexes with bound inhibitors were found to pose significant problems. Some of these could be traced to faulty definitions of geometrical restraints for ligands and to the general problem of a lack of such information in the PDB depositions. Several problems with ligand definition in the PDB itself were also noted. In several cases extensive corrections to the models were necessary to adhere to the evidence of the electron-density maps. Taken together, this analysis of a large number of structures of a single, medically important protein, all determined within less than a year using modern experimental tools, should be useful in future studies of other systems of high interest to the biomedical community.

Characterization of the non-covalent interaction between the PF-07321332 inhibitor and the SARS-CoV-2 main protease

“…In this regard, neutron scattering experiments [ 20 ] conducted at pH 6.6 and room temperature have shown that HIS41 is protonated and CYS145 is deprotonated on the SARS-CoV-2 3CL pro protein also. This is at variance with the results presented on a previous study by the same authors [ 18 ], in agreement with the consensus base catalysis mechanism [ 22 ] and with the results obtained in Ref. [ 23 ] on the homologous SARS-CoV-1 3CL pro , where the measured pKa of CYS (8.3) and HIS (6.4) are consistent with a general base catalysis Chymotrypsin mechanism and cannot be explained by a thiolate-imidazolium ion pair model.…”

“…The 3CL pro dimer has two symmetric extended clefts for optimal (linear) peptide chain adhesion [ 35 ]. The dimer interface involves the N-terminus of domain I + II and the C-terminus of domain III with no participation of the distal and solvent exposed catalytic site [ 22 , 35 ]. When expressed independently, while domain III has no role in the catalysis [ 33 ], the isolated domain I + II is still capable of cleaving a 14-mer peptidic substrate mimicking the N-terminal autocleavage sites of the SARS 3CL pro , although with a much smaller turnover number with respect to the 3CL pro dimer [ 33 ].…”

“…MD simulations were carried out for the Chymotrypsin-like catalytic domain I + II [ 22 , 41 ] of the two isoforms using, in turn, the Amber99sb-ildn [ 42 ] and the OPLS-AA/M FFs [ 32 ] with the GROMACS code [ 43 ] (version 2018.8). In the H–C isoform, H41 was assigned to the tautomer with the protonated N δ [ 25 ].…”

Combining X-rays, neutrons and electrons, and NMR, for precision and accuracy in structure–function studies

“…The divergence of the PDB from the Cambridge Structural Database (CSD) has also led to concerns by the CCDC (Cambridge Crystallographic Data Centre) itself about the quality of ligand structures in the PDB (Liebeschuetz et al, 2012): 'The good, the bad and the twisted: a survey of ligand geometry in protein crystal structures'. This has been reiterated by Jaskolski et al (2021) in a study of nearly 100 coronavirus main protease crystal structures deposited in the PDB, who concluded with a list of problems associated with reliability of the deposited structures, and offered a list of procedural weaknesses of these database depositions.…”

Dr. Alexander Wlodawer—celebrating five decades of service to the structural biology community

“…Several collaborative papers were also dedicated to proper validation of atomic models of macromolecules and the identification of 'bad apples' among published results [25][26][27][28][29][30]. Recently, Alex has been instrumental in validating the structures of proteins from the SARS-CoV-2 coronavirus that are rapidly accumulating in the PDB in response to the COVID-19 pandemic [31][32][33][34].…”